)3 tz 8+ - digital.library.adelaide.edu.au
TRANSCRIPT
)3 tz 8+
STRUCTURES OF
VIROIDS
VIRUSOIDS
AND
SATE LLI TES
Jim Hasel-of f B. Sc. (Hons )
Adelaide University Centre for Gene Technology '
South Aus traÌ i a.
Thesis submltLed to the university of Adelaide in
fulfil-lment of the requirements for the degree of
Doctor of PhilosoPhY.
May, 1 983.
CON TENTS
S TATEMEN T
A C KNOV\¡LED GEMEN TS
SUMMARY
CHA PTE R 1 TNTRODUCTION
A. Viroids
B. Vl rusoids
C. Aims
CHAPTER 2 RNA SESUENCE DETERMINATION
IN TROD U C TI ON
MATERIALS
ME THOD S
A. Isol-ation and sequence determination of
linear viroid or virusoid fragments
A-1 AnalYtical RNase digests
A-2 PreParatlve RNase digests
A-3 5'-32P fabell-ing of RNA
fragments
A-4 3'-32P l-abel1ing of RNA
1
4
5
I
o
fragments
i) Synthesis of Ir'-3'rldpcp
9
11
11
12
12
AEh-)
A-6
ri ) 3'-32P l-abeì,1i-ng
Polyacrylamide geI fractlonation
Sequence determination of RNA
fnagments using the partial enzymic
13
cfeavage technique
A-7 Sequence determination of RNA
fragments using the dideoxynucleotide
chaln termj-nation technique
i) PhosPhatase treatment
ii ) PolYadenYì-ation
ij-i) Reverse transcriPtion
A-B Bolyacnylamide gel elecLrophoresis
B. Sequence determination using cloned viroid
or virusoid sequences
B-1 Synthesls and cl-oning of viroid and
virusoid ds cDNA
i) LinearizaLion and polyadenyl-ation
ii ) First strand cDNA sYnthesis
iii ) Second strand cDNA sYnthesis
iv ) Restrj-ctlon enzyme cleavage and
fractionation
v) M1 3 cloning
B-2 Sequence determination of recombinant
phage M1 3
B-3 Sequence determination of RNAs using
cfoned DNA Prlmers
i) PneParation of Primer
ii ) RNA-DNA hYbnidizatlon
iii ) Reverse transcriPtion
RESULTS and DISCUSSION
A. Techniques
16
16
1T
18
14
1g
20
20
20
21
¿t
¿¿
¿J
23
24
B Partial- enzymic cleavage of viroid and
virusold RNAs
Partial enzymic cleavage of radiol abel-l-ed
RNA fnagments
Dideoxynucl-eotide chain termination
sequencing of RNA fragments
Cloning of vinoid and virusoid sequences
Sequence determination using cfoned viroid
or vinusoid sequences -
Detenmination of complete vinoid or
virusoid primanY structunes
D
CHAPTER 3 CHRYSANTHEMUM STUNT V]ROID
INTRODUCTI ON
MATER]ALS
ME THOD S
A. Primary structure determination
B. Secondary
RESULTS
structure determination
A. Sequence determination
B. Primary sequence and secondary sLructure
DISCUSS]ON
A. Homology between CSV and PSTV
B. Replication of CSV and PSTV
C. Rel-ationship of this isolate of CSV to
other viroid isol-ates
CHAPTER 4 COCONUT CADANG-CADANG VIROID
c
24
25
25
¿õ
27
E
F
G
2B
2g
30
30
3',I
31
33
33
34
36
INTRODUCT] ON 39
M ETHOD S
A. Isolation of the ccRNAs
B. Sizing of the ccRNAs
C. Fingerprinting of the ccRNAs
D. Sequence and structure deLermination of
the ccRNAs
RESULTS ANd DTSCUSSION
A. Sizing of the ccRNAs
B. Fingerprinting of the ccRNAs
C. Sequence and structure determination of
the ccRNAs
D. ccRNAs differ in size but not sequence
complexitY
E. Varlation in sequence between different
ccRNA isoÌates
F. Structural simitarities between ccRNAs and
viroids
G . Re plication of ccRNAs
H . ccR NA s l-ow variants and the time course of
infection
I. Origin of cadang-cadang disease
CHAPTER 5 VELVET TOBACCO MOTTLE V]RUS AND SOLANUM
NODIFLORUM MOTTLE V]RUS
INTRODUCTION
MATERIALS and METHODS
A. Viruses and RNA
41
42
43
44
46
47
48
50
52
53
55
56
5B
60
61
B. RNase Fingerprinting 62
C. BNA sequence analysis
i-) Partial- enzYmic digestion
fj- ) Di deoxynucleotide chain termlnation
D. Synlhesis and cloning of double-strand cDNA
RESULTS
A. RNase fingerprints of VTMoV and SNMV RNA 2
B. Primary structures of VTMoV and SNMV RNA 2
C. Secondary structures of VTMoV and
SNMV RNA 2
D. Possibl-e polypeptide translation products
from RNA 2 species and their compfements
DI SCUSS I ON
CHAFTER 6 SUBTERRANEAN CLOVER MOTTLE VIRUS
INTRODUCTION
MATER]ALS
METHODS
A. Synthesls and restriction endonucl-ease
cleavage of ds cDNA
B. Fingerprlnting of SCMoV RNAs
C. Sequence determlnation of SCMoV RNA 2 and
RNA 2I
Analysis of SCMoV RNA 1 nucleotide
sequences
RNase fingerprinting of SCMoV RNAs 2 and
FNAs 2t
Sequence determination of SCMoV-A RNA 2 and
62
62
63
63
64
66
o/
RESU LT S
A
B
6B
72
73
73
(¿1
74
75
C
76
RNA 2I
DI SCUSSI ON
A. RelationshiPs between the
of SCMoV
B. Sequence homology between
RNA 2I
C. Sequence homologY between
SNMV and LTSV RNAs 2
D. Satellite RNA of TobRV
E.'sequence homoì-ogY between
RNA and vlrusoids
v'arious isolates
SCMoV RNA 2 and
SCMoV, VTMOV,
TobRV s atellite
7B
79
79
BO
B2
B4
B6
B7
B7
8B
CHAPTER 7 VIROIDS VIRUSOIDS AND SATELLITES
INTRODUCTION
ME THOD S
A. tsolation of RNA
B. Blot hybridization
RESULTS
A. Analysls of VTMoV and SNMV RNA 2 sequences
present in vinus and infected tissues
DI SCUSS I ON
A. Multimers of VTMoV and SNMV RNA 2
B. A possible site for RNA processing
C. Vlroid, vlrusoid and satetlite RNAs
8,9
90
92
STAT EMEN T
This thesis has not previously been submibted for an
academic award at this or any other University, and
except where dueis the original work of the author,
the tex t .reference is made in
JIM HASELOFF
AC KN Ol/tr LED GEMENTS
I wish to thank Prof . VÙ. H. El1iot forpermission to underLake these studies in the Department.I aÌso wlsh to thank my supervisor, Bob Syrnons for theadvice and support provlded to me during the course ofthis wonk. In addition, I wish to express my
appreciation to the following people:Dr . Pe ter Palukaitis , for purified CSV and
arouslng my interest in viroids;Drs. Richard Fnanckl and John RandIes, for
heJ-pf ul- discussions and interesting vinuses;Dr. Nizar Mohamed, Julita Imperial and Judith
Rodrigue z, for providing ample amounts of ccRNAs;Dr. George BrueninS, f or unpubl-ished resul-ts
and stimulaLing discussion 1n the J-ab;Dr. Detl-ev Riesner' f or providing unpubl-ished
resufts and eintoPf;Dr. Al,l-an Gould, f or his help and dangerous
sense of humour;Karf Gordon, for his red wine and
socj-o-politics (more fike rosè), and scientificdiscussion;
My fel-1ow viroid/virusoj-d infectedco-workers, Dn. Peter Murphy, Jane Visvader and Paul-Keese, as weIf, as the other numerous faboratory anddepartmenlal lnmates;
Jenny Rosey, Sharon Freund and Lisa Waters,for exceflent technical assj-stance and preparation ofthe figures for thls thesis;
Mrs. To, for her care in typing this thesis;and finally my family, for putting up with
this student.
SUMMARY
The work described in this thesis coticerrls the
establishment and application of technj-ques for the rapidsequence determination of small circular RNAs such as those
of viroids and virusoids. The determined sequences ofchrysanthemum stunt viroid, the vari-ant RNAs of coconut
cadang-cadang viroid and the virusoids of velvet tobacco
mottle virus, solanum nodiflorum mottl-e virus and
subterranean clover mottle virus are presented. The overallconclusions from the work are outlined briefly below.
1. Viroids contain highly conserved sequences centralto their rod-like native structures.
2. Virusoids also contain highly conserved. sequences
central to their rod-like native structures and share the
pentanucleotid.e sequence GAAAC with that of viroids.3. In addition the conserved. seguences of vírusoids are
shared by the linear Rr\A of tobacco ringspot virus.
Presumably the coInmon sequences of each cl-ass ofRIIAs reflect common function, and perhaps suggest some
functional similarity between viroids and virusoids.Sequence homology between virusoids and the satellite RNA
of tobacco ringspot virus al-Iows pred.iction of sites forprocessing of these RNAs from multimeric RIJA intermediates
of replication.
CHAPTER 1
TNTRODUCTION
A. Viroids
Viroids constitute a unique class of
infectious plant pathogens, and as such are a fairly
recent dicovery. The viroid concept vJas first
recognized when the infectlous agents of the spindJ-e
tuber disease of potato (Diener: and Raymer, 1967 ) and
the exocortis disease of citrus (Semancik and lltleathers,
1968 ) , which vrere thought to be viruses, I^i ere shown to
possess unusual propertles : ( 1 ) phenol or other organic
sofvents had no effect upon the lnfectivity of buffered
extracts from lnfected plants; (2) no virus particles
coul-d be isolated or dernonstrated by el-ectron
microscopy;
resistance
( 3 ) the infectious agents displayed a
to nucfeases and an elutlon profile off
double-stranded RNA; andcel-Ìufose columns simil-ar to
(4 ) the inf ectious agents I^/ere always present in high
speed supernatants, possessing sedimentation
coefficients of 1 0- 1 5S. As a more detailed knowl-edge of
the sizes and physlco-chemicaf properties of these two
disease agents became availabl-e (Raymer and Diener'
1969; Diener and Raymer, 1969; Semancik and Weathers
1972a) , it became apparent that these two agents were
the first of a new cfass of infectious nucÌeic acids
(Diener, 1971b; Semanclk and Vrleathers, 1972b; Sånger,
1972); the Lerm virold I^Ias proposed (Diener, 1971b), and
L
the causal agents were renamed potato spindle tuber
viroid ( pSfV ) and citrus exocortls viroid ( CEV ) .
Since that t1me, viroids have been shown Lo
be the causative agents of a further eight plant
diseases, âhd are listed in Table 1 -1 . These viroids
consj-st of infectious l-ow mol-ecul-ar weight RNA species
which are unencapsidated, sì-ngJ-e sLranded,
coval-ent1y-closed circul-ar moleçul-es with a hì-gh degree
of intramol-ecul-ar base-pairing (Diener, 1972; Semancik
et â1., 1975; Sången et âf., 1976). Physj-co-chemica]
sludies of several- viroids (Henco et â1 . , 1977; KJ-ump et
âf., 197B; Langowski et â1., 1978; Domdey et âf., 1978)
cul-minated in a model fon viroid structure in which the
clrcufar RNA mofecuÌes exist as extended rod-l-ike
structures, characterized by series of base-paired
sections interspersed with single-stranded loop
sections. Determination of the compl-ete nucl-eotide
sequence of PSTV ( Gross et â1. , 1 978) , together with
dye-binding expeniments ( Riesner et â1 . , 1 979 ) and
tRNA-binding experiments (v'lild eL â1. , 1 980 ) established
the validity of this model.
Apart from their unique structures ' the
singJ-e feature which distinguishes viroids flrom vlruses
is their apparent Ìack of encoded polypeptlde products.
Viroids RNAs appear to be naked with no associated
protein (Diener, 1971a; Semancik and V'leathers, 1972a),
Table 1 -1 Viroids that are presentlv knownl
Viroid References
1
¿
5
6
potalo spindle tuberviroid
citrus exocortisviroid
chrysanthemum stuntvinoid
'l 0. Lomato planta machovi-roid
( PSTV )
(CEV)
(CSV)
(TBTV) (Vlalter,
( Galindo1 982)
(Diener, 1977 )
( Semancik andÍieathers, 1972;Sänger, 1972)
3
4 chrysanthemum chloroticmottle virold ( ChCMV )
cucumber pale frultvi roid ( CPFV )
coconut cadang-cadangviroid ( cccv )
(HSV)7. hop stunt viroid
avocado sunbl-otchviroid ( ASBV )
tomato bunchy topvi roi d
( Di ener and Lawson ,1973)
( Romaine and Horst,1975)
(Van Dorst andPeters, 1974)
(Randl-es, 1975)
(Sasaki and Shikata,1977 )
( Thomas andMohamed, 1979)
1981 )
et âf . ,
ö
9
( TPMV )
Recent studies indicate that the causative agent ofburdock stunt disease may possess propertles atypicalof those of other viroids (Chen Weì and 'T j-en Po,personal communication), and has been tenativelyomltted from the list.
3
and no viroid-coded transfation products have been found
either in vivo ( Conjero and Semancik, 1977; FIores et
â1., 1978; Conjero et af. , 197g; Camacho and Sången,
1 982a, b ) or ln vitro ( Davies et â1 . , 1 97 4; Semancik et
âf . , 1 977 \ . If viroids do not encode functional
polypeptide translation products, they must rely
entirely on plant host cefl- components for their
replicati-on.
Vlnold repl-icalion has been shown to be
inhibited by actinomycin D, inhibiting DNA-dependent RNA
synthesis (Diener and smith, 1975; Takahashi and Diener,
1975; Uünlbach and Sänger, 197g), and o'-amanitin aL
concentrations which inhibit RNA poJ-ymerase II, and thus
mRNA synthesis (Mühlbach and Sånger, 197g)- Whil-e the
effects of these drugs on vlroid replication may be
indirect, due to general effects on host cell
metaboJ_ism, some evldence suggests that DNA-dependent
RNA polymerase If, the target for o-amanitinr mâY play a
direct rofe in viroid replicatlon. Rackwitz et al.
( 1 981 ) have shown that RNA pol_ymerase If purified from
healthy plant tissues is capable of the o'-amanitln
sensitive transcription of full,-length llnear
complementany viroid RNAs from viroid template in vltro.
However, the in vitro transcription of viroid templates
has also been shown for RNA-dependent RNA polymerases
isolated from healthy plant tissue ( Boege et a1. ,1 982 )
4
and from cucumber mosaic virus infected plant tissue
( D. S. Gllt and R. H Symons, unpubl-ished resutts ) '
So, whil-e the exact naLure ofl the enzymes
involved in viroid replication in vivo remains uncfear,
the f oll_owing detail-s are known. (t ) virolds appear to
repJ-icate through compfementary RNA lntermediates ( Grill
and Semancik, 197B; Zaitl-in et âl ., 1980; Hadidi et â1.,
1 981; ZeIcer et â1., 1982). (2) Longer than unit-length
complenentary viroid RNA intermediates have been
detected in viroid infected tissue extracts ( Bnanch et
â1. , 1 981 ; Rohde anO Sånger, I 981 ; Owens and Diener '
1gB2; Bruening et â1. , 1982 ) (3 ) 0tigomeric series of
RNAs of avocado sunbl0tch vlroid (ASBV) have been
detected in infected avocado tissue (Bruening et al.,
1g82). Various workers have therefore postulated
roll-ing circle mechanisms for the transcription of
larger than unit length viroid intermediates (Branch et
â1., 1gB2; Owens and Dlener, 1982; Bruening a! âI',
1 982 ) . Such mechanisms require that unit-length Iinear
viroid, produced by either specific tnanscniption or
cleavage of ol-igomeric RNAs, b€ ligated to produce the
final circular Product.
B. Virusoids
Four members of a new and unique group of
plant viruses have been recently described ( Randles et
5
âI., 1981; Gould and Hatta, 1981; Tien-Po et â1., 1981 ) '
These vlruses, velvet tobacco mottle virus ( VTMoV ) ,
sofanum nodiflorum motLte virus ( SNMV ) , lucerne
transient streak virus (LTSV) and subterranean clover
mottl-e virus (SCMoV ) were isolated in AusLral-asia (see
Figure 1-1 ) and each consist of 30 nm polyhedral- capsids
containing two maior slngle-strand RNA species. RNA 1
is a l-inear molecule of about 4,500 res j-dues, whereas
RNA 2 is a circufar covalently closed mol-ecul-e of
300-400 residues with a high degree of internal-
base-pairlng. The RNA 2 molecules therefore possess
physical properties simll-ar to those of vlrolds and have
been termed virusoids.
Gould et aÌ. ( 1 981 ) have shown that both RNA
'l and RNA 2 of VTMoV and SNMV are required for viral
infection and Lhat therefore these vi-rusoid molecules
contribute
contras t ,
virusoid
lnfection
RNA. The nature of
components of SCMoV
some function essential-
Jones et al-. (1983) have
for replication.
shown that the
In
of LTSV is apparently not required for viral-
and that it therefore behaves as a satell-ite
the refatj-onship between the RNA
is unknown.
C. AIMS
Three unresofved questions stand behind the
work described in this thesis.
ORIGIN OF VIRUSES CONTAIN¡NG VIRUSOIDS
Adelaide
Melbourne
Brisbane
Perth
Sydney
New Zealando
o
WMoV
SNMV
LTSV
SCMOV
New South Wales
Vict
Queensland
South Australia
Territory
Northern
Western Austalia
e
Tasmania
6
(1)
(2)
How do viroids nepl-icate ?
( if any ) do virusoids contribute toVlhat
vi rus
Does
function
replication ?
address these Seneral- questions of virold
function, the primary and secondary
à number of these molecul-es v'iere
(3 ) the replication of viroids and vinusoids share
common features ?
In order to
and virusoid
structures of
determined.
essential for
mirroned by the
sequences an d/ or
TL was hoped that the location of regions
molecules woufd befunction within these
presence of conserved nucfeotide
structures.
The various techniques required for
viro id/v lnusoid sequence determinatlon are descrlbed in
lhe following chapten. Chapters 3, 4,5 and 6 describe
varlous apptications of these techniques, and the final-
chapter deals with the overall concÌusions from the
I^IOf k.
CHAPTER 2
RNA SEOUENCE DETERMINATION
7
] N TRODU CT I ON
The techniques availabte for lhe rapid
sequence determination of RNA rely on the presence of a
fixedreferencepointwithintheRNA,eg'auniquesite
for primed synthesls of transcnipts (Zlmmern and
Kaesberg, 1978 Symons' 1978 ) or a 5t or 3'
radiolabeÌIed terminus ( Donis-Kel-fer et âI' , 1977 ) ' and
therefore these techniques ane i-deaJ-1y applied to ri-near
RNAs.ModifiedapproacheSmuStbeusedfortheSequenCe
determination of circul-ar RNAs, and the approaches used
in this work fall into two classes'
First, by exploitlng the base-paired nature
ofviroidsandvirusoids,sPecificllnearRNAfragments
may be produced by partial RNase cleavage of the
circufarmolecu]-es.Thelinearvj.roidorvirusoid
fragmentsmayberadlo]-abelled,purifledandSequenced
using the partlal enzymic dlgestion ( Donis-KefIer eL
àI ., 1977) or dideoxynucleotide chain Lermination
(Zimmern and Kaesberg, 19781. Symons, 1978 ) sequencing
techniques. Second, double-strand cDNA can be
transcribedfnomfinearizedviroidorvirusoidRNA'
inserted and propagated in a bacteriophage M1 3 vector'
and the inserted DNA sequenced directJ-y (Sanger et â1.,
1 980 ) o r excised or transcrlbed and used to prime
dideoXynucfeotldechaintermj.nationSequencingofthe
B
ci-rcular viroi-d or virusoid RNA (Zimmern
lgTB; Symons, 1978\ . The details of these
given below.
17-mer primer and a1l
and K.aesbeng,
techniques are
MATERIALS
RNases A and T1, cal-f lntestinal alkaline
phosphatase, E. col j- tRNA, deoxynucl-eoside triphosphates
and isopropylthiogaì-actoside ( IPTG ) were obtained from
the Sigma Chemj-caf Co.
T + pol-ynucleotlde kinase, T,, DNA ligase and
Kl-enow f ragment of E.col-i DNA poJ-ymerase were obtained
from Boehringer.
T,4
RNA ligase, dj-deoxynucfeoside
idere ob tained f rom Ptrlphosphates and d ( TBC )
Biochemicals.
M13 sPecif ic
restrictlon endonucleases htere obtalned from New Engl-and
Biolabs.
RNas" U2I^Ias obtained from Sankyo.
5-bromo -4-chloro-3 - indoyl-gal- ac tos i de ( BCIG )
bras obtained from Bethesda Research Laboratories.
Avian myelobfastosis virus reverse
transcriptase v,/as obtained f rom Lif e Science Inc. ,
Petersburg, Fl-orida.
lo-3zplocrp and lo-32lloRtt, at specific
activities of 1 0OO Ci /mmol , and I v -32p] nrP at a
L
9
speciflc activitY of 2000
R.H. Symons as PreviouslY
1981 ).
Cilmmol- v,/ere prepared bY Dr.
described (Symons, 1977;
E.coli pol-y (A ) polymerase was purif ied
according to Sippel (1973). Phy M RNase !\¡as prepared
( Donis-Ke1Ìer, 1 980 ) from culture supernatants of
Phv s ar um oJ-yce halum, the inocul-um of which was kindJ-y
provided by the school- of Biological Sciences, Flinders
University of South Australia. The extracelful-ar RNase
of Bacill-us cereus r^¡as prepared as descrlbed by Lockard
et al. ( 1 978 ) from a cufture supplied by Dr ' G
Brownfee.
METHODS
A. Isolation and seq uence determination of linear viroid
or virusoid fragments
A-'l AnaÌytical RNase diges ts
Circular RNA vras digested with a range of
RNase concentrations to obtain optimal conditions for
the production of specific finear fragments. Flve
atiquots, each of 0.1 ug to 0.5 ug purified circufar RNA
were resuspended in 9 u1 of the appropriate high salt
RNase digestion buffer (600rnM NaCl, 10rnM MgCl, wiLh
either 2OmM sodium citnate pH 3.5 for RNas. U2, otr 2OmM
Tris-HCl- pH 7.5
pl-aced on ice.
for RNase T or RNase A digestions) and
1 U1 of 100,000 units/m1 RNas" Tt ,
10
I mglm1 RNase A or 1OO unlts/ml- RNas. U 2 v{as added to
one of the tubes, which was to contain the highest
concentration of RNase. The tube conLents \^/ene mixed
and 1 u1 removed to a second tube; this v¡as f oll-owed
subsequently by another two similar 1 0-fo1d RNase
dilutions. No RNase was added to the fifth tube. For
example, if RNase T., I^/as used, the f ive tubes would
contain 10, OOO, 1 ,000 ' 1OO, 'l O and 0 units/ml RNase T',
respectively . After incubation at 0 o c for 60 minutes,
the digestions vrere terminated by extractlon with 1 00 ¡r 1
water saturated phenol : chl-oroform (l : 1 ) and 100 pI
0.2M NaCl, 0.1mM NaTEDTA; the aqueous phases were
removed, washed twice with ether and precipated with 3
vol_umes of ethanol. After 20 minutes at -B0oc, the tubes
\¡,rere centrifuged at lo,ooog for 15 minutes aL 4oc, the
Supernatants I^Iere removed, and the precip j-tates v¡ene
dried in vacuo with 1 O P C1 lr-"Pl nrP.
The dried pellets conlainlng RNA fragments')a
and Iv -"pl RrP were resuspended in 5 u L,o, heated aL
BOo C f or 1 rninute, snap cooled on ice, and 1 U I of
5x pol-ynucleotide kinase buf f er ('l 25mM Tris-HCl pH 9'0,
5OmM MgC1r, 5OmM DTT) added. O.25 units of
polynucleotide kinase was added, and the reaction was
incubated aL 37"C for 30 minutes. 5 Ut of formamide
loading buffer (95% deionj-zed fonmamlde, 'l 0mM EDTA,
O.O2% xylene cyanol FF, O.O2% bromophenol blue) was
11
added, the tubes v\¡ere heated aL B0o c f or 1 minute, snap
cool-ed on ice, and l-oaded onLo a 20x40x0.05 cm 6%
polyacnyl_amide geI containing TBE (9Oml¡ Tris-borate
pH 8.3, 2mM NaTEDTA) and 7M urea (Sanger and Coulson,
1978 ) . Af ter el-ectrophoresis, the gel was
autoradiographed.
A-2 Preparative RNase digests
Purlfied circular RNA (2 to 20 Ug)
resuspended in 50 p1 of RNase T., and or RNase
v, as
sal-t digestion buffer (see above) and cooled on
The appropriate amount of RNase Tt, RNase A or
U Z high
1ce.
RNase U 2'
as determined by analytical ribonucl-ease dlgesLs, h/as
added and incubation continued for 60 minutes at 0oC.
Digestions hlere terminated by extraction with 1 50 pl
water saturated phenol : chloroform (l : 1) and 100 pJ-
H^0. The aqueous phase was removed, washed twice wlth 1
¿
mf ether, and 450 pl ethanol I^ras added: Af ter 20
minutes aL -BOo C the sample I^ras centrlf uged at 1 0,000g
for 15 minutes at 4"C and the supernatant discarded. The
precipitated RNA fragments coul-d be either 5' or 3 I
radiol-abell-ed.
J¿A-3 5 ' - P-labe11ing of RNA fragments
The pellet, with 20O uCi of added ty-32PlATP
(2OOO Cilmmol), b/as dried in vacuo' resuspended in B U1
1.5mM spermidine'
cool-ed on ice. 2
buffer ( see above )
kinase v,rere added
30 minutes.
32
12
heated aL
pl of 5x
and 1 Ul
and the
B0oC for 1 minute and snaP
1, pol-ynucf eotide kinase¿+
(4 units) T, polYnucleotide4
reaction incubated al 37oC for
A-4 3 r - P-Iabel-l-ing of RNA fragments
i) Synthesis of Ir' -32PTopcp
5oo uci Iv-32P -]nrP
in vacuo,
dcMP, 2 Ul
( 4 unlts )
reaction
90oC for
Ci /mmol ) vras dnied
2 VI'l 0 mg/m\ 3r
buffer and 1 Ul
(2000
32
resuspended in 5 Ul ,aO, and
5x Tq poÌYnucleotide kinase
T,4
poJ-ynucleotide klnase r¡i ere added. The
r^ras incubated aL 37oC for 30 minutes, heaLed at
-l minute and stored aL -15" C bef ore use.
P-labelJ.ingii ) 3 ' -
vacuo, FeE
The preclpitated RNA fragments
spended in 20 Ul i0mM Tris-HCl
were dried in
pH 9.0 I
containing 0.01 units cal-f
phosphatase, and incubated
intes tinaf al-kali-ne
aL
reacti-ons I^Iere then extracted
saturated phenol- : chl-oroform
NaCI, 0.1 mM NaTEDTA. The aqueous
washed twice with'1 mf ether and
with 450 Ul ethanof at -80oC for
for 20 minutes. The
1 00 Ul water
1 ) and 1 00 Ul 0.2M
phase was removed,
the RNA precipitated
20 minutes. The
500c
wi th
(r :
reaction tubes vüere centrifuged at 1 0,0009 for 15
13
minutes aL 4"C and the supernatant hlas discarded. The
precipitated r phosphatase treated RNA fragments were
dried in vacuo, tr€suspended in 5 Ul ,rO, heated at B0oC
f or one minute and snap cooled on j-ce. 1 U1 of
l¡'-ttt,ldpCp (50 uCi), 6 ¡r1 of 2x T,t RNA ligase buffer
('1 OOmM HEPES pH 7 .5, 6.6mM DTT, 3OmM MgCl, , 20% (v/v )
redistlll-ed DMSO, 100 UM ATP) and 1 U1 Tq RNA J-igase
( 4 .6 units, 1 .5 Ug ) vüere added, and the reaction v{as
incubated aL 4"C for 16 hours.
A-5 Polyacrvl-amide se1 frac tionation
1 O pl of formamide loading buffer (95%
deionized formamlde, 10mM NaTEDTA, O.O2% bromophenol
blue, O,O2% xyl,ene cyanol FF) was added to each reactlon
mixture containing 5t - or 3 I - radiofabelfed RNA
fragments. The reaction mixtures vùere heated at B0oC for
3O seconds, snap cool-ed on ice and Ioaded onto an
80x20x0.05 cm 6% polyacrylamide gel containing 90mM
Tris-borate pH 8.3, 2nY1 EDTA and 7M urea. After
el-ectrophoresis for 6 hours aL 25 fiA, the gel was
autoradlographed aL room temperature for 5-30 mlnutes
and the resultant autoradiograph used as a template to
focate and excise the 32p-l-abel-led fragmenLs. Excised
bands v,rere eluted by soaking overnight aL room
tempenature in 500 pì- of 500mM ammonium acetate, 1mM
Na^EDTA, 0.1% SDS, whlch contained 60 ug E.coli tRNA as¿
14
canrier if the fragments \^Iere to be Sequenced using the
partial_ enzymic cleavage technique . After soaking, the
efutlon buffer i.Jas removed and the RNA fra6Ements were
precipitated by the addltion of 1 mI ethanol and storage
aL -80oC for at f east 30 minutes. After centrlfugation
aL 10,0oog for 15 minutes at 4oc, the pelleted fragments
v,rere resuspended in 'l OO pJ- 0.2M NaCt, 0.1mM NaTEDTA and
re-ethanol precipitated with 300 pI ethanol-. After
centrif ugation the pellets i^¡ere dried in vacuo '
Punlf ied 5t o r 3 | radiof abel-led RNA f ragments \^Iere used
for sequence determinatlon by elther the partial enzymic
cleavage on dideoxynucfeotide chain termination
technì-ques.
A-6 Sequence determinalion of RNA fragments using the
partial enzvmic cleavage technique325r- or
60 UC E.coli LRNA,
)l P-labelfed RNA
were resuspended in
fnagments, with
12 pf HZO and six
aliquots of 'l u I dispensed . The f ollowing buf f ers b¡ere
added to the six tubes.
Tube N (No enzyme) 9 Ul 20mM Na citrate pH 5.0
1 mM NaTEDTA
7M urea
Tube T (RNase T") 9 ul 20mM Na citrate pH 5.0
1 mM NaTEDTA
7M urea
15
Tube U ( RNase 9 Ul 20mM Na cltrate pH 3.5
1mM NaTEDTA
714 urea
Tube L (alka]i ladder) 5 ut 50mM
pH 9.0
Tube D (RNase PhyM) 9 yl 20mM Na citrate pH 5.0
'lmM NaTEDTA
7M urea
Tube B
(BaciÌ1us cereus RNase ) 5 ill 20mM Na citrate pH 5.0
1mM Na ED TAa
U, P, and B were heated at B0oC for I minute,
'z)
NarCO, / NaHCO,
Tubes N, T,
snap cool-ed
Tube N
Tube T
Tube U
Tube L
Tube P
Tube B
Tube L hlas
on ice, and the ribonucleases added.
1 U1 10,000 units/mL RNase T.,
'l Ul 5 units/ml- RNase ïl¿
'l UÌ RNase PhyM extract
ceneus RNase extract
heated at 1 00oC for 90 seconds while the
pl B
remalning tubes \^Iere incubated at 5 0o C f or 20 minutes .
At compfetion of the seQBencing reactions, the tubes
v\,ere stored at -B0o C or were kept on ice while being
prepared lmmedlatel-y f or ef ectrophores j-s. Bef ore
polyacryl-amide gef electrophoresis, formamide l-oading
buf f er (95% de j-on ized formamide, 1OmM EDTA , O.02% (w/v )
16
blue, O.02% (w/v ) xylene cyanol FF) vlasbromophenol
added to the
Samples were
on ice before
samples to a final
heated aL B0oC for
e l- e c t n o P h o r e s i s .
volume of 12
1 minute and
pl.
snap cool-ed
A-7 Sequence de termination of RNA fragments us ing the
dideoxynucleotide chain termination technique
i ) Phosphatase treatment
Purif ied 5'-32p-l,abell-ed RNA f ragments,
obtalned by RNase T.'| digestion, were each resuspended in
20 uI 5OmM Tris-Hcl pH 9.0, heated at B0oc f or 'l minute
and snap cool-ed on ice; 1 Ul ( 0.0'l units ) cal-f
intestinal afkal-ine phosphatase was added, and the
reactions incubated at 50oC for 2O minutes. The
reactlons were extracted with 1 00 ul water saturated
phenol : chforoform (l : 'l ) and 100 Ul 0.2M NaCl, 0'imM
EDTA; the aqueous phases were washed twice wlth 1 ml
ether and the fragments precipitated wlth 300 Ul ethanol
aL -BOoC for 20 minutes. After centrlfugation at 10'000g
for 15 minutes at 4oC, the pellets b¡ene dried in vacuo.
f i ) PoJ-yadenYlation
Phosphratase treated RNA fragments were
resuspended in 1o uI water, heated at B0oc for 1 minute
and snap coof ed on ice. 2 VI of 2mM ATP ' 4 u l- of
5x E.col-i poly(A) polymerase reaction rnixture,
comprising 105 U1 H.O, 50 U1 1M Tnis-HCI pH 7.9, 25 pI
17
O.1M MnClr, 'l O Ul 1M MgCJ-Z and 1O pI O'1M DTT, and 6
and
p1
theof E. col-i poly (A ) poJ-ymerase extract were added,
reaction incubated aL 37"C for 60 minutes' The reactions
vüere extracted with 1oo u1 water saturated phenoJ- :
chloroform (l : 1 ) and 100 u] 0.2M NaCl, 0.1mM EDTA'
washed twice with 1 ml ether and precipitated with
300 Ul ethanol at -BOoC for 2O minutes' After
centrifugation at lO,OOOg for 15 minutes aL 4oC, the
pell-els were dried in vacuo.
fii) Reverse transcriPtion
Polyadeny]-atedRNAfragmentswereresuspended
in 1O ilr H,O with '1 ul 0.25 mglml dT8C, heated at B0oC¿
and al-f owed to cool- to room ternperature over 20 minutes.
2.5 U1 aliquots \^Iere dlspensed into f our tubes to give
reaction mixtures of 5 Ul, contalning 5OmM Tris-HCl
pH 8.3, 5OmM KCI-, BmM MgClr, 1OmM DTT, 2 unlts of avian
myelobl-astos j-s virus reverse transcriptase, 2 yCi
I o-"p_loRtp or dcTP, 5o irM of the remaining
deoxynucl-eoside triphosphates and a single
dideoxynucl-eoside triphosphate species essentially as
described by Symons ( 1 978 ) . Re actions blere incubated at
3ZoC for 30 minutes; 5 Ul of formamlde loading buffer
(95% deionized formamide, 'l OmM NaTEDTA, O-02% (w/v)
bromophenol b1ue, O.O2%(w/v) xylene cyanoÌ FF) hlas added
to each reaction, âhd the tubes vJere heated at 1 00oC for
2 ninutes and snap cool-ed on ice before
1B
e 1 e c t r o p h o r e s i s .
A-B Pol-y acnyÌamide gel efectrophoresis
Radio]-abel]-edproductsofthepartiaJ-enzymic
cleavage and dideoxynucfeotide chain terrnination
technlques vJere fractionated by el-ectrophoresis in
BOxZOx0.O5 cm B% polyacrylamide gels or 4Ox20x0'05 cm
20% polyacrylamide gels containing 90mM Tris-borate
pH 8.3, 2mM NaTEDTA and 7M urea (Sanger and Coulson'
1978).
The sequence determination of some RNA
fragments\dascompl-icatedbythepresenceofband
compression artif acts (Kramer and MiJ-l-s, 1978 ) arising
from incompJ-ete denaluration of RNA or cDNA fragments
during electrophoresis. In order to el-iminate band
compression, f ragmenLs \^lere f ractionated in
potyacryl-amide geJ-s conLaining 98% f ormamlde '
Sequenclng reaction mÌxtures were
preclpitated by adding 'l 0O p I O ' 2M NaCl, 0 ' 1mM NaTEDTA
and 300 Ul ethanol, and the sampl-es I^Iere kept at -B0oC
for 20 minutes, centrifuged at 1 0,000g for 15 minutes aL
40c, and the peJ-lets dried 1n vacuo. The samples were
resuspended 1n 5 ul formamide loading buffer, heated at
SOoC for 1 minute, cooled on ice and loaded onto a
40x20x0.05 cm 20% polyacryfamide 8el contaì-ning 98%
formamide buffered with 1 6mM NarHPOO '
AmM NaHzPo,-r
dò
19
described by ManiaLis and Efstratiadis ( 1 980 ) '
Following electrophoresis, gels b¡ere autoradiographed aL
-BOo c, using cal_cium tungstate inLensifying screens.
B. Sequence determination using cl-oned viroid or
vírusoid sequences
B- 1 Syn thes is and cloning of vinoid and vinusoid
ds cDNA
i) Llnearization and polyadenylation
Purified cj-rcul-ar RNA (2-10 Ug) in 10 U1
distilled water v,¡as heated at 1 00o C f or 30 minutes in a
seal-ed capil-Iary to Élenerate randomfy cleaved
full-length linean molecules. As described above for
radiol-abeffed RNA fragments in A-7 ( i ) , terminaÌ
2, (3 t )-phosphate groups vüere removed from the cleaved
molecules by the addition of Tnis-HCl pH 9.0 to 50mM and
O. O1 units calf intestinaf alkaline phosphatase followed
by incubation aL 50oc for 20 minutes. Reactions I^Iene
phenol-chl-oroform extracted, twice ether washed and
ethanol precipitated.
Phosphatase treated RNA mofecufes I^/ere
resuspended in 50 U1 water, heated aL B0oC for 1 minut,e
and snap coofed on ice; 10 ul of 2mM ATP, 20 pJ- of
5x E.col-i poly(A) pol-ymerase neaction mixLure (see
A-7 (i j- ) ) , âhd 30 U] E. coÌi poly (A ) pol-ymerase extract
r^rere added and the reaction incubated at 37"C for 60
20
minutes. Reactions vJene extracted with 100 pJ- water
satunated phenol- : chloroform, twlce ether washed and
precipitated with 3 volumes of ethanol-.
ii ) First strand cDNA sYnthesis
Polyadenylated RNA vüas resuspended in a 5O
reaction mixture containing 50mM Tris-HC1 pH 8.3, 50mM
KC1, 1OmM MgCJ-.,,'l OmM DTT, lrnM each of dATP, dGTP and
p1
drrP, zoo uM Io-32r-locrr (60 uCi), 0.6 ug (dT)to and 30
units avian myel-oblastosis virus reverse transcriptase,
and incubated at 42oC fon 60 minutes. The reaction
mixture r^ras heated aL 100oC for 1 minute, snap cool-ed on
ice, 'l Ul
incubation
(10 Ug) heat-treated RNase A added and
continued at
hrene extracted with 'l 00
1 00 U1 water saturated
twice ether washed and
50oC fon 20 minutes. Reactlons
Ul 0.2M NaCl, 0.1mM NaTEDTA and
phenol : chl-oroform (t : 1),
precipitat,ed with 3 vol-umes of
ethanol.
i j-i ) Second strand cDNA synthesis
Si-ng1e strand cDNA v¡as resuspended in 10 Ul
H^0, boiled for 1 minute and snap cool-ed on ice, added¿
to make up a 25 Ul reaction mixture containing 5OmM
Tris-HCl pH 7.5, 1OmM MgClr, 10mM DTT, 1mM dATP, dCTP,
dGTP and dTTP and 2 units Klenow fragment of E.coli DNA
polymerase 1, incubated for 4 hours at 37oC and kept aL
-2Oo C b efore use .
j-v ) Restriction enzyme cl-eavage and f ractionation
21
Doubl-e strand cDNA I^/as subj ected to digestion
by various restriction endonucleases under conditions as
specif ied by t,he suppl-iers of the enzymes. The cleaved
ds cDNA bras fractionated by electnophoresis in a
20x40x0.05 cm 6% polyacrylamide gel (Sanger and Coulson,
1977 \ containing TBE buffen ( see above ) and 2M urea.
Following electrophoresis, the gel vlas autoradiognaphed,
the ds cDNA fragments ütere exclsed and each eÌuted in
400 ut 0.5M ammonium acetate, 0.1% SDS, 0.1mM NaTEDTA aL
room temperature overnight, âhd ethanol precipiated
tw1ce.
v) M1 3 cloning
Purified ds cDNA fnagrnents r^/ere llgated into
an appropriate restriction site of the repllcative form
of the phage M13 mp7 using phage Tr* DNA J-igase (Goodman
and MacDonald, 1979; Messing, Crea and Seeburg, 1 981 ).
Further speciflc details are provided in fol-lowing
chapters. Ligated M13 RF and ds cDNA uras used to
transform competent E. coli JMl 01 and the celfs vùere
plated on agar media with BCIG ( 5-bromo-4-chl-oro
-3- indoy I-P-D - gal ac tos ide ) and I PTG ( Is opropyJ- -p
-D-thlogalactopynanoside ) . Recornbinant M1 3 phage v\rere
screened by sequencing as described bel-ow.
B-2 Sequence deterrnination of recombinant phage M13
M13 phage containing cl-oned ds cDNA inserts
22
r^iere selected, âs judged by insertional- inactivation of
ß-galactosidase activity (Messing, Crea and SeebüPBr
1 981 ), âtrd the inserted sequences hrere determined using
the dideoxynucleotide chain termination technique as
described by Sanger et al-. (1980 ) with a 17-mer M'l 3
specific oligonucl-eotide primer ( GTA, CGACGZC2AGT ) .
B-3 Sequence determination of RNAs usln cl-oned DNA
pr]-mers
i ) Preparation of primer
The DNA primers used in this work were of two
types, being either fragments restricted from
recombinant phage M1 3 RF DNA or transcribed using
recombinant phage M13 ss DNA as a tempfate. In the
former case, recombinant M1 3 RF \^/as isol-ated using the
method of Birnboim and DoJ-y (1979) , restricted with the
appropriate enzyme, âhd the fragment ( s ) containing
vlrold or virusoid sequences purifed by efectrophoresis
through a 6% polyacrylamide gel containing 7M urea
(Sanger and Coul-son, 1978). In the l-atter case,
recombinant M1 3 ss DNA containing viroid or virusoid
sequences of the same polarity as the RNA sequence r^ras
transcrlbed uslng an M1 3 specific ol-igonucfeotide primen
and the Klenow fragment of E.coli DNA polymerase 1 with
lo-ttplocrp and l-o-32plonrp (specific activities of
1 000 Ci /mmol ) essentially as described by Bruening et
23
a1 . (1982). The resulting partially double-stranded DNA
mol-ecules v,rene sub j ected lo restnictlon enzyme
digestion, âtrd the l-abelled fragments fractionated by
polyacrylamide gel el-ectnophoresis. In both cases the
purlfled DNA primers were eluted from polyacrylamide
gels by soaklng (Maxam and Gil-bert, 1980 ) .
ii) RNA-DNA hYbnidization
RNA-DNA hybnids were prepared as f ol-lo\^IS.
The purified restriction fragments and 1 to 2 ve of the
appnopniate RNA were resuspended in 25 ¡rI of 0.18M NaCl,
lOmM Tris-HCl pH 7.0, 1mM EDTA, 0.05% SDS, heated aL
1000c for 2 minutes, and incubated aL 600c for 2 hours.
The RNA-DNA hybrids I^Iere twice ethanol precipitated and
dried
1]-IJ
ln vacuo.
Reverse transcriPtion
The RNA-DNA hybrids hiere reverse transcribed
in the presence of dideoxynucleoside triphosphates
essentiaJ-1y as descnibed above in A-7 (ii j- ) . However, if1t"P-radiof abef f ed transcripts of recombinant M1 3 ss DNA
\^rere used as primens, Io-32p]ONTPs I,\,ere omitted and
replaced by the unl-abefled dNTP species. Revense
transcripts v/ere fractionated by polyacrylamide gel
electrophoresis as described above in A-8.
Al l experimen ts invol-ving the use of
recombinant DNA molecul-es were performed within safety
guidelines set out by the Austrafian Academy of Science
24
Committee on Recombinant DNA mofecul-es (ASCORD).
RESULTS AND DISCUSSION
A. Techniques
The various rapid geI sequencing techniques
viroid and vÍnusoidused for the determination of
sequences are outlined in Figure 2-1. These techniques
share the advantage of requiring onl-y smafl amounts of
purified RNA for thelr use and, while there are
disadvantages inherent 1n the use of each procedure, the
combined use of different techniques alfows rapld and
reLiabfe RNA s equence anal-ysis. The various approaches
are revlewed bnief lY bel-ow.
B. Partial- enzymi-c cl-eavage of viroid and virusoid RNAs
( 60OmM NaCl, 1 OmM MgCl, )
highJ-y base-paired natj-ve
virusoids are stabil ized
strand specific RNases Tl
Under conditions
and
of high saÌt concentration
Iow temperature ( 0oC ), the
structures of viroids and
and so cJ-eavage by the single
and A 1s initialJ-y limited
sites on the molecules.
polyacryl-amide gel
main disadvantage in
uz
to relatively few accessibfe
Thus, pârtial- ribonucfease digestion of the native
cincul-ar RNA moÌecuf es glves rise to rel-atively f ew,
speciflc linear RNA fragments which may then be
radiol-abelled and fractionated by
ef ectrophoresis (Figs 2-2ß1. The
Figure 2-La
purified. circular RNA
A-I, A-2 Partial RNase , A or U2 cleavage
32
lt
32 A-4 3r- P'1abe11ingA-3 5r- P-labelling
A-5 Polyacrylamide gel fractionation
A-7 r) Phosp.hatase tre atment
A-6 Partial enzymiccleavage sequencing
fI) Polyadenylation
I111) RTase dideoxy-
sequencing
A-8 Polyacrylamide gel electrophoresis
B-t t) Linearization, phosphatase treatment, polyadenylation
Figure 2-Lb
B-2 Klenow dideoxy-sequencing
purified circular RNA
11) 10 strand cDNA synthesis, tu\ase treatment
lrr ) 20 strand. cDNA synthesis
fV) Restrict.ion, gel fractionation
V) Ligation into M13 vector, transformation
B-3 I) excision or transcriptionof primer
I11) R.irrA - DNA hybridization
III) RTase d.ideoxy-sequencing
A-8 Polyacrylamide gel electrophoresis
Figure 2-2
RNA 2.
Analytical RNase A digestions of SNMV
As described 1n the text, 0.5 pg purified SNMV RNA 2
v,/as variously digested with 0, 0.1, 1, 10 or
100 u g/nI RNase A unden conditions of high sal-t and
1ow temperature, and lhe resulting l-inear RNA
f nagments 5'-32p-,-abelted and f ractionated by
denaturing polyacrylamide gel electrophoresis. The
presence of a band in the Lrack containing SNMV RNA
2 untreated with RNase A corresponds to a smal-l
amount of full-length l-inear breakdown product (377
residues in size ) present with the i-ntact circufar'l and 'l p g/ml
RNase A hrere used to obtain fnagments suitable for
sequencing.
RNA. Concentrations of between 0
RNa'se A 0¡g/ml)
o o.1 1 10 100I
SNMV RNA 2LINEAR I
-¡p
-.,-
7*O
ii.ri'ç
6% TBE
7M UREA-i''
Flgure 2-3
digestlons
Preparative RNase A and RNase T
of VTMoV RNA 2
5 Ue amounts of purified VTMoV RNA 2 were digested
wlth 0.2r 0.1 or 0.05 Ug/m1 RNase A and 150 or 75
units/ml RNase T., under condi tions of high salt and
1ow temperature. 5r-radlolabelled products are
shown fractionated on an B0 cn long 6% poly
-acrylamide gel containing 7Vl urea. OnIy the bottom
portion of the gel vras autoradiographed and the band
corresponding to full-Iength 1j-near VTMoV RNA 2
(365/366 resldues) mlgrated about 30 cm from the
origin. Followlng a 5 mlnute autoradiographic
exposUr€ ¡ bands r^rere excised and eluted for sequence
detenmination.
II
1
.Èf
RNase A RNaseT1
50 100200 75 150ng/ml U/ml
\I
ì
LINEAR VTMOVRNA 2
6% TBE7M Urea80 cm
ifl\
\ Ë
{
þ.&
tÇ
Ë
a
ü
:.,.kIri*
_1.
È.
-1 20b +
25
using this technique is that' if the native circular RNA
molecul-e possesses an exposed singl-e-stnand negion (such
as a termlnal hairpin l-oop) containing accessible sites
f or aIl- nibonucl-eases, 1t is dif f icult t'o obtain RNA
fragments spanning such an exposed region. This
disadvantage is not shared by those sequencing methods
which rely on cloned viroid or virusoid fragments.
C. Partial- enzymic cleavage of nadiof abel-l-ed RNA
fnagments
The purlfied 3r- or 5r- radiofabelled RNA
fragments obtained after partial RNase cleavage of
intact vlnoid or virusoid molecules were sequenced uslng
the partial enzymic cleavage method ( Donis-Ke11er , 1977;
Lockard et â1., 1978; Krupp and Gnoss, 1979;
Donis-KefÌer, 1980). An example of one sequencing gel
1s given in Figure 2-+. In particuJ-ar, the use of
fragments l-abelled separately aL either the 5r on 3r
terminj-i alfowed the sequence determination of long RNA
fragments and, with shorter fragments, resol-ved gel
compression artefacts ( Kramer and Mi11s, 1978 ) when the
relevant nucfeotide sequences v\rere determined from both
directions.
D. Dideoxynucl-eotide
fragments
chain terminalion sequencinA of RNA
Figure 2-4 Partial enzymi-c dlgestlon sequencing
technlque.'
A 5'-32p-labelred
RNase T, digestio
non-denaturlng co
d iges t ion un d.e r d
RNases as describ
RNase T,, ; U, RNas
PhyM; B, Bacil-1us
RN.A f ragment, obtalned by partial
n of SNMV RNA 2 under
ndltlons, v,ras sub j ected to partial
enaturing conditions with various
ed in the text (N, no enzyme; T,
e U17 L, aIkaI1 ladder; P, RNase
cereus RNase). The resulting
f ragments brere separated by I0 cn, B% polyacryJ-amide
geI elecLrophoresis and the resulting autoradiograph
is shown with residues 132 to 175 of SNMV RNA 2.
NTUL PB-
UC
--t--
AG
-17OGA
AGu-160
ta GUU
c
aaa
a
3t¡
A
a
-t) "'
-t-
o
1r.
t
UG
150
140
-
aq
It
o
Jt
:
2
too
G
G
G
A
Gc
U
A5
26
' All- 5'-32p-radiol-abel-l-ed fragments pnoduced
by RNase T.'| cleavage of viroid or virusoid RNA possess a
3r proximal- guanine residue, together with a
2t (3 ' )-phosphate group. Treatment of such fragments
with calf intestinaÌ phosphatase ( Efs LraLiadis et âf . ,
1977 ) removed both the radlofabefled 5' phosphate and
2, (3t)-phosphate groups, and the RNA fragments couÌd
then be used as templ-ates for polyadenylation usinpg
E. col-i poly (A ) poJ-ymerase (Sippel, 1973; GouId et âI' ,
1978 ) . The synthetic ol-igonucleotide d ( TBc ) blas then
used as a specific primer for reverse transcrlption in
the presence of dideoxynucfeoside triphosphates. The
polyacrylamide gel fractionated products of such a
transcription neaction are shown in Figure 2-5. The
dideoxynucl-eotide chain termlnation and partial enzymic
digestion techniques are complementary, âflowing further
confirmation of sequences and the resofution of
occasional band compressions.
E. Cfoning of viroid and virusoid sequences
Purified circufar RNAs I^/ere hydrolysed by
prolonged heating aL 1 00oC to generate nandomly nicked
full-length finear mofecufes. Terrninal- 2' (3' )
-phosphates were removed by treatment with caLf
intestinaf phosphatase and the RNA mol-ecules
polyadenyl-ated with E. coli poly ( A ) polymerase. First
Flgure 2-5
s eq uen c 1ng
Dldeoxynucleotide chain terminatlon
of RNA f.ragmen ts
An RNase T,, cleaved fragment of VTMoV RNA 2 was
phosphatase- treated , polyadenyl-ated arid reverse
transcrlbed in the presence of dideoxynucl-eoside
triphosphates as described in the text. Two
Ioadings of the resultant radiolabelled franscripts
are shown -fractionated on an B0 cm B% polyacrylamide
ge1 containing 714 urea. Residues 355 to 190 (3';5')
of VTMoV RNA 2 are shown.
I
I
I
I
1
- 280
- 290
Ç.Õ{ ao - 300
TtOöÒ
ACGtt
c(t190 -
200 -210 -220 -230 -
240 -250 -
{
lç
t(,
rrÛ
-:*
ì
\
\ì\
\
\\ì
Ê.4
I)
-:
ra
-tI
I o -310
æ,
- 320
- 330
- 340
:I-Ir
e260 -
210 -
\
280- \
\290 -
ìl
\300 -
C - 350ç
'r..]
- 355
8% TBE
7M UREA
SOcm
--Q
(I
r¡-(T
ITI
I!i
rDIT
-ìr¡ ItJat
I
C3
t
ì---a
ìr
¿l
32strand P-cDNA r^las synthesized using reverse
transcrj-ptase and oligo- ( dT ) 1 O as pnimer ( Gould and
Symons, 1982). The RNA.DNA hybrids hrere heat denatured
and the tempfate was removed by RNase A treatment.
Immediately prion to the synthesis of the second DNA
s trand , the cDNA hras heat-denatured; this step I^Ias f ound
necessary to avoid the formation of anomalous ds cDNAs
with Iarge apparent mol-ecuf ar weights. The sel-f -prlmed
second DNA strand was transcribed using the Kfenow
fragment of E.coIi DNA poJ-ymerase 1, and the resulting
population of circularly penmuted ds cDNA molecules \^ras
then digested with an appropriate restriction enzyme ( s )
to pnoduce specific DNA fragments for cloning (see
Figure 2-6\. The restriction fragments were then
purlfied by non-denaturlng polyacrylamide ge1
electrophoresis and ligated into a bacteriophage M1 3
vector, tnansformed and propagated in an E.co1i host
(MessÍng, Crea and SeeburB, 1981 ; Sanger et âf ., 1980).
F. Sequence determination using cfoned viroid or
virusoid sequences
Viroid or virusoid sequences vüere determined
using recombinant M13 phage in either of two b/ays.
First, recombinant phage ss DNA !ùas sequenced using the
dideoxynucl-eotide chain termination technique with an
M1 3 specific primer ( Sanger et â1. , 1 980 ) ( see Figure
Figure 2-6 Synthesis and nestriction endonuclease
cleavage of SCMoV RNA 2 ds cDNA.
As described 1n the textr PUrified SCMoV RNA 2 was
finearized, phosphatase treated, polyadenyl-ated and
used as a tempfate for first strand cDNA synthesis
( 1 o ). The ss cDNA vüas denatured and treated with
RNase (1o + RNase) and second strand cDNA was
synthesised by seff priming- (2o). Doubl-e-strand cDNA
was digested with Hae III, Hha I, Sau96 I, Hpa II +
Sau3A I or Hpa II + Taq I. Samples taken aL each
step r^rere fractj-onated by 6% polyacrylamide gel
efectrophoresis in the prêsence of 2M urea. SampJ-es
1o, 1o + RNase and 20 were loaded wlth (+H) and without
(-) heating aL'l 00oC for 2 minutes. Specific bands
produced by restriction endonuclease cleavage v.tere
suitabl-e f or cf oning.
HãHfO
Hsi F
ItH 3iår¡l<Ð<<r <TIU'TI
FULL.LENGTHLINEAR
SCMoV RNA2sr DNA
?
J
i
rt
6% TBE2M Urea
2B
2-7 ) . In this wâV, cl-oned sequences corresponding to
either viroid/v irusoid or its compÌement, depending on
orientation of the cloned fragmenLs, coufd be
determined. Second, cl-oned sequences were excised or
transcribed for use as primers for reverse transcrlbed
dideoxynucl-eotide chain termination sequencing of intact
vinoid or vlnusoid RNAs (Zimmern and Kaesberg, 1978;
Symons, 1978). The primed transcripts obtalned in this
way are representative of the whole popul-ation of RNA
templates, and thus this sequencing technique has been
used to estimate the rel-ative proportions of RNA
sub-species within heterogeneous populations ( see Figure
2-8).
G. Determination of complete vlroid or virusoid primary
stnuctures
Construction of the complete prlmary
structures of each cincular mofecul-e depended on the
obtaining of numerous overlapping sequences by one or
more of the techniques described above. Once the entire
base sequence of a molecule had been determined by
merglng the various overlapping sequences, secondary
structure model-s b¡er?e constructed uslng the methods of
Tinoco et al. (1971 ) . The following chapters pnesent
the determi-ned structures of several viroid and virusoid
RNAs and outl-ine some of the interesting features of
these unique mol-ecules.
Figure 2-7
sequencing.
M1 3 dideoxynucl eotide chain termination
Single-strand DNAs
bacteriophage M1 3
dideoxynucleotide
a 17-mer specific
isolated from recombinant
hrere sequenced by the
chain termination technique
d.
us 1ng
cl-onedprimer.
of VTMoV
The sequence of
RNA 1 ds cDNA is s hownSau3A I fragment
determined from
s trands.
both indivldually cloned DNA
Sau 3A I
SITE - 160
- 150
- 140
- 130
-120
- 110
-70
Sau 3A I
S !TE -01
- 101
- 20'
- 30¡
- 40r
- 501
- 60r
- 701
- g0l
- g0l
-100r
- 1101
-1201
+
(+)ACGT
(-)
ACGT
It:
.+
q,t
oö
.lI
)
t -100
o
t -e0o
. -80
a
a(,-¿
a
-a,
aô
I
a¡a
rt
iaa't Ò'
aI.ö
a'a
La'tt
i¡t
-60
-50
-40
\
T
IÊ
aa
31 -1291
Figure 2-B Dideoxynucleotlde chain termination
seq uencfnA of intact RNA using cloned DNA pnimers.
Recombinant M1 3 ssDNA containing sequences
corresponding to SNMV RNA 2 residues 131 to 216 vüas
transcribed to produce a 1abelled complementary
strand as described in the text. The cÌoned insert
v{as excised, punified and the l-abelÌed transcript
hybri_dized to intact VTIt4oV and SNMV RNA 2; this
prlmer was elongated uslng reverse tnanscriptase in
the presence of dideoxynucleotides. The transcripts
r^rene fnactionated by B0 cm 6% potyacryl-amide gel
el-ectophoresis and the tracks A, C, G and T
correspond to the dideoxynucleotj-de specles present.
Note the sequence heterogeneity evident in VTMoV RNA
2 aL residue 108 which results in band doubling, and
in the termination of reverse transcription aL
residue 49.
T G CA TGCA
I
6tAl
19
ú
6u
6U A6*E
h.blr¡
tA
ìAG- ¡lD 6c
16
c,A
I.¡iteit
=lr!r
;;- (
tE)t
il
It "..': II;
Ëäõ
l}t¡a{tÒ-
ea{rt
h ouo'
,oo"
ooÉ
\*l*-.i!
77
G
A
A
-, oo
-, oou
uur_c
_6'
-r"u'
Èr_.j-_ì
ì= _,bf¡ _ __
r¡-\._
ìi¡ i3ì l¡ì-
\¡
U I-
-3ot ç!u
, oo'O(I
-
VTMoV RNA 2
tc
rll6
GA Ut
G-ú 6o"' A
125t
\-
SNMV RNA 2
At126
CHAPTER 3
CHRYSANTHEMUM STUNT VIROID
29
INTRODUCTTON
Chrysanthemum stunt
described by Dimock (1947 ) and vras epidemic among
cul-tivated Chrysanthemum morifol-ium varieties during
1945-1947 in the U.S.A. (Srierly and Smith, 1949). At
that time the production and distribution of
chrysanthemum varieties in the U. S. A. \^/ere highly
centrallzed, allowing the rapld spread of the disease
( KelÌer, 1 953 ) . Transmission of the chrysanthemum stunt
disease by both grafting and sap inoculation (Brierly
and Smith, 1949; 0lson, 1949; Brierl-y and Smith, lt951 )
demonstrated its infectious nature and al-l_owed the
development of control measures to prevent accldental_
spread of disease (fefler, 1953). Dlener and Lawson
(l9lZ ) demonstnated LhaL the causative agent of
chrysan themum stunt disease r¡ras a viroid, a l_ow
mofeculan weight RNA species with properties slmil_an to,
but distinct from potato spindle tuber viroid ( pSTV ) .
Chrysanthemum stunt viroid ( CSV ) was shown to possess a
greater electnophoretic mobility Lhan PSTV, and the host
range of CSV hras shown to be mostJ_y confined to some
plant species in t,he compositae family whereas pSTV witl
replicate in species from a number of plant famil_ies
(Diener, 1979; Hollings and Stone, 1973). Ribonuclease
fingerpninting has al_so shown that the primary sequence
of CSV differs significantly from that of PSTV (Gross,
disease bras first
30
Domdey and Sanger, 1977 \ . However, cDNA-RNA
hybridization techniques have indicated that aL Ieast
20% of the PSTV primary sequence is common to that of
CSV (Owens, Smith and Diener, 1978). RNA sequence
debermination studies vlere undertaken to obtain primary
and predicted secondary structures of CSV and to compare
these with the structures previously determined for PSTV
(Gross et âl ., 1978).
MATERIALS
The CSV isol-ate used in this work I^ras originalJ-y
obtalned from infected Chrysanthemum cuftivars, Charm
type, âhd was kindly provided by T. C. Lee, Adelaide
Botanic Gardens, via Dr. R. I. B. Francki, University of
Adelaide. CSV, purified from infected chnysanthemuns as
described by Pal-ukaitis and Symons (1 980 ) , vras kindly
provlded by Dn. P. PaIukaitls.
ME THOD S
A. Primary structure determination
The primary sequence of CSV was determined using
techniques described in Chapter 2. Linear viroid
fragments vrrere obtained by partlaJ- digestion of purified
CSV unden conditions of high saÌt and l-ow temperature
using 3750 units/rnÌ RNas" T.l , 2 pg/n)' RNase A or
2 units/ml- RNase U Z. The I j-near RNA f ragments \,rere
31
532 P-radiolabelled using |-
"-32p l nrp T,
+and
polynucleotide kinase, f?actionaLed by denaturing
polyacrylamide gel el-ectrophonesis and sequenced using
onJ-y the partial enzymic cleavage method, as descrlbed
in Chapter 2. Some pantial enzymic digests of virold
fragments r^iene fnactionated in polyacnyÌamide gels
containing 9B% formamide, as descnibed in Chapter 2 A-8,
in order to etiminate band compression artifacts (Kramer
and Mil-l-s, 1978 ) arising f rom incomplete denaturation of
the fragments during electrophoresis. The compÌete base
sequence of CSV üras assembl-ed from sequence data of a
l-arge number of RNA fragments obtained by partiaJ- RNase
diges t ion .
B. Secondary stnucture detenmination
A possible secondary structure model- Ì,ùas
constructed from the compfete CSV sequence using the
matrix method of Tinoco et al-. (1971, 1973) and the
predicted thermodynamic stabil-ity of the modef was
calcul-ated using vafues provided by Dr. D. Reisner
(Steger, Gross, Randles, Sanger and Reisner, in
preparation ) .
RESU LTS
A. Sequence determination
Purified circufar CSV vías subjected to partlal
32
dj-gestj-on with RNase T., UZ or A under conditions of high
sal-t concenLration (60OmM NaCl, 'l 0mM MgCJ- Z and at 0oC in
order to limit cleavage by the single-strand specific
RNases to rel-ativeJ-y f ew accessible si tes on the highty
base-paired RNA mol-ecul-e. The resulting viroid
f ragments hrene 5'-32p-labetl-ed in vitro using T q
poJ-ynucleotide kinase ano Iv -32p] nre and f ractionated by
size on a denaturÍng polyacrylamlde gel. Figure 3-1 a
shows the gel patterns obtained fon partial digestions
of CSV with RNa.ses T1 , UZ and A. Digestion with either
of the sì-ngì-e base specif ic RNases Tl (G specif ic ) or
U^ ( A s pecif ic ) gave ri-se to f ewer f ragments than¿'digestion with the C and U specific RNase A.
The gel fractionated 5 I - fabeffed fragments
obtalned by pantial- RNase digestì-on of CSV wene excised,
efuted and sequenced using the partial enzymic cl-eavage
method as described in Chapter 2 A-6. An example of one
sequencing gel is given in Figure 3-1b. For some
regions of the viroid molecul-e, sequencing u/as
complicated by band compression ( Kramer and MiJ-1s, 1978)
due to the presence of stabfe base-paired halrpin
structures. However, these band compressions coufd be
el-iminated by the use of sequencing gels which contained
98% formamide rather than 7VI urea in order to ensune
complete denaturation of the RNA fragments.
Figure 3- 1 Purification and nucleotlde sequence
determination of CSV fragments.32(a)
the
Autoradiogram of the
partial digestion of
trt)- P-labell-ed products of
, U2 and TlCSV by RNases A
after fractionation by electrophoresis
polyacrylamide slab gef as descnibed in
A-5. The largest radiolabell-ed fragment
I ength l-inear C SV ( CSVL ) which migrated
fnom the onigln. XC is the posltion of
ona
Chapter 2
is full
about 30 cm
the xylene
cyanol FF dye marker which corresponds to fragments
about B0 residues long. A number of the shorter CSV
f nagments (including band X ) 'were excised and ef uted
for sequencing by pantial enzymic digestion.
(b) Autoradiogram of part of a sequencing gel (g%
polyacnylamide ) contalning the various partial
enzymic digests of fragment X. Digestions, as
described in Chapter 2 A-6, r^¡ere with RNase T.,' (G),
RNas. UZ (A) alkal-i (N) to produce the reference
l-adder, RNase Phy M (A+U) and BacilÌus cereus RNase
(C+U). Part of the nucleotide sequence of fragment X
from residues 207 to 265 is given.
c) U)
r: I
o
>< c) I
ffiffi
,Jlif
fft t
{ù¡Þ
üt I
t\l
RN
ose
É'
RN
ose
Ll2
RN
ose
1-1
"¡ It
¡r'Þ
,
I ô X
\\
\ \
úN
)l.
Ll.l
r.)
È
L¡r
I
\\
G) Þ z_
c+>
c +
c)
r-ì
(-
Gì
Cì
Ì-,'
r- 6
.ì 61
cr-ì
C C
'ì C
Cì'r
rì'ìC
)''
6ìI
l--.
sl(
6l
rì
ì(
I
rF
..)O
LJ O
33
B. Primary Sequence and Secondary Structure of CSV
The compl-ete base sequence of CSV I¡¡as assembled
from sequence data of a large number of RNA fragments
obtained using the pantiat RNase digestion technique
(Figune 3-2lt. The 356 residues are numbered according
to the scheme of Gross et a1. (1929) ror PSTV and the
main overtapping sequences used for the pnimary
structure determination are shown in Figure 3-2.
A possible secondary stnucture modef h/as
constructed from the CSV sequence using the methods of
Tinoco et a1 . (1 97 1 ; 1 973 ) and 1s compared with the
published structure for PSTV ( Gross et â1. , 197e\
(Figure 3-3). The refative number of G.C base pairs in
the predicted CSV stnucture (64 G.C, 44 A.U, 16 G.U) is
l-ower than that of PSTV (7 3 G. C, 37 A. U , 16 G. U ) and,1
using val-ues kindly provided by Dr. D. n/diÀ"er (Steger, illGnoss, Randles, Sanger and R1eìi\sner, in prep araLion ) , the l,)
thermodynamic stabitities of the proposed models for CSV
and PSTV brere calculated to be AG (25oC, 1M NaCl) = -540
KJlmol- and -610 KJlmol respectively.
DTSCUSSION
A. Homology between CSV and PSTV
The striking feature of both the primary and
postul-ated secondary structures of CSV is the extent of
homology with the previousJ-y sequenced PSTV molecule
Flgure 3-2 Primany sequence of CSV.
The sequence
residues
of the 356 residues
the numbered according
of CSV is given and
to the published
1978l,. The 247
boxed. !{i thin the
s equence
residues
of PSTV ( Gross et âI. ,
homologous wÍth PSTV are
circular sequence are given the l-ocations
overlapping sequences obtained from RNase
of CSV; these sequences do not represent
J-ength of these fragments. Each sequence
with the RNase (4, T1, or Ur) which gave
from which that sequence vüas derived.
of
fragments
the entire
is labelled
the fragment
Figure 3-3 The predicted secondary Structures of CSV
and PSTV (Gross et a1. 1978).
The boxed areas contain residues homologous between
the two viroids.
1601 PSTV
359 350
CSV
^F-o¡
ffifou^"""""3oo
^AAOOUUUCC
ouo
cAc
CO OCU' UCUO
50 100c c c
ocucoo
u^c^o@l
250
100
250
^oo^ccuucu oo
uooc
ouco oGcco
c cocoA
^o^ucc$o,c
cA
c uu
200U
60 150
UU
uC
qFã4 cup
^ ucc
356uu
350 - 3oo 200
34
(Gross et â1., 1978). Of the 356 residues of csv, 247
residues (69% ) are homologous with those of PSTV, and
occur in two main areas in the primary structure (Figure
3-2) extending f rom residues 247 to 'l 10 and 148 to 206.
These areas are separated by two regions of about 40
residues each containing only two small areas of
homology. The postulated secondary structure model for
CSV (Flgure 3-3) shows that the two main areas of
homology each correspond to one base-paired end of the
native mofecuÌe. These are separated by the two regions
of lesser homology which are positioned almost exactly
opposite each other in the native mofecule and are
predominantl-y base-paired. Thus, Lhe conservative
arrangement and base-palring of such non-conserved
regions in the pnimary sequence al-lows the CSV mol-ecufe
to form a stabl-e secondary strucLure simll-ar to that of
PSTV.
B. Replicatlon of CSV and PSTV
Although the host ranges of PSTV and our isolate
of CSV differ significantly ( see Introduction ) , they do
overlap in such plant hosts as the composlte Gynura
aurantiaca (llenerr1979; Palukaitis and Symons, 1980;
Niblett et âf., 1980). It is feasible that replication
of the two viroids in these plants will occur by simil-ar
mechanlsms in view of theln similarities in size and
35
sequence.
Assuming the existence of translatable l-inear
forms of the RNAs (Kozak, 1979; Konarska eL âl ., 1981 ),
the possibl-e polypeptide products of both the virold and
putative complementary RNA stnands of csv and PSTV can
be predicted from their known primary structures. Maj or
diffenences are found between the possible polypeptide
tnanslation products of CSV and PSTV (see Chapter 7),
suggestlng that neither viroid codes for proteins
involved in their replication. This is consistent with
the l-ack of evidence fon any viroid transl-ation 1n vivo
( conj ero and Semancik, 1 977 ) and in vitro ( Davies
et âf . , 197 4; Semancik et al- . , 1977 ) .
In contrast, the overall secondary structures of
CSV and PSTV are conserved despite diffenences in
sequence. Given the fack of evidence for functlonal
viroid-coded translation pnoducts, the replication of
CSV and PSTV rnay invofve necognition by host enzymes
which are capabl-e of RNA-dependent RNA synthesis. Thus,
the sequence and structural- features common to both CSV
and PSTV may play a role in such recognition processes.
An example of such a conserved feature 1s si-tuated aL
the centre of the native mol-ecules (Fig. 3-3) (CSV
residues 74-110, 247-284; PSTV residues 76-112, 247-284)
and incÌudes two relatively Ìarge single-stranded
reglons which are compl-etely conserved between CSV and
PSTV .
36
C. Rel-ationshiP of thls isol-ate of CSV to othen viroid
isol-ates
Both Owens
(1 977 ; 1 982 ) have used
origins from ours, for
lsol-ate obtained from
this PSTV i sol-ate has
1978).
et al-. ( 1978 ) and Gross et al-.
isolates of
comparative
Dn. T. 0. Dienen. The
been determined (Gross
CSV, of different
studies with a PSTV
sequence of
et al,. ,
PSTV to show that bY hYbridization
isofate of CSV contained about 2O% sequence homology
whereas a viroid isolated from Columneawith PSTV,
ery throphae contained about 40% sequence homology.
the Columnea viroidaddition, thein
electrophonetic
Owens et al. (1978) used DNA compfementary to
anal-ysis, their
In
hadCSV isofate and
mobilitles in a non-denaturing
polyacnyl-amide gel which wene rnarkedly faster than that
of PSTV, indicating appreciably different sizes and/or
secondary structures. In contrast, our isol-ate of CSV
shares 69% sequence homology wlth PSTV, is only 3
residues shorter, and possesses a similar secondary
structure. The data suggest that, even allowing for
some errors in estimates of sequence homology determined
by cDNA-RNA hybnidization analysis, our isolate of CSV
may be,more closely reÌated to PSTV in size and sequence
than are the Owens et af. (1978) isolates of CSV and
37
Columnea viroid.
Gross et al . ( 1 977 ) have compared the
oligonucleotide fingenprints of an isol-ate of CSV'
obtained from Dr. M. Hol-lings, with those of PSTV and
obtained distinctl-y dif f erent patterns. I t v'Ias
concfuded that CSV and PSTV differ significantly 1n
sequence. Subsequent sequence determination of that
isol-ate of CSV, white confirming its non-identity with
PSTV, showed 73% sequence homology with PSTV (Gross
et al-., 1982). Flgure 3-4 shows the predicted
structures of the two isolates of CSV, together with
that of PSTV. It can be seen that both isolates of CSV
sharing 346are closeJ-y rel-ated,
whi-le the CSV isolate of Gross et
residues in
aI. (1978)
common,
i- s only 2
differences
in the left
mol-ecu1es.
residues smal-1er in s1ze. The f ew
between the
seq uenc e
located
nat ivehand sides
I t is unknown
isofates are mainly
they are drawn) of the
whether the differences in structure
two
(as
between these CSV isofates corresponds to differences in
biol-ogical properties.
Three independent isol-ates of CSV have been
characterized and it seems likeJ-y that a1l differ.
These three CSV isolates, together with the Cofumnea
vinoid, are closeJ-y related to PSTV. It therefore
appears tikel-y that there exists a group of viroids,
Figure 3-4 Comparison of the secondary structures of
two sequenced isolates of CSV.
The secondary structune model-s for the Australian CSV
isolate (CSV-R; this work) and English CSV isolate
(CSV-E; Gross et ãL., 1982) are presented. The two
viroid isolates share over 97% base sequence
homologyr âhd the few residues not shaned by both
isol-ates are shown boxed.-
PST V
^cuô^ACU AACU OUOOUUCC
A
u c oc u
oouu Ac^ccu cucc 0^GcA0A A A
^o oc o
^^6^ AOAAOOCGO CUCOO O^OC UUCAO
50 100 *-.^AA^
A G C AC ^
A AC
ucc.ccooo cuoG^ocoA uooc A^Aog ôougooo ^ c ^ c ^c u
ouo cc ocoo co Aoo^o
150
Iuu ccccco GA^^
u
1
\
¡00ôu u
lill :uc(c¡clJr
I
I
GUUGG'UUCA.CGCCAÀOO CCG^ USUGOO OOOg UUCGUU UUCU
c c c^o
uuuuucocc oAocc'cuco ^Aôuc
AOg qoccc
^uAAUcc
oocuuc0cu'cuco uuucc ccocucc cAc oo cocc oc uccuucG c c c^ A C OC C
rcu
oooc cuuuU IJU C
o\200
GgAOGGU
uccc^uc
c
u
u
^cUUc
cU
A
0
250359 350
CSV- Ao)o^
U U U U U OC O C C
ooô^c uacu oouucc ocu Acuccu ccuG uoc
100
c c co c u
cuoo^oG^AOU cO^ ^O^ucc
o@þôocsc
oouuuccuuco ocu.ucuooc'Gco^uccco
UC
^GO^CC
UUCUOO
U
cccuA ^
AOGGU
o^uuu ucccAu cuuc
cucc^c
^c u
o^o Aoo^o
50I ¡r
uþl^ ^ ^ u ^^ uct@l'Ã'þ.ero ^o^^^
6Aoocs o^Ao c
^O^o^ o
uuc^o ucc.ccoog
c AOO OOCCC
^A U
^^^ uCç
300
^c
150
c o
UC
o^Go cuc.uccuu
U
U
ï"ccuuoñuo^
la
f " u
350
cc^Aoo cco uoAgoo oooc ^cô^u'uu uuc uuuuu'uuccoc
uc ^fil ^cotr fttccuuc . o^^ou
cc
ll c0¡u cc c o0^00^a0u
I
250
cc
U o 200 u300356
CSV-E @ @
I
^Âl0A^lil|loô¡
@
r
@ @.lO
I.UrC
I
@
@UCUcc 0cu0 0rllllllilllo0 c0^u cuu
CA UUc
uorA 00c
||U. Cc0
UUC
lila^0
ilUC
A
^
ill illll ll lll00t
illcco
côr.0^uc0
ct
¡00^ccUU AU
cuctl^ u0 0^10
lllllc CUUC.l^
^O UcC'cC000
c
l^uuc
¡001G cu
ilill llucl uu 0ruu
¡l
iluu
cGAGA
illtcucu
00
IUC
C
l
I
tc
uuI
Il
U
001 ^Cil rlccu u0u@
ouu
c
ilil1|ccuuc0 0
^l
I
rïil
00
lt
0
I
c
¡c
I0
U
Il
UUUU
cc 0u00 0c ' ¡
il ulll I
oo.Ùokc.c 0 u
-t €I
cÞ
0ctu
ill0Gc I
l^^@
c
I0
lllrilcu tu00c lil ill lllu00 0^00
^t
¡00AU^
c,
I
@uu ET
UUUUUUc
c
@
U
@
u uc uc
O
-)0
lncluding PSTV, whì-ch share common
possibly secondary structures, and
fnom a common ancestnal viroid.
sequences and
which may be derived
CHAPTER 4
COCONUT CADANG-CADANG VTROID
39
]NTRODUCTION
Cadang-cadang is a serious and economicalJ-y
important disease of coconut palms which vras first
neported in 1927 on San MigueI IsIand in the Phillppines
(ocfemia, 1g37). In the fol-lowing years, incidence of
cadang-cadang disease I^Ias reported in surrounding areaS
of the Phil-ippines and nor^r, 56 years af ter its f irst
known occurrence, the disease j-s found widespread over
the south-east part of Luzon and many neighbouring
islands ( Figure 4- 1 ) . By 1 96?, only 1 00 of the 250, 000
coconut palms on san Miguef Island had survived the
disease (Bigornia,1977). It is estimated that
cadang-cadang dlsease is still responsj-bl-e for the death
of 5OO, OOO palms each year in the Philippines ( e '
ZeLazny, personal communication). Recent work has
indicated that tinangaja disease of coconuts on Guam, âD
island 1 ,500 miles east of the Philippines' has the same
aetiology as cadang-cadang disease (Boccardo et â1.,
1981 ). Hohrever, cadang-cadang dlsease has not been
found in any other coconut growing area.
The first symptom of the cadang-cadang
disease is the devel-opment on the affected palm of
smal-1, rounded coconuts whlch are distinctively
scarified. Laten, the fronds develop characteristic
yellow spots and as the disease advances the cro\^In of
Figure 4-1
coconuts.
Incidence of cadang-cadang disease of
Regions of the Philippine islands whene bhe disease
is widespnead are shown cross-hatched, while
isolated incidences of the disease occur in
surrounding areas (Zetazny, 1979). The different
ccRNA i solates used in thi-s work hrere each obtained
from separate diseased coconut palms in one of the
following locations : 1, San Miguel Island; 2,
Sorsogon; 3, Llgao; 4, Lake Baao; 5, Tinambac; 6,
Guinayangan; and 7, San Nasciso.
1a7
oo Burtas
U $¡
ol "
5
km
Samar
o
Masbate
Leyte
ß
anes
50 I
t3N
123 E
Southern Luzon
40
the palm is reduced to a tuff of short yelÌow fronds.
The course of the disease invariabl-y ends with the death
of the infected palm, which occurs from 3 years to more
than 15 years ( usual-1y about 1 0 years ) after the
appearance of fhe first symptoms (Zelazny and Niven,
1980 ) .
Li ttle r^ras known of the nature of the
pathogenic agent responsible for the cadang-cadang
disease until Randl-es (1975) showed the existence of two
RNA s pecies that r^rere present only in diseased paIms.
The RNA species ürere named ccRNA 1 and ccRNA 2 in order
of increasing size, âhd wene shown to share thermal-
denaturationr nucl-ease sensitivity, centrifugation and
electrophoretlc properties with viroids ( Randfes , 1975;
Randl-es et âf . , 197 6) . The ccRNAs hrere also
subsequently shown to be cincul-ar (Randles and Hatta
1979) , like viroids. Randfes and Pafukaitis ( 1 979 ) ,
using cDNA-RNA hybridization techni-ques, demonstrated
that ccRNA'1 and ccRNA 2 shared common sequences and
not found inthat the sequences of
healthy palms.
Recently it
ccRNA 2 occun as fast and slow efectrophoretic variants
and that the occurrence of the variants is nel-ated to
the stage of disease development in the coconut palms
( Imperial et ãI., 1 981 ) . The fast electrophoretj-c
the ccRNAs \^Iere
has been shown that ccRNA 1 and
41
varlants, ccRNA 'l f ast and ccRNA 2 f asL are present 1n
infected palms aL early stages of the disease, and as
the disease progresses over a period of years the ccRNA
1 slow and ccRNA 2 slow variants first appean and then
predominate ( Imperial et âf., 1 981 ; Mohamed et âf' ,
1982).
All four ccRNA species have been recenbly
shown to be infectious (Mohamed and Imperial'
unpubl-ished results ) and ' as the ccRNAs are
slngle-strand covalentl-y closed circul-ar RNAs with high
degrees of secondary stnucture ( Randles et âf., 1976;
Randles and Hatta, 1979), they possess both biological
and physical properties simil-ar to those of viroids. In
order to further investigate the intrigulng
rel-ationships between the cadang-cadang disease, the
vaniant ccRNAs and viroids, the sequences and structures
of the dif f erent ccRNAs I^¡ere determined and compared.
METHOD S
A. Isol-ation of the ccRNAs
Purif ied ccRNAs \^Iere kindly provided by Dr.
Nizan A. Mohamed. Fnonds were harvested from naturally
infected coconut palms from a number of sites in the
Philippines. Nucleic acids I^iere extracted f rom the l-eaf
tiÀsue as described by Imperial et a1. ( 1 981 ) using
their Method 1. Tndividual- ccRNAs LIere purified by 3
42
cycfes of polyacrylamide gel efectrophoresis ( Imperial-
et âf ., 1981 ).
B. Sizing of the ccRNAs
Sizes of the ccRNAs I^rene
el-ectrophoresis in 6% polyacrylamide
cm) containing 9B% formamide (Maniat
1980 ) fne f olJ-owing vrere used as mo
mankens - solanum nodlfl-orum mottle
377 residues ( Haseloff and Symons, 1
mottl-e virus (VTMoV ) ntln 2, 365 resi
Symons, 1982); chrysanthemum stunt v
residues (Hasel-of f and Symons, 1981 )
cucumber mosaic virus ( CMV ) RNA 4, 1
and Symons,l982); CMV satel-lite RNA,
( Gordon and Symons, 1 983 ) ; chicken 1
residues ( Spohr et â1., 1976); alfal-
(AMV ) nl¡R 4, BBl residues (Brederode
yeast 5 . BS RNA, 1 5B nesidues ( Rubin,
RNA, 121 residues (Vtiyazaki, 1974);
estimated by
gels ( 4OxZO
is and Efstr
lecufar weig
virus ( SNMV )
982); vefvet
dues ( Haselo
iroid (CSV ) ,
; I strain o
027 residues
336 resldue
BS rRNA, 180
fa mosaic vi
et âf., 198
1 97 3) ; yeas
Escherichia
x0.05
adlatis,
ht
RNA 2,
tobacco
ff and
356
f
(Goutd
e
0
rus
0);
r 5s
col-i
phenylalanine tRNA , 7 6 nucleotides ( Banrefl- and Sanger,
1969).
The circular RNAs (SNMV RNA 2, VTMoV RNA 2,
CSV and the ccRNAs'ürere boil-ed for 15 minutes in
distilled water before electrophoresis to produce the
l-inear f orms. Af ter ef ectrophoresis, gels I^Iere stained
43
r^rith 0.01 % tol-uidine bfue and destained with water.
C. Fingerprinting of the ccRNAs
Purified ccRNAs (0.5 Ug) vüere dried down,
resuspended in 5 UI 5mM Tris-HCI pH 7.5, and digested
with 0.1 UC RNase A at 37oC for t hour or with 20 unlts
RNase T.', aL 56oC for 30 minutes. The resultant
oligonucleotide fragments urere transferred to another
tube contaì-ning 5 uci dried down Iv-32p] arP (2ooo
Cilmnol ) and 1 .5 U1 of 5x Tr* polynucfeotide kinase
buffer ( 25OmM Tris-HC1 pH 9.0, 50mM MgCIr, 5OmM
dithiothreitol-), and 0.5 pI (0.5 units) Tq
polynucl-eotlde kinase added. The reaction Idas incubated
aL 37oC for 20 mj-nutes and 5 Ul formamide l-oading buffer
(95% (v/v ) Oeionized formamide, 10mM NaTEDTA, 0.02%
(w/v) bromophenol bIue, O.02% (w/v) xylene cyanol FF)
added.
Radiol-abel-Ied ollgonucl-eotides h¡ere
fractionated by two dimensional- polyacryl-amide gel
el-ectrophonesis. For the first dimension, preparatlons
!,rere ef ectrophoresed in 40x20x0.05 cm 10% polyacrylamide
gels containing 25nM sodium citrate pH 3.5 (leWachter
and Fiers, 1972; Frisby , 1977 ) . After the xyl ene cyanol
FF dye marker had migrated 14 cfl, electrophoresis \^ras
stopped r and gel strips r^¡ere excised and embedded aL the
bottom of 40x20x0.05 cm 25% polyacrylamide gels
44
contalning B9mM Tris-borate pH 8.3, ZmM NaTEDTA (Frisby,
1977). Polymerization of the second dimension gels r^ras
catalysed by the addition of 300 Ul 10% (w/v) ammonium
persulphate,30 Ul TEMED, 50 Ul 10% (w/v) ascorbic acid
and 70 llf 30% (w/v ) HZOZ per 50 ml of gel soÌution to
ensune compfete polymerization in the region of the
f irst dimension gel strip. Samples v\rere electrophoresed
upwards until the bromophenol bl-ue dye marken had
migrated 1B cn,
oJ-igonucfeotides
32
were detected by autoradiography.
D. Sequence and structure determination of the ccRNAs
Punified ccRNAs \^rere sequenced essentially as
described in chapter 2. Purified ccRNAs ( 5 ug) isorated
f nom sì-n91e inf ected coconut paJ-ms i^Iere sub j ected to
l-imited digestlon by 3 units/ml_ RNase UZ, 5 ng/nl RNase
A or 2000 units/mI RNas. T.l under non-denaturing
conditions ( 600mM NaCf, 1 OmM MgCl Z aL 0oC ) . The
res.ulting l-inear RNA f ragments brere 5'-radiof abef led. r 32--using I V---P_IATP and Tq polynucfeotide kinase or, after
treatment wi-th calf intestinal phosphatase,
3r-radiolabelled using [¡'-3'rldpCp and Tq RNA t-igase,
and fractionated by denaturing polyacryl_amide gel
el-ectrophoresls as detailed in Chapter 2 A-3 r4 r5.
Radiolabel-l-ed f ragments b¡ere located by autoradiography,
excised, el-uted, and sequenced using partial enzymic
and the fractionated P-labeÌed
45
cleavage methods. The sequences of numerous overlapping
fragments vüere assembled to give the compl-ete primary
structure of each circul-ar mol-ecuf e.
Secondary structures of the ccRNAs vüere
mappgd using St nuclease ( Wurst et âf . , 1 978 ) . Futl
J-ength 5t - on 3 | -radiol-abelled ccRNAs, obtained as
descrj-bed above by RNas" T1 digesLion, were suspended in
20 Ul 200mM NaCl-, 0.05mM ZnSOO, 50mM sodiurn acetate
pH 4.6, containing 5 Ug Escherichia coli tRNA c arrier,
and incubated aL 37"C for 10 minutes with 0.1, 'l or 10
units of S" nuclease ( Boehringer ) . The reactionI
mixtures r^i ere extnacted wlth phenol, precipitated with
ethanol- and fractionated by el-ectrophoresis in a
polyacrylamide gel containing BM urea and TBE buffer
( 9OmU Tris-borate pH 8.3, 2ml4 NaTEDTA ) . Products of
partial enzymic sequencing reactions of the same ccRNA
species vüere run as markers, thus allowing sifes of S1
nucl-ease sensitivity to be l-ocated. Data so derlved
r^rere used during consLructj-on of the secondary structure
models of ccRNA 'l f ast and ccRNA 1 slow.
Secondary structure modeÌs for the native
ccRNAs hrere constructed
Tinoco et
using the
al-. (1971;
base pairing
1 973 ) and the
pnedlcted RNA
values kindl-y
. Gross, J.W.
matrix
provided
Randles,
procedure of
thermodynamic stabilities of the
s tructures r^rere calcul-ated using
by Dr. D. Reisner (C. SLe65er, H.J
46
H.L. Sanger and D. Reisnen, unpublished results).
RESULTS and DISCUSSION
A. Sizing of the ccRNAs
The sizes of the finear fasl and slow
variants of ccRNA 1 and ccRNA 2 were estimated by
electrophoresis in polyacrylamide geJ-s containlng 98%
formamide, using Iinear RNAs of known mol-ecular weight
as markens (Figure 4-2). No differences in mobiJ-ity
were observed between the different ccRNA isolates. The
sizes of the RNAs were estimated to be : ccRNA 'l f ast,
250 residues; ccRNA 1 slow, 300 residues; ccRNA 2 fast,
500 residues; ccRNA 2 slow, 600 resldues. Therefore the
ccRNA 2 fast and ccRNA 2 sÌow forms are approximately
twj-ce the size of the correspondlng ccRNA'l forms and
the sl-ow forms of both ccRNA 1 and ccRNA 2 are 20%
larger than the corresponding fast forms. These
estimates of size differ from those of ccRNA 1 (31013
resldues) and ccRNA 2 (438t5 residues) as obtained by
length measurements made after el-ectron microscopy
(Randl-es and Hatta, 1979).
(1979) Oid not differentiate
Randles and Hatta
the fast and sfow
However,
between
ccRNA variants which mâV, together with perhaps
under-estimated experimentaf error, have resulted in the
differences observed.
Figure
forms
4-2 Slze estimation of the fast and sfow
of ccRNA 1 and ccRNA 2 by eleclnophonesis in a
6% polyacrylamide ge1 containing 98% formamide.
148, 620C and X2 vlere different isol-ates of ccRNA
obtained from Ligao, PhiJ-ippines. Sizes of the
ccRNAs wene determined frorn a standard curve of
mobillties of the RNA markers (described in Methods)
pl-otted against their known sizes on a logarithmic
sca1e. 0nJ-y the Iinear RNAs, hot circuf ar RNAs vüene
used for size estimation. Fon SNMV RNA 2, VTMoV RNA
2, CSV, and the ccRNAs, the linear forms are
satellite (sat)indicated by arrov\rs.
RNA, the bands are (in
2, and 3 runnlng as a
sateflite RNA. Marker
Fon CMV
order
broad
RNAs,
p lus
from the
band, RNA
in order
yeast 5.8S
TRNA.
top) RNAs 1,
4 and
from the top,
RNA, yeast 5Sare chicken 185, AMV RNA 4,
RNA and E.coIi phenylalanine
It *i*
'f,+
SN
MV
RN
A 2
VT
Mov
RN
A 2
csv
CM
V +
sat
MA
RK
ER
S
¡ir+
a I
"""*
o t-
t"",
e zo
clr¿
e'l
o2oG
l ccR
NA
t-s
low
x2 I
62O
C c
cRilA
2-f
ast
X2
ccR
NA
2-s
low
Il, rlI,
^lI o 2 e z
SIZ
E (
no.
ot b
ases
)
êc
ê c= o g I = a 3
N ê ê
/o Ð z I Þ ¿ q o o ú
o o ¡ 2 I Þ I N o o t
o o ! z N I ø_ o É ¿ o o o d
o u z I o f I o o o
47
B. Fingerprinting of the ccRNAs
The sequence rel-ationships between the four
ccRNAs \^rere further investigated using RNase A and RNase
T, fingerprints. The individual purified cincularI
ccRNAs isolated from a single infected palm vùere
digested to compl-etion with RNase A or RNase T., , 5'-32p
radj-ol-abelled, and the resulLanb oligonucfeotides
f ractionated by two-dimens j-onaI gel el-ectrophoresis.
The RNase A fingerprints of the four forms of ccRNA
extnacted f rom the same tree (isol-ate Ligao t.,, ) show
essentiaì-1y identicaf patterns of l-abelled
oligonucleotides ( Figune 4-3 ) . This indicates that the
three larger ccRNAs contaln the same sequences as the
smalf est ccRNA 1 f ast. SimiJ-arJ-y, essentiaJ-ly identical
patterns of radiolabell-ed oligonucfeotides vüere obtained
af ter digestion with RNase T.,' ( dat'a not shown ) , however
three extra oligonucl-eotides \^Iere found in the RNase T.,
f ingenprints of the f ast ccRNA f orms, which I^iere not in
the fingerprints of the sfow ccRNA forms; the
significance of this is not known, but may be rel-ated
either to sequence heterogeneity obsenved in ccRNA fast
forms (see below) or to difficulty experienced in
ensuring the RNas" T1, in contrast to RNase A
digestions, always go to compl-etion.
The ccRNA 2 fast and slow forms are estimated
to be twice the sizes of their respective ccRNA 1 forms
Figure 4-3 RNase A fingerprints of ccRNAs.
The fast and
digested wi th
fnactionated
sl-ow forms of ccRNA l and ccRNA 2 were32RNasê A, 5r- P-label-1ed and
by two dimensional gel
is
e I e c t r o p h o r e s i s .
from left toMigration
right and
top.
in the first dimension
in the second dimension from bottom bo
CCRNA 1 FAST CCRNA 1 SLOW CCRNA 2 FAST CCRNA 2 SLOW
t
a o oaa aO
a a(a a
la.D
raO
.D
DID
oqtt o
- D- o-(o:oo-.
1at)
.Da o Ja a
loe a
at:r-
baa aa. a
- 1o .D aoaDO
eoo¡
aa
(D-¡rD
( Figure
ccRNA 1
4-2l, and,
and ccRNA
it is
it is possible that
dimers of the ccRNA
48
since the RNase fingerprints show that
2 possess similar sequence conplexity,
the ccRNA 2 fast and slow forms a"e
1 f ast and sl-ow forms respectively.
J-ikely that each ccRNA s l-ow speicesFurthermore,
contains only
fast species.
the sequences
repeated sequences of the respective ccRNA
In order to extend these observaLions,
of the ccRNAs lrere determined.
C. Sequences and structure deterrnination of the ccRNAs
Native circufar ccRNAs vüere subjected to
limited digestion either by RNas" T.l , which catalysed
cleavage of ccRNA'l species at singl-e sites and ccRNA 2
species aL elther or both of two sites to pnoduce
specific full- length Iinean ccRNAs (Flgure 4-4), or by
ribonucleases A or U2, which produced smafl-en
overl-apping RNA fragments. These l1near RNA molecules
vüere 5 r - or 3 ' - radiol-abelled and then purif ied by
polyacryl-amide gel electnophonesis. The sequences of
these 5'- or 3 r - l-abelled f ragments vüere determined by
the partial enzymic digestion technique. The use of
f ragments f abel-1ed separatel-y at both the 5 r - and 3 r -
ends al-l-owed the sequence determination of long RNA
fragments up to 574 nesidues long and, with shorter
fragments, resol-ved gef compression artefacts ( Kramer
and Mills, 1978) when the rel-evant nucLeotide sequences
Figure 4-4 Partial RNase T . digest of ccRNAs.
Purif led ccRNAs (lsoIate Llgao T., ) hrere digested
with 2000 units/ml RNase T., under condltions of hlgh
salt and 1ow lemperature as descnibed in the text.
Resulting 11near RNAs were 5,-32p-tabell-ed and
fractionated on a 5% polyacrylamlde geI containing
7M urea. Di-gestion of ccRNA 2 species gave rlse to
linear RNAs corresponding in sizes to those of the
ful-1-length ccRNAs 2 and ccRNAs 1.
ccRNAl ccRNAl ccRNA2 ccRNA2
f ast slow fast slow
Origin
tpr+.ff - ccRNA2 slow linearccRNA2 f ast linear
##r-..t+r ccRNA 1 slow linear
ccRNAl faEt llnear
5% TBE7M Urea
è.
49
b/ere determined from both directions.
assembl-ed
The sequences of
to construct theoverlapping fragments r^rere
complete primary structunes
molecules.
of the circul-ar RNA
Secondary structure models for the native
ccRNAs were constructed using the base-pairing matrix
pnocedure of Tinoco et aÌ. (1971), val_ues for the
thermodynamic stability of the predicted RNA structunes(C. Steger, H.J. Gross, J.!ù. RandJ_es, H.L. Sanger and D.
,,1Rþli)sner, unpubrished results), and experimental evidence
for the l-ocation of ribonucfease sensitive single
stranded regions on the native mol-ecul-es. rn addition,
specific fulf-rength linear ccRNAs, ppoduced by rimited
ribonuclease T,, cf eavâge, vJere eithe r 5, - or 3r-32p
fabeÌ1ed and the susceptible singJ-e strand regions in
the native structures rocated by the s1 nucl-ease mapping
procedure ( Vùurst et â1. , 1978) . The sltes of cleavage
were detenmined by co-el_ectrophoresis of the
radiol-abel-led fnagments of the s1 nuclease digest withproducts of sequencing reactions using the partial
enzymic digestlon procedure (Figure 4-5). Thus the
possibility of speclf ica1ly l-1ne arj zing the circul_ar
ccRNAs by limited cleavage with RNase T., both
facilitated sequence determination of the mofecul-es and
afl-owed s1 nucl-ease mapping of the renatured l_inearized
mol-ecuf es.
ltJ
Figure 4-5
S1 nuclease
Partial enzymì-c digestion sequencing and
mapp ing of the ccRNAs
Full--ì-ength l-inear lsolate Baao 54 ccRNA 1 fast was
pnoduced by partlal RNase T., digestion under
non-denaturing condiLì-ons, 5'-32p-rabelled and
purified by polyacryl-amide gel efetrophoresis as
descnibed in the text. Punified radiolabell-ed RNA
vras,subjected to treatment by no enzyme (N), RNase
T. (T), RNase U. (U), alkali (L), RNase PhyM (P),t¿
Bacillus ceneus RNase ( B ) and 1 0 units nucl-ease S1
(S). The products were fractionated by B0 cm 6%
polyacryl-amide gef electroporesis, and cleavage
sites for nucl-ease S1 are shown anrovJed on the
predicted secondary structure modef of ccRNA 1
f ast.
2A
'\^'^î,.r"1()
I sll^il""^
AU
;;
"r'íÌf"iI ltcâÞi ¡iaR"3i
R
GCAGG. AGA GCCGCACUAC AC
(¡
CC UCU.CGGCG
AU
ccAGGG.CACC
UCCC GUGG
U
U
2.+c
1àU
g7
J\GGGG
ccccA
A
218
9*.'t^-ilrIIII
-
5?89È g6lñÂìNôt ñilllll-.1
\
ú'tfe?ar¡O co¡-(oÂlôtôlñÂtN dÈiôINÂìÑôIN NôIN
illllI ill
\
U(¡
zta
U
A226
ì@câôl
-l^I,,
224
æb<oô6t¡ùi¡Àl
tl I If,
st.t-
,õoo-JrtFz
tII
,
t\ \llllltI I
\\t{att {
t0
titl. ¡ '
50
D. ccRNAs differ in size but not sequence complexity.
TheSequencesandpredictedstructuresofthe
ccRNA 1 fast and sl-ow forms isolated fnom a slngJ-e
infected coconut palm (isol-ate Baao 54, Figure 4-1) are
shown in Figure 4-6 together with the known structures
of four viroids, PSTV ( Gross et âf. , 1978) , CSV
(HaseIoff and symons, 1981 ), CEV (Visvader et al ., 1982)
and ASBV (Symons, 1981 ). The two native ccRNAs 1
possess extensive regions of intramolecul-an base pairing
and can form rod-tike native structures similar to other
viroids. The ccRNA 1 fast and ccRNA 1 slow possess 246
and 287 nesidues respectively, âDd have cafculated
stabililies of -320 and -360 KJmol- -,1thermodynamic
respectively.
sequence and
differs by an
struc ture of
fast ) which
The ccRNA 1sl-ow contains the entire
structure of the smal-ler ccRNA'l fast but
additionaf
nati-ve mofecul_e between resldues 123 and 124 0f ccRNA',l
fast ( Figune 4-6) . Thus , the rod-1ike, base-paired
nat j-ve structure is maintained in the larger molecul-e.
The nucl-eotide sequences of ccRNA 2 fast and
ccRNA 2 slow, consisting of 492 and 574 residues
respectiveJ-y, are perf ect dimers of the respect j-ve ccRNA
'1 f orms. A schematic summary of the rel-ationships
41 residues
is added at
duplicated sequence and
(residues 'l 03-143 in ccRNA 1
the right-hand end of the
Figure 4-6
structures
Sequences and redicted secondary
of the Baao 54 isol-ate of ccRNA 1 f as t
and ccRNA 1 sl-ow ane shown with those of PSTV, CSV,
CEV and ASBV. The structures are aligned under the
central conserved negions of these viroids (boxed).
Cadang-cadang RNA 1 fasti20\ u¡ I
"\, r.\ oooo ¡u
t^2ao
Cadang-cadang RNA I slow
2a€
ao t@
^l80 !ao
cccc uc uccc ouúo
20t"l"
320
2AOr60
rto
2A7
PSTV
r20 tao
"t^!
\.
t"I
35e2ao
êc^l¡220 "ìo
r20 tao
I
lAO
r80
I
CSV
\,
m
oo¡ucu Jcu aouucc oou uc¡c
ü ilo
ASBV
80
I E¿LUo ¡uo.I c uu Itu
u 3ao "_32o ^.._ gæ
cEvl0 a0 tæ ttot
I
Il7
40
t"
u8P!æræ:J I I
^ ^/ u !
s
".1' , J¡I t t lct
rao c r0o r¡o
51
between the pnimary structures of all
their predicted secondary structures
foun ccRNAs and
is glven in Figure
can base4-7. V,¡hile each of the monomeric ccRNA 1 forms
pair intramoÌecul-arJ-y to forrn a single rod-like
conformer, the ccRNA 2 forms, due to their dimeric
nature, can each form either of two rod-like conformers
(A or B, Figure 4-7) and a large number of intermediate
cruciform-shaped structures, one of which is given 1n
Figure 4-7 .
The ccRNA 1 fast and ccRNA 1 sfow mol-ecufes
each possess, under experiemental- conditions, a highly
accessibl-e site for cleavage by RNase T.,, at the
right-hand termlnal hairpin loop of the predicted native
structure (between residues 124 and 125 1n Baao 54 ccRNA
1 fast and between residues 145 and 146 in Baao 54 ccRNA
1 slow ) . Limited RNase T., digestion of each ccRNA 2
species produced specific finear RNA fragments
corresponding to both the respective full-length linear
ccRNA 2 and ccRNA 'l mofecufes. Sequence determination
of these fragments showed that cleavage of the ccRNA 2
molecules occunred aL two sites located at the same
sequences as for the two ccRNA 1 molecul-es. This
suggests that either the predicted confonmer A of the
two ccRNA 2 mol-ecufes on possible cruciform
intermediates exist in sol-utlon, whereby the appropniate
terminal hairpin loops are exposed. However, the
Figune 4-7 Schernatic representatlon of the
sequences and pnedicted structure relationships
between the ccRNAs.
The circular sequences of the four ccRNAs are shown
wibh bIack, âhd cross-hatched boxed negions
representing the sequences highly conserved between
ccRNAs , PSTV, CSV and CEV. The white, and stippled
boxed regi-ons represent those sequences duplicated
within the ccRNA 'l sl-ow species. Positions
cornesponding to residue'1 of ccRNA fast or ccRNA 1
slow are indicated by black dots. Both ccRNAs 2 are
dimers of the respective ccRNA 1 forms and can
potentially form either of two rod-like conformers A
or B as wel-l- as a Iarge number of cruciform-shaped
intermedlates, of which one is shown. Each ccRNA 1
species possesses a single, highly accessibl_e site
for RNase T. cleavage Iocated on a terminal hairpinI
loop; these sites are indicated by arror^rs. Each
ccRNA 2 species possesses two such accessible sites
for RNase T. cleavage and arrows indlcate whereI
these sites al-so occur on hairpin loops in the
different ccRNA 2 conformers.
ccRNA I last
5 I
+-
ccRNA 2 last
ccRNA I slow
ccRNA 2 sl
-Hr
J e
B
f-
-lA
B
j a-
52
existence of type B conformens cannot be precluded.
E. Variation Ín sequence between different ccRNA
isol-ates
Diffenent lsol-ates of cadang-cadang RNAs hlere
each obtained from single infected coconut palms from
different locafities in the Philippines ( figure 4-1 ) .
Sequence differences between the isotates consist of two
types. First, the sequences of the ccRNA 1 sl-ow forms
can differ. While afl ccRNA 1 fast forms are
essential_Iy identical ( see below ) , ccRNA ',l sl-ow f orms
can diffen in the length of the repeated sequence
inserted between resi-dues 123 and 124 of ccRNA 1 fast.
Three different repeated sequences found in nj-ne
sequenced isol-ates of ccRNA 1 slow are given in Figure
4-B; these vary in length from 41 to 55 residues but ane
al-l internalty base-paired to produce dupJ-icated
structures as well- as Sequences al the right-hand ends
of the natlve molecul-es. Interestingly, the right-hand
ends of the mofecules of PSTV, CSV, CEV and ASBV (Figure
4-6) are similarJ-y distanced from the centraf conserved
regions of these molecules. In contrast, the right-hand
side of the ccRNA'l fast mol-ecuIe 1s shorten while those
of the elongated ccRNA 1 sfow mol-ecufes are cfoser in
size to those of PSTV, CSV' CEV and ASBV.
Second, four of the six isoÌates of ccRNA 1
Figure 4-B Sequence variatlon between ccRNA 1 sl-ow
of three ccRNA isol-ates.
The sequences and
and ccRNA 1 slow
structures of various ccRNA'l fast
ASisol-ates were determined
text. As essentially alldescribed
variation
in the
occurred aL the right hand end of
sequence
the
ccRNA 1 slow molecul-es, only this region is shown.
Boxed regions represent those sequences which are
duplicated in the ccRNA 1 slow mol-ecul-es and which
are 41 (isolate Baao 54), 50 (isol-ate Ligao 148) or
55 residues ( isolate Ligao T1 ) long. AIl sequenced
ccRNA 1 slow isol-ates correspond to one of these
forms ( Table 4-1 ) .
ccRNA I last'120
GCG
cGc
ccRNA I slow Isolatc Ligao T,
GCG
cGc
'160
uccG
cGcc
ccRNA I slow
100
lsolate Ligao l4B
120
120
I
200 160
140
180
ccRNA I slow lsolate Baäo 54
100
ucGCG
coc
CUGGG
GGCCC
53
fast sequenced each consist of two populations of
mofecules, one of 246 nesj-dues and the other of 247
residues, which differ in the presence or absence of a C
aL residue 1 9B ( Figure 4-9, Table 4-1 ) ' Simil-ar
sequence heterogeneities have al-so been reported fon CEV
(Gnoss et â1., 1982 ) and the viroid-like RNA of vTMoV
( Hasel_off and symons, 1982) . The nefative proporfions
of the two ccRNA 1 fast subspecies vary between
different lsol-ales as Iisted in Table 4-1. For the two
ccRNA 2 fast isol-ates sequenced, the relative
proportions of the Lwo forms are the same as those of
the corresponding ccRNA 1 fast. In contrast to the
'ccRNA 1 and 2 fast species, no similar sequence
heterogeneity has been observed in nine isol-ates of
ccRNA 1 slow and the one sequenced lsolate of ccRNA 2
slow (TabIe 4-1), Each isolate of the ccRNA slow
species thus consists entirely of either one subspecies
or the other; in all except one case, the C at the
position corresponding to ccRNA 1 f ast residue 'l 9B was
absent. The - various sequence differences between the
ccRNA isolates do not seem to correlate with differences
in geographic focation.
F. Structural- simil_arities between ccRNAs and viroids
The ccRNAs shane two regions
nucfeotides,
of sequence
with the viroidshomology, each of abouL 2O
Figure 4-9
ccRNAs.
Sequence hetenoEeneit y r^ri thin the
Purified ccRNAs were found to consist
or a mixture of two RNA
the presence or absence
position corresponding
species which
of an extra C
of either one
differed in
residue at the
ccRNA 1 fast residue 197
predicted native stnuctures
shown with stars to
to
or 198. Port j,ons of the
two
the
species are
sequence differences.
those sequences common to
of these
indicate
indicate
CEV.
Boxed regions
PSTV, CSV and
U
GCCGC U
CG
60
60
CG A
c*200
U
GCCGC U
CGGCG A
cc**o
U
2
G
G cUCC CC
r ccRllÀ isolates were purified from nucleic-acid extracts of
single, infecteci coconut Palms. Ì
t ReI¡tive proportions of sequence variants were <letermined by
sequence analysis ()f RNase Tl-digested, ¡5'-32p¡radiolabelled
fulJ--length linear ccRNAs. Íf a ccRNA consisted of a mixture of
variants, band doubling was observed on sequencing gels after
ccRl.¡À I f.rst rr:sir'lue 197. Relative proportions of the two sets
of banrì doul¡.Lets; wcrc takcn ar; estimates of the molar proportions
of the two varianE ccRNA sPecies.
* These ccRNA I fast s.pecies consisted of a nixture of two
species, one of 246 ar,ð the other ot 247 residues.
*r Ligao Tl ccRNA 2 fast species contained sequence heterogeneity
at the positions corresponcling to ccRl¡À I fast resl<lue 198. Due
to limltaÈions of sequencing Èechnigue, it was not determined
whethcr climeric ccRlt^ 2 fast consistecl of a 492 (2 x 246) residue
s¡recies togathcr with a 494 (2 x 2411 residue species or cnly a
493 (246 + 2471 residue species.
Table 4-1 Propertìes of RNÂs of various ccRNA isolates'
Re l ati ve ProPorti ons
ccRNA lsoìate* of sequence variantst
ccc
Totaì length
of ccRNA
(resi dues )
Length of sequence
dupl i catl on
( res I dues )
ccRNA I fast
Baao 54
Tinambac
Li qao l4tÌ
Li gao 620C
Li 9ao 1910
Li gao Tl
ccRNA 2 fast
Baao 54
Lì gao Tl
ccRNA 1 slow
Baao 54
Ligao'l48
Li gao 620C
Li gao 19lD
Lì gao Tl
Li gao 5
Gui nayangan
San Mi grreì
San Nasci so
ccRNA 2 slow
Baao 54
1.0
1;0
0.8
0.6
0.4
o.2
0
0
0.2
0.4
0.6
- 0.8
?46
246
246 1247+
246 /247
246 /247
246 /247
492
A92-494**
287
296
296
296
301
?96
?96
296
297
574
1.0 0
0.2 0.8
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0
0
0
d
0
0
0
0
0
I 0
4t
50
50
50
55
50
50
50
50
4t1.0 0
54
PSTV, CSV and CEV (Figure 4-6). The latter three
viroids are closely rel-ated, sharlng abouL 50% sequence
homology. The two conserved regions are base-pained in
the predicted structures of the native molecul-es to form
highJ-y conserved secondary structures.
The conserved regions shared by the ccRNAs
correspond to regions of PSTVTCSV and CEV postul-ated to
be involved in base-pairing of viroid compl-ementary RNAs
with a plant smal-l- nuclean RNA (snRNA ) in a manner
anal-ogous to that proposed for the interaction of mRNA
intron-exon splice junctions with mammal-ian Ula snRNA
(Gross et âf ., 1982; Diener, 1981), and in the formation
of a stabilizing stem-1oop structure in the viroid
complements ( Gross et â1., 1982) . The proposed
lnteraction between viroid compl-ements and snRNA is
postulated to reflect the origin of vlroids from an
lntnon ancestor (Diener, 1981) or as a basis for
pathogenesis ( Gross et âf. , 1982; Diener, 1 981 ) , but has
not been proposed to be directJ-y invol-ved in viroid
neplication. However, the RNA or compl-ementary RNA of
aL least one viroid, ASBV, is incapabl-e of base-pairing
wi-th a UIa-like snRNA or of formatlon of the conserved
stern-loop structune, despite up to 1B% sequence homology
betwen ASBV and other vlroids (Symons, 1981 ). It is
possible that the central- conserved regions of viroids,
including ASBV, tr€f lect f unctional- simll-arities rel-ated
55
to viroid replication rathen than to the postulated
snRNA b inding.
G. Replication of ccRNAs
As PSTV,CSV,CEV and the ccRNAs are capable of
autonomous repJ-ication, the enzymes involved are of
considerabl-e interest. Ho\^Iever ' no viroid-encoded
translation products have been found in vitro (DavÍes et
âl . , 197 4; Semancik et aJ . , 1977 ) or in vivo ( Conj eno
and Semancik, 1977). Although PSTV, CSV and CEV share
around 50% sequence homology' none of these vlroids nor
thein putative complementary RNAs can theoretically
encode similar transl-ation products (Hasel-of f and
Symons, 1981; Vlsvader et al-., 19BZ), even assumlng the
existence of LransLatabfe l-inear viroid RNAs in vivo
(Kozak, 1979; Konarska et â1 . , 1981 ) . Possibl-e
protein-coding regions simifan to those of other viroids
are not found in the ccRNAs on their complements nor are
there any AUG initiation codons present. It therefore
seems highly unlikely that the ccRNAs can code for any
functional- polypeptide product. All evidence indicates
that ccRNAs and other viroids must rely entlreJ-y on host
components for their replication.
Larger that unit-length compfementary
intermediates have been detected in PSTV, CEV and
infected tissues ( Branch et âf . , 1 981 ; Rohde and
(-) RNA
A SBV
ItSanger,
56
1981; Owens and Diener, 1982; Bruening et â1., 1982) '
In addition, âtr oligomeric series of RNAs of ASBV (+)
have been detected in infected avocado tissue; the dimer
of ASBV has been purifled and characterized as a
single-stnand, circul-ar molecule slmifar to the ccRNA 2
molecules. Rolling circl-e mechanisms have been
postul-ated for the synthesis of oligomeric compl-ementary
RNAs from circular viroid templ-ates (Branch et a1',
1981; Owens and Diener,19B2; Bruening et â1., 1982),
and oligomeric viroid (+) sequences could be simply
generated by transcniption of multimeric ( - ) s trand
templ-ates. Unit length viroid pnoduced by either
specific transcription or cleavage of oligomeric viroid
RNAs must be ligated to produce the finaÌ circufar
product. Such a model for viroid replication, invoÌving
oJ-igomeric RNA intermediates, could readily account for
the formatÍon of the dimeric forms of both ccRNA 1 fast
and ccRNA 1 slow; that is, ccRNA 2 fast and ccRNA 2 sl-ow
nespectively. Rate-Iimiting steps during the
transcription or the possible processing of viroid
transcrJ-pts would al-1ow the dirneric ccRNAs 2 to
accumul-ate over the monomeric ccRNA 1 species.
H ccRNA slow variants and the tlme course of infection
In the initial stages of cadang-cadang
disease, only the fast forms of ccRNA 1 and ccRNA 2 are
57
present in infected Pa1ms and it is onJ-y after a furthen
of ccRNA 1 and ccRNA24-30 months that the sfow variants
2 first appear and in the foll-owing yeans predominate
(Mohamed et al ., 1982]}. These data, pl-us prelJ-minary
evidence that the ccRNA fast species are more infectious
that the ccRNA slow species (ImperlaI et âf., 1981 ) ' are
consistent with the de novo generation of the ccRNA sfow
varlants durlng each cadang-cadang disease Ìnfection.
This proposition is supported by the following sequence
data.
1) The ccRNA 1 sf ow forms dif f er f rom ccRNA 'l fast by
the insertion of a single repeated sequence (Figure
4-6) and coul-d be simply generated from the ccRNA 1 fast
by processing and/ or transcription mechanisms '
2) The ccRNA 1 slow isolates can differ in the size of
their inserted sequence repeats (Figure 4-8, Table 4-1 )
suggesting separate orlgins for these ccRNA slow
variants.
3) !,Ihi1e most ccRNA fast isolates contain a sequence
heLerogeneity aL residue 1 98, and consist of varying
ratlos of the 246 and 247 residue species, each of the
nine sequenced ccRNA slow isolates consists of a singl-e
homogeneous poputation, either with or without a C
residue at the position homologous to ccRNA 1 fast
residue ,l 98, and with only one slze of repeated Sequence
5B
(Figur" 4-9, Table 4-1 ).
These data are consistent with the generation
of ccRNA sl-ow forms from ccRNA fast by single, rare
sequence duplication events occurring separatefy in each
cadang-cadang inf ected paIm. AII ccRNA sl-ow mol-ecul-es
wou1d, therefore, origlnate from single panent moÌecul-es
and may accumulate in pneference to ccRNA fast species
due to a competetive advantage in repJ-ication.
I. 0rigln of cadang-cadang disease
The ccRNAs share biological- properties and
sequence and structuraf homology with viroids so that
application of the term coconut cadang-cadang viroid
( CCCV ) i-s fully justified. However, whereas other
viroids consist of a single predominant infectious RNA
species, CCCV consists of several variant RNA species.
It is feasible that CCCV may have arisen from a
pre-existing viroid and that mutation or infection of
ner^/ hosts, such as the coconut palm and rel-ated host
specles ( Randles et â1 . , 1 980 ) , resulted in the
production of the variant ccRNAs by abberant
transcrlption and/or pnocessing mechanisms whlch
nonmally occur faithfully 1n the replicaiton of other
vinoids. The outbreak and subsequent apparent rapid
spread of the cadang-cadang disease in the Philipplnes
this century (ZeIazny, 1979) is consistent with such an
59
origln of the ccRNAs.
As viroids do not appear to encode functional
polypeptide products, it seems llkel-y that these
pathogens rely entirel-y on the interaction of the viroid
RNA with host celf components for replicatlon. If so,
the homology between cccv, which replicates in several
species of the monocotyl-edonous plant family Pal-maceae
(Randl-es et al- . , 1980 ) , and other viroids, which
replicate in dicotyledonous plant hosts ( Dienen, 1979) ,
may mj-rnor simifar homology between cell-uIar components
responsibl-e for viroid repl-ication in these different
hosL plants. The exact nature and function of these
possibly conserved host cel-l- components is as yet
unknoü/n.
CHAPTER 5
VELVET TOBACCO MOTTLE VIRUS
AND
SOLANUM NOD]FLORUM MOTTLE VIRUS
60
INTRODUCT]ON
As outlined in Chapter 1, a nev'r unique group
of plant viruses has been reponted in Austral-asia
(Randfes et â1., 1981; Gould and HaLLa, 1981; Tlen Po et
â1., 1981; Francki et â1., i983). The viruses consist
of 30 nm diameter pofyhedral capsids containing two
major single-strand RNA specles; the RNA 1 species ane
l-inear rnolecules of abou L 4 ,5OO residues (Mr 1 .5 x 1 O6 )
wheneas the RNA 2 spec j-es are circular, covalentl-y
closed mol-ecules of 300-4OO residues ( Mr 1 .25 x I O5 )
with a high degnee of internal base-pairing and have
been tenmed virusoids as they share physical- propertj-es
simil-ar to those of viroids.
So f ar, there are f our members of this ner^/
group of plant viruses; vel-vet tobacco mottl-e virus
( VTMoV ), solanum nodiflorum mottle vinus ( SNMV ) , l-ucerne
transient streak virus (LtSV) and subterranean cl-over
mottl-e virus ( SCMoV ). The best characterized of these
ane VTMoV and
since both RNA
for infection.
two vi ral- RNAs
of coding for
VTMoV and SNMV
hybnidization
homology fon
SNMV which possess a bipartite genome
1 and the virusoid RNA 2 are necessary
The genetic functions provided by the
have not been determined except for that
the coat protein ( Gould et âf., 1 981 ).
are serofogicall-y reÌated wh1le cDNA-RNA
analysis gave estimates of sequence
the vlral- RNAs 1 of between 20% and 50%
61
depending on the stringency of the assay conditons
(Gould and HaLLa, 1981). 0n the other hand,
hybridization anal-ysis indicated that the complete
sequence of VTMoV RNA 2 (Mn 1.2 x lO5) is contained in
SNMV RNA 2 (l4r 1.3x 105) (GouId and Hatta, 1981).
Despite the cfose sequence simil-arities between the RNAs
2 of VTMoV and SNMV, helther RNA will suppont the
replication of the heterologous RNA 1 (Goufd et â1.,
1981 ) which indicates a hi-ghJ-y specif ic relationship
between the RNA 1 and RNA 2 of each vlrus.
Al-though viroids and virusoids appear to
share similar physicaÌ charactenistics, viroids are not
encapsidated and repl-icate autonomousl-y (Diener, 1979;
Gross and Riesner' 1980). In order to further
lnvestigate t,he intriguing nel-ationshlps between the
RNAs of VTMoV and SNMV, we have sequenced the RNA 2
species of each vlrus and compared their structunes with
those of viroids.
MATERIALS and METHODS
A. Viruses and RNA
R.I.B. Francki, J.V,l
b¡ere purified from
viraf RNAs isolated
Randles and A.R
lnfected Nicotiana
VTMoV and SNMV I"Iere kindJ-Y pnovided by Drs.
Goul-d. Viruses
cLevelandii and
and purifled essentiai-1y as
et al-. (1981).descnibed by Randles
VTMoV and SNMV
CCCV in Chapter
B. RNase FingenP rinting
The RNase
RNA 2
62
A and RNase T.,, fingerPrints of
were determined as descrlbed for
digestion \^rere al-so sequenced using
chain terrnination technique.
'-P-1 abetled fragments vJere
cal-f intestinal phosphatase and
E.cot1 poly(A) pol-ymerase.
4
C. RNA sequence determínation
1) Partial- enzvmic di estion
Specific Iinear RNA fragments vrere obtained
from clrcular RNA 2 molecules by partiaJ- RNase digestion
under non-denaturi-ng conditions as descnibed 1n Chapter
2, except that 150 units/mI of RNase T.' and 0-25
unlts/ml- RNas" U2 were required for VTMoV RNA 2 and 300
units/ml of RNase Tl and 0.25 units/ml RNas" UZ fon SNMV
RNA z. The resul-tant RNA fragrnents were 5'-32p-labell-ed
in vitro , f?actionated by polyacrylamide geI
el-ectrophoresis and sequenced by the partial enzymic
digestion technique as described previously.
2) Dideoxynucleotide chain termination
As described in Chapter 2, RNA fragments
produced by RNas" T1
the dideoxynucleotide
Specific purified 5t -
dephosphorylated with
polyadenylated, using
Sequencing reactlons were carried out using d ( TBC) as
63
the speciflc Primer-
D. Synthesis and cJ-oning of double-strand cDNA
Double-strandcDNAwaSsynthesizedfromSNMV
RNA 2 as described in chapter 2, and dlgested with the
restriction endonucfease Sau3A I. The DNA fragment
corresponding to residues 'l 31 to 216 of SNMV RNA 2 was
purified and ligated into the BamH f site of the
repticative form of phage M13 mp7 using Tr* DNA Iigase as
described in Chapter 2, Recombinant phage vüere scneened
by sequence detenmination using a specific M1 3 primer
( cTA, CGACG^C^AGT ) and the dideoxynucleotide chain4¿¿
termination sequencing technique. Recombinant M1 3
repJ-icative f orm was isol-ated (Birnboim and Dof Y, 1979) '
digested.with Sau3A I and the cfoned insert purified on
a 6% polyacrylamide gel (Sanger and Coulson, 1978; Maxam
and Gilbert, 19BO) and used as a primen fon the
sequencing of RNA 2 of VTMoV and of SNMV by the
dideoxynucleotlde chain termination technique (Zlmmern
and Kaesberg, 1978; Symons, 1978; 1 981 ).
RESU LTS
A. RNase fingerPnints of VTMoV and SNMV RNA 2
Figure 5-1 shows the
flngerpnints of both the
The fingerPrints of the
RNase A and RNase T.,'
and SNMV RNA 2 molecules.VTMoV
two RNAs share many sPots in
2
Figure 5-1 RNase fingerprints of SNMV and VTMoV RNA
Purified circular sNMV and vrMov RNA 2 were digestedwith RNase A or RNas" T1, 5'-32p-tabetled and
separated by two dimenslonar ger er-ectrophoresis.The direction of finst dimension erectrophoresis isleft to right, and the direction of second dimension
el-_ectrophoresls 1s bottom to top. A, RNase A
dlgested vrMov RNA 2; B, RNase A digested sNMv RNA
2; C, RNase T., digested VTMoV RNA 2; D RNase T.,
digested sNMV RNA z. origonucreotides unique to thevrMov or sNMV fingerprlnts are indicated by arrohrs.
i¡
B
(t{î\
Ia
oo
t*
Ia
O,,oa
ao'o t'ioì.o\ I
+o
A
a
Ia
-anoa
a
t_
O
Oo
ì'
'¡Oz\
.1
î':oo
D III
O
o
t*
O
ooa
ID
(D
c tO
IDo
r¡r'\-
I\
o
L
ao
O
I\
ì
oo
..(\
a
64
common, which confirms the exlsLence of sequence
homology between VTMoV and SNMV RNA 2 suggested by
cDNA-RNA hybridization studies (Gould and Hatta, 1981).
However, the fingerprj-nts of each RNA species contain
unique oligonucleotides showing that the smalfer VTMoV
RNA 2 is not wholly contained within SNMV RNA 2 and that
the RNAs are related but distinct species.
B. Primary stnuctures of VTMoV and SNMV RNA 2
The base sequences of VTMoV and SNMV RNA 2
r^rene determlned by using both the partial- enzymic
digestion and dideoxynucleotide chain termination
techniques with linear RNA fragments derived from
partial RNase cl-eavage of the native circular RNAs 2.
The sequence determination of RNA fnagments from both
5t-Lerminii, using the partial enzymic digestion
technique, âod 3 r - terminii-, using the dideoxynucl-eotide
chain terminat j-on technique, âf l-owed conf irmation of
sequences and the resolution of occasional band
compressions ( Kramer and Mi11s, 1978 ) which i^Iere seen on
sequencing in one direction but not in the other. The
complete sequences of the two RNAs wene evenbualì-y
obtained from the sequences of numerous ovenlapping RNA
fragments.
The complete base sequences of the two RNAs
Al- though the RNAs areare given in Figure 5-2.
Figure 5-2 The
the 365 residue
l1near form and
primary sequences of SNMV RNA 2 and
form of VTMoV RNA 2 are shown in
aligned for maximum sequence
366 residue form of VTMoV RNA 2 has an
RNA
homology.
extra UMP
sequence
The
residue aL position 108 (arrowed). The
differences between VTMoV RNA 2 and SNMV
2 are boxed. R.esidue 1 1n each case corresponds to
the left-hand end of the secondary structure model of
Figure 5-3.
SNMVRNA2 . zo . ¡to . 60
GUUccUGcccuUGGGGAcUGAUuuUUGGuucGccuGGuccGUGUccGUAGUGGAUGUGUAGUUccuGcccuUGGGGAcUGAUUuUUGGuucGccuGGUccGUGUccGUAGUGGAUGUGUÂVTMoV RNA 2
UCCACUCUGAUGAGUC
UC.CACUCUGAUGAGUC :B:
80 . 100
AAGGACGAAACGGAUGUACCGCU UCUUG
AAGGACG AAACGGAUGU ACCGC UUC UUG80 . 100
. 120
UCGACCUCGAC
CUCGACCUCGACI lle
c u GG ac u AG; G au c G rccc lcGc u c a c
CUGGACUAGUGAUCGAGGG AGGCUC' 110
. 200
C UCC AAUGACUUGGGGUC ACUGUGUAA
CUC C AAUGAC UUGGGGUC ACUGUGUAAt80 200
?60
GGGAGCUGGAC C CUCUCACC AC
GGGAGCUGG ACCCUCUC ACCAC
clõ]ccauåucrtõlccl4lccAUGUGAluaic GCG
238
239
GCG
. 299
GGGAGUCAAGGACGC
GGGAGUCAAGGACGC' 298
c
. 160
U C ACGCCC GC U GIAIGU AGA UGU AGUtt
u c AcGc c cGc uGlqGU aG AUGU AGU
' loo
clqc.ld.
179
AU A
AU A
t?8
. 357
ccc[E 6çuF-¡T| -66c,o¡culccu[v_.1
310
220
olloAIgG
UAC
UAC2?0
UACUACAG
UACUACAG
260
320
G G u a o, o, u G A a co rïo.¡e1olt
G G U A G u GU U G A AGG U C G C[gjA280
310
210
CCGGCAUCAGAG
CCGGCAUCAGAG300
360
ET.cAGGcUGGcAGGUAAcUCCÀGGCUGGCAGGUAAC
' 300 365
AUUGCAC ACCACCGGU AUC ACG
GAUUGCACAC CACCGGUAUCACG320
377
UA
65
covalentl-y closed circular molecuÌes' the sequences are
presented in linear forrn fon convenience and ease of
comparj-son. sNMV RNA 2 consists of 377 nesidues while
vTMoV RNA 2 consists of two approximately equimoÌar
species, one of 366 residues which, like SNMV RNA, has a
u at residue 108, and another species of 365 residues
where this residue is defeted. This sequence
heterogeneity within RNA 2 of VTMoV was determined by
sequence anafysis of individual purified fragments which
differed in size by one residue and which I^Iere denlved
from either the 366 or 365 residue species. Further
confirmation of this sequence hetenogeneity and an
estimate of the nefative proportlons of the two species
vrere obtained using a cloned DNA fragment (derived from
residues 131 to 216 of SMNV RNA 2) as a primer on the
mixture of the intact vTMov RNA 2 species (see Figure
2-B\. This 365 residue species is arbitraril-y presented
in Figure 5-2 and numbering of the VTMoV RNA 2 sequence
witl refer to this sPecies.
The extensj-ve sequence homology beLween VTMoV
and SNMV RNA 2, originalJ-y reported on the basis of
hybridization anal-ysis with cDNA ( Gould and Hatta, 1 981 )
and show by RNase fingerprinting, is confirmed by the
sequence data. As suggested by RNase fingerprinting
data, each RNA 2 species contains unique sequences, thus
95% of VTMoV RNA 2 is homologous with SNMV RNA 2 and 92%
66
ofSNMVRNA2ishomologouswithVTMoVRNA2.The
sequence diffenences are unevenly scattered throughout
the two RNAs with a cluster of base differencés around
residues 339-359 0f SNMV RNA 2 and residues 333-347 0f
vTMoV RNA 2, while there is. al-most compÌete sequence
homology between nesidues 360-146 of SNMV RNA 2 and
residues 348-1 45 of VTMoV RN A 2.
C . Secondary s truc tures of VTMoV and SNMV RNA 2
Secondary structure models for the two RNAs
were constructed as described by Tinoco et al- . ( 1 97 1 )
and are shown in Figure 5-3. Both RNAs form extensively
base-paired rod-like structures which are simifar to
those described for viroids (Sangen et â1 ., 1976; Gross
et af. , 1gB2; Hasel_off et â1., 1982) . The structures
are consistent with the known sites of high sensitivity
to ribonuclease under the conditions of high sal-t
concentration used to generate specific RNA fragments
from the circular RNAs for sequencing ' Thus, the
terminaÌ singl-e-strand hairpin loops and the centraf
singJ-e-strand regions of both RNAs ( residues 70-1 00 and
285-305 ) \^rere especial-1y susceptible to RNase cf eavage.
The properties of the proposed structures are
summarized (Table 5-1) and are compared lo those of the
pubJ_ished structures of four viroids. The vTMoV and
sNMV RNA 2 mofecules possess proportions of G:C base
Tab I e 5-1 Properties of proposed secondary structures for Rl'lA 2 of VTMoV andSNMV compared with those of several viroids
RNANo.ofresi-dues
No. of basepai rs
A:U G:C G:U
G:Cbase pai rs
as%of total
Res i duesbase
pa'iredlo
^G*(K¡/mol at25"C inlM NaCl )
VTMoV RNA 2
VTMoV RNA 2
SNMV RNA 2
ASBV
PSTV
CSV
CEV
365
366
377
247
359
356
371
3B
38
4l43
37
44
34
72
71
76
28
73
64
72
l3l420
12
l6l6l8
59
58
55
34
58
52
58
67
67
73
67
70
70
67
- 350
- 345
-455
-280
- 610
-540
-590
*Parameters for calculation províded by Dr. D. Riesner (Steger, Gross,Randles, Sänger and R'iesner, personaì communicatjon).
Figure 5-3
RNA 2 and
the segment of the
residue fonm
366 residue
Pnedicted secondary structures of SNMV
the 365 of VTMoV RNA 2 plus
form of VTMoV RNA 2
contaì-ning the extra
sequence differences
UMP nesidue (residue 108). The
between the RNAs are boxed.
Ic
l^
UU ccuGcc CUUGGGG UG UUGGUUCGCCUGGUC GUG CCG GUGG UGUGU.AUC.CACUCUGAUG
cAO
AG100
G
120
I
140I
UG C UUCUU ccAccu.ccAc.cuc cu.AGuG G^GGG
AcGGcuc ccEluc^ . cGc
160
" \uElGCU GUAGA
't80
"^ \r" ^ucuecu.c@ ,^t "^ uo^"
SNMV RNA 2
c
20luu
ec \r u
G
,aoru[]e r
cgG
280
U c
40Ic
c cc I
UGGUU GCCU UôCGUG
60
cacc.acAcG u
U UA A
o c crþþ
" u .1ooGGA U CC U U
UG AC UUCU GC
AC.UG GGGA CG
Et
GA
260
G^CCU.CG^C'CUG CU^G.UG
U
CC GG CG
GG çC.GCcl
I
300
E.go
c
cc
c
ou ^gug
U
oo.cAc Goc
UU- cq^cco corccu@ e$cs{þFrucrþ^u uc I \"^\"" I
360 - 340 .l;rc Æ-uìe¡c¡curc\9
o¡ l-ülc¡c cuccc uco^G.qqo scEl suco é)l"u ^o l" \à^ u
"l¡c o I
260 240
ceucB
l^220
u/,20,0
^c.uocþþ'c rcruce oþ ruo
o u AA
377gG
SG
VTMoV RNA 2
ugu cu G
CCUGCC GG
GGACGG. UC
G
c AUUCUGAUG C GG CG
rIuc cc
c ucg^G.gcc G ous
^sGcG A
@
't80I
A cA uc ^ucurou'{ u^c c^ u3^c
20ulu u
60 Gcc
'r20
240
r@l ^" cuccccuI
^c260
140
^l:::
160U c
u.c coc.ueþ1. oueo
;;; :i;; ;;;;;; lä ;;; :; l;;;, Eu lE^n u-^^ ,1"22O 2OO
cGAGOG
UU
s
cuG CCG GUGG UGUGU.AUCC
GGC. C^CC'ACACG U^GG AG^CU^C GG CC GC
o
^^r l
aee.[þccec CUGGA SUUG GAU
AUGUUI
300365 360 320
100G
uucu fl erccu
GGG CG CUGGA
El
67
pairing which are l_ower than that of cccv and higher
than that of ASBV, but which are similar to those of the
similarly slzed PSTV, CSV and CEV. The thermodynamic
stabilities of the proposed model-s were cal-culated using
vafues kindly provided by Dr D. Riesner (steger, Gross,
Randles, Sanger and Riesner' unpublished data) ' The
val-ues of -455 KJlmol for SNMV RNA 2 and of -345 and
-455 KJlmo1 for the Lwo forms of vTMov RNA 2 (Table 5-1 )
are consistent with their thermaL denaturation
properties; thus VTMoV RNA 2 gave a Tm of 57oC in 0.15M
NaCl, O.O'l 5M sodium citrate, pH 7, while SNMV RNA 2 gave
a highen Tm of 64"C under the same conditions (Goul-d and
Hatta, 1 981 ; Goul_d, 1 981 ) . The pnedicted stabilities of
the RNA 2 0f VTMoV and of SNMV are lower than those of
the simitarl-y sized viroids PSTV, CSV and CEV but highen
than those of the smaller CCCV and ASBV (TabIe 5-1 ).
D. Possible PolY peptide translation Products from RNA 2
species and theÍr conPl-ements
Since the RNA 2 species of VTMoV and SNMV are
nequÌred with the homologous RNA'l for viraÌ infection
(Gould et â1 ., 1981 ) , the RNA 2 mol-ecules must code for
some protein product ( s ) and/or contain structuraÌ
information essential for viraf replication. Evidence
suggests that an AUG codon, hot necessaril-y that neanest
the 5t terninus of the mRNA, functions as the initiation
6B
signal- f or eukaryotic mRNA translation (Banal-1e and
Brownlee , 1 97 B; Ko zak, 1 982; Lomedlco and McAndrew,
1982) and that eukaryotic ribosomes do not interact with
cincular RNAs (Kozak, 1979; Konarska et âf ., 1981).
Therefore, Lranslation of the RNA 2 species woul-d
require the existence of specific Ìinear RNA fonms.
condition is met, the extensiveAssuming that this
sequence homology
form of VTMoV RNA 2, and their
RNA sequences, aIl-ows them to
polypeptide products ( figure
small polypeptide product is
RNA 2 and the 366 residue
putative complementary
code for several- slmil-ar
between SNMV
5-4).
shared
However, only one
between SNMV RNA 2
and the 365 resi-due form of VTMoV RNA 2 and their
compl-ements. AIl possible transl-ation products are l-ess
than 100 amino acids in length and therefore the genes
coding for the viral- coat protelns (approximately 300
amino acids (Randles et â1 ., 'l 981 ; Hollings et âl .,
1979) ) must reside in the RNA 1 species.
DISCUSSION
In overal-1 structune, VTMoV and SNMV RNA 2
resembLe viroids in being smal-1 single-strand covaJ-ent1y
cfosed circufar RNA mol-ecules which form rod-like native
structures with extensive base-pained regions
interspensed with single-strand regions. Howeven, in
contrast with viroids which replicate autonomously and
Figure 5-4
product is given with
base of the initiation
codon ( s )
RNA
the residue number of the flrst
codon plus the termination
Possible polypeptide products of SNMV
365 and 366 residue forms of VTMoV RNA 22 and
(A),
s hown
the
and their putative comlementary RNAs ( B ) are
in schematic form. Each possible transfatlon
in parentheses.
the same residue
compl-ementary
ane retained andsequences,
therefore run in the 3 '
aneas represent regions
homoJ-ogy and the bl ack
in the RNAs shown. The
regions of sequence
products of the two
For the
numb e rs
to 5t direction. The cl_ear
of amino acids sequence
areas regions of non-homology
cross-hatched areas are
homology between different
VTMoV RNAs which correspond to
2. l¡'lhere annon-homology in SNMV RNA
product is obtained fnom the two forms of
VTMoV RNA 2, only one is shown.
reglons of
identical
LENGTH OF POLYPEPTIOE PFOOUCT (amino acids)
FNA 2
Spec'as
lst nosrdu"ol
Coóon
30 ao 60 t0
(UAG)
90 ro0
(u^c)
(UGA)
(UAG)
(U^G)
(uGA)
(UGA,
!o
(U^G)
soto 20
(u^G)A
(uGA)
(uG^t
(u^G)
(UAG,UGA}
(UAG UGA)
(uA G)
(UGA)
(UAG)
(UAG)
184
185
303
30¡
226
r75
r76
117
54
5a
51
93
93
93
70
?o
?o
VIMoV (365)
VIMoV (366)
SNMV
vIMov (365)
vlMoV (366)
SNMV
vIMov (365)
VIMoV (366,
(365)
(366)
(366)
(f,66)
vtMov (366)
SNMV
SNMV
(365)
sNvv
(365)
SNMV
69
are not encapsidated ( Diener, 1 979; Gross and Riesner,
.l gBO ) , the RNA 2 species are essential components of a
bipartite genome and are encapsidated (Gould et â1.,
1981 ) . !,¡hi1e viroids do not appear to code f or
functional_ protein pnoducts ( conj ero and semancik, 1 977 ;
Davies et â1., 1974; Semancik et â1., 1977; Haseloff et
â1., 1g82]t, thene 1s no information available for the
RNA 2 speci-es.
The fack of conservation of possible
translation pnoducts between SNMV RNA 2 and the 365 and
366 residue forms of vTMov RNA 2, desplte greater than
go% base sequence homology, suggests either that the 365
residue form of vTMoV RNA 2 may be non-functional or
that RNA 2 coded translation products may have no
f unction in viral repl-ication. Although the invol-vement
of RNA 2 coded transl-ation products in vlral replication
cannot be excluded, 1t seems likeIy that the unique
viroid-like structunes of the RNA 2 molecul-es encode
some function besides that of a templ-ate, and that this
function is required for the replication of both RNA 1
and RNA 2 species.
Since neither VTMoV RNA 2 nor SNMV RNA 2
supports the replications of the heterologous RNA 1
species (Gould et âI ., 1981 ), the bioJ-ogical-
speclficities of the RNA 2 species must be determined by
differences in primary and/or secondary structunes. The
70
only extenslve
VTMoV and SNMV
region of sequence differences between
RNA 2l-ies around VTMoV RNA 2 residues
333-347 and SNMV RNA 2 resdues 339-359. Hence, this
reglon may be involved in determi-ning the specificity of
the relationship between the RNA 1 and RNA 2 species,
although the involvement of othen structural differences
cannot be excluded.
An unexpected complication during the
sequence determination of the RNA 2 mol-ecul-es v{as the
occurence of sequence heterogeneity in VTMoV RNA 2 whÍch
consisted of two RNA species differing in the presence
or absence of a U residue at posltion 108 (Figune 5-2,
5-3 ) and existing in approximatety equlmolar amounts.
The two RNA species may have arisen either by a single
rnutation in a panent molecule fol-l-owed by independent
repllcation of the resultant two RNA species, or a
mixture of the two species may be produced during each
cycì-e of repJ-ication by transcriptional and/ or
processing events. In each case, iL is possible that
one of the two RNA species may be non-functional.
Sequence heterogeneity within RNA populations has been
reported for RNA phage Oß (Domingo et a1., 19781,,
vesicular stomatltis virus ( Holland et â1. , 1979) ,
satellite tobacco necnosis vinus (Donis-Kel-l-er et âl . ,
1981), citrus exocortis viroid (Gross et âl ., 1982) and
coconut cadang-cadang vinoid ( Hasel-off et â1., 1982).
71
In the secondary structures of the viroids
PSTV, CSV, CEV and CCCV there is a central reglon of the
native rod-like structures which is highl-y conserved in
both sequence and structure ( Hasefoff et âf. , 1982) -
ASBV (Symons, 1981 ) does not share this common structure
except for the residues GAAACC (ASBV nesidues 45-50)
which, as in the other vlroids are present on a single
strand l-oop in the central- region of the native
molecule. Interestingì-y, VTMoV and SNMV RNA 2 also
contain the sequence GAAAC (nesidues B6-90 in both
mol-ecul-es ) which is al-so present in a singJ-e-strand
region in the centre of the proposed secondary
structures. Howeven, there 1s no extensive base
sequence homology or compl-ementarity between the vinoids
PSTV, CSV, CEV, ASBV and CCCV and t,he VTMoV and SNMV RNA
2 mol-ecuf es.
It, will be of considerable interest to
determine the exact mechanisrns by which VTMoV and SNMV
support the replication of the homologous RNA 1 species
as wel-1 as the molecular basis for the speclficity
between the RNA 1 and RNA 2 species, and to determine
whether the common structunal- features of viroids and
virusolds mirnor some common function. Chapter 6
outÌines work with subterranean cl-over mottle virus
which al-l-ows some definition of the virusoid sequences
involved in such relationshiPs.
CHAPTER 6
SUBTERRANEAN CLOVER MOTTLE VIRUS
72
INTRODUCTION
Several lsolates of subterranean cfover
mottle virus ( SCMoV ) from Western Austral-ia have been
described ( Francki et âf. , 1 983 ) . As judged by electron
microscopy, pFeparations of SCMoV consist of honogeneous
populations of polyhedral- virus panticles about 30 nm in
diameLer, and serological tests using antiserum to
punified SCMoV failed to reveal- any antigenic
diffenences between the isol-ates. However, the various
SCMoV isolates appear to contain differi-ng encapsidated
RNA components, i¡ühile al-1 isol-ates contained a J-inear,
singJ-e-stranded RNA species of approxlmateJ-y 4500
residues in size (RNA 1, Mr 1.5 x 106), each isol-ate
contained either one or both of Lwo viroid-like clrcul-an
RNA species, RNA 2 (approximately 400 resides) and RNA
2t ( approximately 300 residues ) .
The SCMoV isol-ates used in this wonk v,/ere the
isolates A, B, D and E of Francki et aI. (1983). Some
time after i-ts isolation SCMoV-A Idas
both RNA 2 and RNA
contrast, SCMoV-E
2t with the RNA 1
found to contain
species. In
1 and RNA 2 whil-e
'l and RNA 2t. The
isol-ates are shown
isol-ates A, D and E
SCMoV-D contain
onJ-y RNA
onJ-y RNA
of theseRNA components
in Figure 6-1. Interestingly, while
urere obtained as f ield isoJ-ates,
from SCMoV-A by passage through
l-eaves.
contains
SCMoV-B and
fractionated
SCMoV-B was produced
single l-esions on pea
Figure 6-1 RNA components of SCMoV isolates.
RNAs extracted from SCMoV isolates E, A, BandD
a4%
R NAs $Ie re
r^rere fractionated by electrophoresis 1n
polyacryl-amide geI containing 7M urea.
detected by slaining with tol-uidine blue. RNA 1 and
the circular forms of RNAs 2 and 2t are shown. The
linear forms of RNAs 2 and 2t.migrated from the
gel.
SCMoV ieolates
EABD
-fl-{--li-it--.'-
*s- t
4S TBE
+7M uroâ
73
The finding of SCMoV isolates which differed
in their RNA 2 and/or RNA 2t components suggested either
( 1 ) the existence of two serologically indistinguishabfe
viruses which each possessed different RNA components or
(2) the existence of a single virus whlch could support
the repì-ication
The
of either or both RNA 2 and RNA 2t.
fol-lowing work describes efforts to
distinguish between these possibllities and to determine
the structures of SCMoV RNAs 2 and 2t .
MATERI A LS
Isol-ates of SCMoV were kindJ-y provided by Dn.
Richard Francki. Viral propagation, purlfication and
extraction of viral RNAs I^¡ere perf ormed by Mr. Chris
Davies as described ( Francki et âI. , 1 983 ) . SCMoV RNA
species vüere purified by polyacryl-amide gel
el-ectrophonesis (Gould, 1981 ) , frostly by Mr. Dav j-es.
ME THODS
A. Synthesis and restriction endonucl-ease cl-eavage of
ds cDNA
Random-primed first strand cDNA was
transcribed from SCMoV RNA 1 essentially as descrlbed by
TayJ-or et al-. (1976l, . Purified SCMoV RNA 1 (2 Ue) bras
resuspended in 25 p1 containing 50mM Trls-HC1 pH 8.3,
5OmM KC1, 'l OmM MgClr, l OmM DTT, 1O0pM Io-3tr] acrr
74
(fO ¡rCi), 5OOUM dATP, dGTP and dTTP' 2 mg/mI
treated salmon sperm DNA and 20 units avj-an
myelobl-astosis virus reverse transcriptase.
incubation aL 37oC for 2 hours, the reaction
and treated wlth RNase A befone synthesis of
second-strand cDNA as described in Chapter 2,
DNase 1
After
I^¡as boiled
Me thods
B-1. Synthesized doubfe-strand cDNA was digested with
varlous restriction endonucfeases and fractionated by
polyacrylamide gel el-ectrophoresis as descrlbed al-so in
Chapter 2, MeLhods B-1,
B. Fingerp rinting of SCMoV RNAs
Purified circul-an SCMoV RNAs 2 and 2t were
RNase A fingerprinted
Chapter 4.
uslng techniques outlined in
C. Sequence determination of SCMoV RNA 2 and RNA 2l
Purified SCMoV RNA 2 and RNA 2t were each
subjected to partial- digestion under non-denaturing
conditions, âS described in Chapter 2, using 150
units/ml RNas" T1 ' 0.1 vc/nl- RNase A or 0.25 units/ml-
RNas. UZ. The nesulting linear RNA fragments were
eithe r 5' - or 3'-32P radiolabelled, fractionated by
polyacryl-amide gel el-ectrophoresis and sequenced using
the partial enzymic digestion technique essentiaJ-Iy as
descrlbed in Chapter 2, except that sequencj-ng gels
75
contained TBE buffer, 714 urea and 25% (v/v ) Oeionized
formamide. The sequences of overlapping RNA
wene used to obtain the primary stnuctures of
circular RNAs and the methods of Tinoco et a1-
used to predict the native secondary stnuctures
mol-ecules.
f n agmen ts
the
(1971)
of the
RESU LTS
A. Analysis of SCMoV RNA l nucl-eotide sequences
In order to analYse the sequence
rel-ationships between the RNAs 1 of the SCMoV isolates,
double-strand cDNAs I^Iere transcribed from purified RNAs
1 and then digested with the various sequence-specific
restriction endonucfease, Alu I' Hae III, Hha I, Hpa II
(see Figure 6-2) and Acc 1 (data not shown).
PoJ-yacrylamide get electrophoresis of digested ds cDNAs
results in gef pattenns which reflect the anrangement of
nestrlction enzyme recognition sequences on the original
RNA 1 molecules. As shown in Figure 6-2 ' all- four
isol-ated RNA 1 species gave rise to indistinguishable
patterns of ds cDNA restriction fragments for all
enzymes used. llhi)-e, ( 1 ) the synthesized ds cDNAs may
not be wholJ-y repnesentative of the respecti-ve RNA 1
species, and/or (2) small nucleotide sequence
differences between the RNAs 1 may exist which do not
affect the size or number of observed ds cDNA
Figure 6-2 Restriction endonucl-ease digestion of
SCMoV RNA 1 ds cDNA.
Double-strand cDNA was synthesized from RNA 1
purifled from four dlfferent isol-ates of SCMoV.
Isolates 1, 2, 3 and 4 correspond to SCMoV-8,
SCMoV-4, SCMoV-B and SCMoV-D, F€spectlvely. The ds
cDNAs vüere untreated or dlgesfed with AIu I, Hae
III, Hha I or Hpa II and fractionated on a 6%
polyacrylamide gel containing 2M urea.
3,-32p-tabell-ed Hpa Ir digested M13 mp7 RF vras
incfuded as size markers, with sizes of 1596, 829,
B1B, 652, 545, 543, 472, 454, 357 t 183, 176, 156,
129, 123, 79 and 60 base pairs.
RESTRICTION DIGESTS 0F SCMoV RNA 1 ds cDNA
UNCUT Alu I Hoe III Hho I Hpo II
SCMoV IS0LATES 123 4123t+W|TTTTÑ--axa- -!rI
IlíIå
.1
a
t-
IIi
IIII
ì\
.Ç
tÉ
tta
F
Ii.
FF
t
f'
ia
?I
It I
L
I\
ì
\
\
tI
tI¡
I
{
-¡iÒ p
.l'-lr
-lr
76
nestriction fragments, the
four isotated RNA 1 sPecies
results do suggest that the
are certainÌy closely
and may be identical.rel-ated in nucf eotide sequence t
B. RNase fing erprinting of SCMoV RNAs 2 and RNAs 2t
In addition, the sequence reÌationships
between the RNAs 2 of the SCMoV isol-ates were
investigated using the RNase fingerpninting technique.
Purified cincul-ar RNA 2 and RNA 2t species vüene digested
to compl-etlon by RNase A, 5'-3 2P-radiolabelled and
fractionated by two-dimensional polyacryÌamj-de gel
electrophonesis as descnibed in Chapter 4. The
resulting oligonucleotide patterns are glven in Figure
6-3 and show the f ollowing. ( I ) f rre ol-igonucleotide
patterns obtained from the RNAs 2 of SCMoV-E and SCMoV-A
are essentially identical . (Z ) fne ofigonucleotide
patterns obtained from the RNAs 2t of SCMoV-4, SCMoV-B
and SCMoV-D are af so essentialJ-y identical-. (3 ) Sorne
ol-igonucleotides appear to be common to both the RNA 2
and RNA 2t specì-es (see Figure 6-3).
Although RNase A (C and U specific)
fingerprinting may not reveal- minor sequence differences
present in pyrimidine-rich regions of the molecules, the
data suggest that, l-ike the isol-ated SCMoV RNAs 1 , each
RNA 2 and RNA 2t species is eithen closely rel-ated or
identlcal to similan species from different SCMoV
Flgure 6-3
2t.
RNase A fingerprj_nts of SCMoV RNAs 2 and
Circular RNAs 2 were purifled from SCMoV-E and
SCMoV-A r âhd circular RNAs 2t r^rere purif 1ed f rom
SCMoV-4, SCMoV-B and SCMoV-D. AlI RNAs r¡¡ere
digested with RNase A, 5,-32p-tabell-ed and
fractlonated by 2-dlmensional polyacrylamide ge1
el-ectrophoresis. The resulting oligonucleotide
flngerprints are shown with the dinections of
el-ectrophoresis in the first dimenslon being left to
right, and, irt the second dimension, bot,tom to top.
,ZYNU
o-^oncs,ZYNU
8-^oncs,ZYNU
Y-^onc8
ZYNU
Y-^onc8
ZYNU
3-^onc8
o
a
o
aOr
,J.
¡O
o
a
I
o
a
¡
I
o
O
t
I
0
o
a
o
J
o
a
a
o
a
o
|¡
0.
4Ç
oa
J
O
,o
a
O
o
tII
,o
o
o
tO
77
1soÌates. different RNA 2
or RNA 2t the identical
fragments
cJ-eavage under non-denaturing conditions (see Methods,
this chapter) of RNA 2/2t fnom different isolates, and
by preì-iminary sequence data (not shown).
In contnast, there are considerable
differences between the oJ-igonucleotide fingenprints,
and thus primary sequences, of the RNA 2 and RNA 2t. So
while some degree of sequence homol-ogy 1s indicated by
lhe number of shared oligonucfeotides, RNA 2 and RNA 2t
each contain unique sequences and do not differ simply
in the possession of repeated sequences.
From the evidence presented, it seems 1ikely
that the different isol-ates of SCMoV, which are
serologically indistinguishabl-e ( Francki et âf., 1 983 ),
contain essentially identical RNA 1 species and differ
only in containlng either or both RNA 2 and RNA 2t
specles. Th is is al-most certainlSr the case f or SCMoV-4,
containing both RNA 2 and RNA 2t and SCMoV-B (which hlas
derived directly from SCMoV-A) containing only RNA 2t.
Furthermore, although RNA 2 and RNA 2t appean to diffen
significantly in nucleotide sequence, no sequence
differences v\rere observed between simil-ar RNAs 2/21
obtalned from diffenent SCMoV isolates.
The possible identity of the
species is also supported by
obtained after partÍaI RNase uzT orA
7B
C. Sequence determination of SCMoV-A RNA 2 and RNA 2t
Linear RNA fragments v¡ere obtained from the
RNA 2 and RNA 2t of the SCMoV-A isolate by partiaì-
ribonuclease digestion under non-denaturing conditions.
These fragments v¡ere radiolabell-ed and sequenced using
the partial- enzymic cJ-eavage method, and the sequences
of overlapping fragments vlere assembled to glve the
compl-ete primary stnuctures of the circul-ar molecul-es.
The RNA 2 and RNA 2t specles each consists of 388 and
327 nesidues respectively. In addition, the two RNA
a singJ-e common region of abouL 220species
residues
pr imary
share
of aÌmost complete sequence homoÌogy. The
structures of SCMoV-A RNA 2 and RNA 2t are
in Fi6Sure 6-4 in a convenient linear form with
sequences shown boxed. Secondary structure
these mo.l-ecufes were
Tinoco et at. (1971)
constnucted using the
R NAs to
presented
the shared
models for
methods of
6-5. Both
and are shown in Figure
fo rm
hel-ical rod-like
may base-pair intramolecularl-y
structures similar to those of VTMoV
and SNMV RNA 2 and viroids. Strikingly, the nucfeotide
sequences conserved between SCMoV-A RNA 2 and RNA 2t are
located so as to form the entire base-paired left-hand
sides (as drawn) of the native mol-ecul-es. Consequently,
it is the differing lengths of the unconserved
right-hand sides of the natj-ve molecul-es which account
f or their dif f erence in si-ze.
Figure 6-4
RNA 2I .
Primary structures of SCMoV RNA 2 and
Nu cfeotlde sequences common to the two RNAs are
s hown boxed .
ItO
SCMoV
SCMoV
R NA 2
RNA 2'
A6A.66CAU A
A6A66CAU UU
UAUUC CACG C U6 UC U6UACUU
UAUU C C A C6 C U6 UC U6 UAC UU
AC6AAACA6C6CACC6CAAAC6AAACA6CGCACC6CAA
6U UATUAUA
UACUAUAUC
l¡a
66CCCCA(CUTACUUUCG66CCCCACCUCACUUUC6
UAU C A6 UACAC UGACGA6 U CC C UAAA66UAUC A6 UA C A C U6 A C.6 A6 U C C C UAAA66
1ú
C U U 6 G C C'A 6 A C C U
A
6
6AAG6CUA66AA66CUA6
A
cccccAcAc.UU6CUU66A
c6ccAAuC6AUUC6
TUAC6U6UUACCUAC6U6UUAC
¡o¡
UC
CU
l6
tJO
ct+0
A
AC,la
UCUAC6UAUACCCtú
ta, tr,
C AA6 C CAAAAACC 66 U CC C C AAC6 C A6 UUUAG UAU C AA6 UC6 U CG C AUC C
6CUA6C6UUC6ACA6A6U6CtLo
zØ 1¡0
AC6 CUC C CGA6G6 466 AA6 UUUG C6 C C UU6A66 U
40UCU6CAC66UCGU66U
6GAC6CGGUUCU6GUClro
cGc6r2aD lío
AACA66 AAAA6 U6 UU66AAU6 UUU6 AAG6 U CU U6 C 6
A C A C U C A C C C 6.G.6 A G 6 C C A U C G 6 6 C A 6 A U U A U A C U A2oo 1ú
6UUGUCAA66ACC6UUGUCAA66ACC
tt
A
c
A
U
c
UA
U
UU6UUA66UUU6UUA66U
úollo
c 6A
A6
AAC6UCCAU
AAC6UCCAU
C CC UC C UC6 C6 6AUUUU6AA66 UG UU
C C C UC C UC6 C66AUUUU6 AA6G U6 UU
AGCUACCCAAA6CUACCCAA
6UC6UUAGU
6UCGUUA6U
AUUACUAC
AUUACUAC
)æU
!¿o tz7
Figure 6-5
RNA 2I .
Secondarv structures of SCMoV RNA 2 and
NucÌeotide sequences common to the two RNAs are
shown boxed.
SCMoV RNA 210 ó0 80 100 120 t1o,
CAcc!c-LULCA6AC CCAAU
c AAAcc Gcca ACC0
rtAA.
180 200
^r8
oouur* u'., ^oor'nu"o'
^ u rr^ (
;; ;;;;;. .;;i;;^*o^iii^iå..c6 I
220
1ó0
ccccOIJ A
cGuA IJC
20
20
300
'o7'*o***ou^^oouuol
280 260
^0c- OG UOGU
CA
2r{,380 3ó0 3t0 )20
2
SCMoV RNA 2'80rc ó0
1¿0
.^/uc cuuG
W ^u "7u^;ooo"-r9o^ooJ\^i'*"'**T. ¡c GCG c
220 200 -100
120 60
\corou^ c accoo,,
c u uun^Gau^ocoru
ACG
Uc
280 2lr0321
79
DISCUSSION
A. Rel-atlonships between the various isolates of SCMoV
The four SCMoV isolates used in this wonk
have nov\r been shown to be indistinguishabfe
serologicaì-l-y (Francki et âl . , 1983 ) , to contain
indistinguishable RNA 1 species and are likeJ-y to dlffer
only in containing either one or both RNA 2 and RNA 2t.
Glven this, then irrespective of whether SCMoV RNAs 2/21
are required for viral infection, âs appears to be the
case with the RNAs 2 of VTMoV and SNMV ( Gould et âf. ,
1981 ), or are satellite RNAs, as appears to be the case
for LTSV RNA 2 (Jones et âf., 1983), it seems that SCMoV
RNA 2 and RNA 2t must be functionally equivafent, LhaL
is, interchangeable.
B. Se uence homolo between SCMoV RNA 2 and RNA 2t
The native structunes of SCMoV RNA 2 and RNA
2t show remarkable conservation of the left-hand sides
of the mofecufes (Figure 6-5). It therefore seems
reasonable to propose that these common sequences and
structures mirror the apparent interchangeabl-e functions
2 and RNA 2t aneof the mofecules. Thus, if RNA
satell-ite RNAs, the conserved left-hand sides of the
molecules may contain recognition
replication by viraf and/or host
RNAs are functionally simil-ar to
signals required fon
componen ts
VTMoV and
If the
SNMV RNA 2,
BO
the left-hand sides of the rnolecul-es may al_so contaj_n
some undefined function required for viral replication.
InterestingJ-y, the conserved sequences of SCMoV RNA 2
and RNA 2t share homotogy wlLh VTMoV and SNMV RNAs ?.
C. Sequence homology between SCMoV, VTMoV, SNMV and LTSV
RNAs 2
The determined sequences and pnedicted
secondary structures of the viroid-like RNAs of SCMoV,
VTMoV and SNMV and two isolates of LTSV ( Keese and
Symons, unpublished resutts) are shown in Figure 6-6,
and common sequences indicated. First, the sequence
GAUUUU 1s present on al_l_ RNAs in a simitar position on
the native structures (startlng at approximately resj-due
number 20 in atI cases). The conservation of this
sequence suggests that it may play some rol_e which is
common to the repl-ication of aÌ1 six RNA.s, including the
LTSV RNAs 2, Second, the vj_rusoids of SCMoV, VTMoV, and
SNMV all contain conserved sequences whlch are centnar
to their native structures. These consist of two
regions, one of 24 resldues and the other of 9 residues,
which are postioned on opposite sides of the rod-like
molecul-es. SimiJ_arJ_y, viroids af so contain highly
conserved sequences which are centraÌ to their rod-like
native sturctures (chapter 4), and the pentanucreotide
sequence GAAAC is present, predorninantly
2
Figune 6-6
SCMoV RNA
Seouence and structural homology between
2 and RNA 2t and SNMV, VTMoV and LTSV RNAs
The pnoposed secondary stnucture model-s fon the RNAs
ane shown wiLh conserved sequences indicaLed in
gneen.
60I
€GU ^u c G A
^cc cu uccA ccu cc^
,, "
"Fj3;å:"--"-"1/ | '-'324 I
320
:icu
c
cu uc ^Ecu^[crftcco ucu
uo ", I c \J ^c ^oI ./t300 280
ccu^cc
c^GC
c ^ ^ c I u. c s
^. GG u c
^ c u c o c rê ð¡-c-uf, c fu?t'dcl cu u^
^ c uc
u u r cc A !---J ^tto/or'^ I
^^l"l\þ"1260 ', 240 220
GU t_.,d:.?iù,."t"-r"I! I
CAAIJUGAG ACOUUUCGGCC'UGCC GGCC.UC UCAOUG^G CGGU
ucAo
LTSV-A RNA 2 40I
^t:ii"l"liìr+ ^ ""
140120 60I
U
cpþ cue þþcu.[þco
GGAU
uc ccu^c GA ACGU.uu I
I
260
GGA.GOUc
CAGUc
GCG
C UGCAA^GCCGG AUGGAG
ccGG AG ^CUCACUC.GCCcu^
'140
:i.l',:+r¿:;i¡^wg';'^
ccG
1-cu
324
"û"": c
U
oc
U
160I
Ic
u
UA ccc rccrculuc
^ ñì--'I
I
240
GUUAA CUj
I
20
CA AGU AGUC
l, cc u
l"80
I
200
^G ^ C
GG
2ao'U
qu ls c
300 2
LTSV-N RNA 2 402lc cl
" ^+-,¡ñ?¡-s:{"" "l +:,uþFlì'q"^""c
ÀU C G A
cG cu ucc^ ccu ccA
80 1O0 12Ol_l^t^
" "díïìt:" "Fr:+-s ! :i"$;'¡. " I ^,, u
^
jo^ "
o u,,. o o "
j. u o
" "^
60I
^o'^ cflt
: ::::: :o
@"^ ucAsl-c
UGCAAAGCCGG UGSG
ccGG ^G
AGUC^CUC cc ^l.c
I
oc
U
G
200 180
StMoV RNA 2
20
I60
SCMoV RNA 2
300
SNMV RNA 2
40 ó0 80 100 120 160
CA OU
CG
Àcc0
io* .;;i ^;;;, ^;;,.; Yo^ ^oo^ ^uoo,o./ A A / AA ca//
'i'280 260 24 220
140,Llcalc c
c--c clc A AAcÂGac ccAAU Aa cc 0c
,180 2 00
/r/t, otoo /uou co oc,AÁc- JLUao- cAAGg uc n"t^ct 3u
ccuccoo oc c
GA Â C
ô 340 3r0 120
6020 t0
280
80 120 110
^l
tó0
ccu¡u^ c ,ccoou cc u uc o^oau^otnru
\- .tccu oc c
¡oococ
u
U ^ u'
7 u^;oo
o
"u^, E o
^ oo
J\ ^crê
c^cu Go *"ufc
220 " 2oo 1oo)27 ?60 260
1
IG
c
l^I
377
20 40
UAC . GG
100 120
I
160
c" \o[180
"^ \,. ^uourou.cþl u^c c^ uo^c
c¡ucft ¡c¡uc¡ cEì ruolro u
^aI
220
Bu
140I
cc
^c uucuuo c
UC
c^qGo cGcuc ccPlucr ' ccc ccu GUAGAAc
Ac
oCCUGCC CUUGGGG
c uF¡-îl cfã¡lc o s Gf,ihF¡-tã¡c.L\ U UgI \.^\,. I
360 ! 340
c^cc.^c^co u¡c,6ü1c¡c¡clu d"fl-
320
GqNlt,8,ß,
c o cr[þ OCUOOA GUUO GAU CA
I
280
üi260
c cuccc ucc^q'c6o oo[\ ouo cþþcu ^c lc \g^ vc
^c o I - a
240
ou Acuo oo
GGACGC GGAC
^ u uc UU
VTMoV RNA 2 40uql
cu c ^
c GS I
ccuqcc s6 c oquu Gccu uçccuc
ccc.[þccec ccG^ cc. cucG^ ouuo oru oe@l rc CUCCC UCg^C'3qg q qUO ^cC
ul'200
180I
A cA uc a U
cueo uoueou'{ u^c c^ uoAc
c^uc ^c^uc^ c@ r.uo ou ecuo
o lfãlo u -^^ ul t o
l-^l2zO 2OO
clI
300
UO
120 140
GAGGO G
rIGCUC C
c 160cc I
^clc coc.uGlg u
o
oU
G
c100
A U CC U
UG AC UUCU OC
Et C C U AGI AC G
260U
365 360
u U
C^CC.ACACG UAGG.^GACU^C'GG CC'G
lucll
32O 30O 2AO 240
B1
singÌe-stranded ' in the central
SCMoV, VTMoV, SNMV virusoids and
However the virusoid mol-ecules do
the conserved stem-J-oop structure
or postulated Ula snRNA homology
conserved sequences of
all sequenced virolds.
nol appear to contain
( Gross et â1., 1982)
( Diener, 1 981 ; Gross et
âf., 19BZ ) which
except ASBV.
are present in all- sequenced vinoids
The GAUUUU and central conserved sequences
which are shaned by the SCMoV RNAs 2/2t and VTMoV and
sNMV RNAs 2 are focated within the left-hand sides of
the native mol-ecules which, in SCMoV RNAs 2/21, are
highJ_y conserved. In contrast to the SCMoV virusoids,
VTMoV and SNMV RNA 2 will only replicate in conjunction
wlth the RNA 1 species from the same virus, and the RNA
2 species are thenefore not ínterchangeabl-e (Gould et
â1 ., 1981) desplte greater than 90% sequence homoJ-ogy.
It is therefore interesting to note that the main
sequence differences between VTMoV and SNMV RNA 2 1ie
clustered opposite the GAUUUU sequence and that the
remainder of the left-hand side of the native structure
is al-most completely conserved ( Chapter 5 ) . Thus, these
obsenvations are consistent with both the functionaÌ
simitarity of the SCMoV, VTMoV and SNMV virusoids and
the importance of the left-hand sides of these mol-ecul-es
in replication.
It is tempting to speculate that as lhe SCMoV
82
virusoids share common structures with VTMoV and SNMV
RNA
Th at
2, they may afso share common biological properties.
is, SCMoV RNA 2
infection in a
and/or RNA 2t be required for
that shown for
may
to
the RNAs 2 of VTMoV and SNMV; ând the central conserved
sequences of these mol-ecul-es may play some nole in
fulfil-ling this requirement. However, this proposition
must be viewed with some scepticj-sm; fon while LTSV RNA
2 (which has been shown to behave as a satellite RNA
(Jones et âf., 1983) and is not required for viraÌ
infection) does not share the central- conserved
viraf
sequences
satel I i te
does.
manner simil-ar
of SCMoV, VTMoV and SNMV RNAs, another
RNA, that of tobacco ringspot virus (TobRV),
D. SateIlite RNA of TobRV
TobRV belongs to the nepovirus group and
consists of 28 nm isometric particles containing two
slngle-strand RNAs of molecular weighL 2.7 x lO6 (RNA 1 )
^and 1.3 x 10" (RNA 2) which comprise the entire genome
of the virus. RNA 1 and RNA 2 each possess a 3r-polyA
tnact (Mayo et â1., 1979a) and a 5'-covalently l-inked
protein (Vpg) (Mayo et al-. , 1979b) . In 1969, a novel-
RNA species hras found in cultures of TobRV that had
pneviously been apparently fnee of it (Schneider, 1969l'.
This RNA species, which was dependent on TobRV for
B3
reptication (Schneider, 1971), shaned almost no
nucleotide squences in common with the supponting RNA
(Schneiden, 1977) and hlas cl-assified as a satellite RNA.
Since that time, ãL leasL 24 distinct isolates of TobRV
have produced a satellite RNA of unknown origin during
l-aboratory propagation (Kiefen et ãI ., 1982). The
satellite RNA consists of a singl-e linear species
( Olener et âf . , 1 97 4; Schneiden, 1 977 ) of approximately
350 residues (Sogo et al., 1974), and does not share the
3'-poJ-yadenyJ-ate and 5 | -l-inked protein that are
characteristic of the TobRV genomic RNAs ( Kiefer et âf. ,
1982) , but lnstead bear 5'-hydroxyl and 3r-phosphate
groups ( Kiefer et â1. , 1982; G. Bruening, personaf
communication ) . Kiefer et a1 . (1 982) have shown that
TobRV also encapsidates a muÌtimenic series of larger
than unit length TobRV satetlite RNA sequences and that
doubfe-strand RNA fractions isolated from infected
plants can be denatured to pnoduce simil-ar mul-timeric
series of both satellite and compl-ementary RNA
sequences. Schneider and Thompson (1 977 ) have shown
that the doubl-e-strand RNAs purffied from infected
tissue are infective only after denaturation and
addition of TobRV, and Sogo and Schnieder (1982)
demonstrated that while the double-strand RNA
preparaLions conbained predominantly l-inear mol-ecuIes,
circular and I racket' shaped molecul-es vJere also
B4
detected. Thus Kiefer et a1. ( 1 982) have proposed a
rolling circle type mechanism for the replication of the
satelllte RNA of TobRV which would account for the
production of circul-ar and longer than
sequences of the sateltite RNA and its
unit length RNA
compl emen t .
E. Sequence homoJ-ogy between TobRV satellite RNA and
virusoids
The compì-ete sequence of a satell-ite RNA
associated with the budblight st,rain of TobRV has noh¡
been determined ( Bruening, unpubl-ished results ) and was
kindly pnovided by Dr. George Bruening. The sequence
which consists of 357 nesidues is shown in Figure 6-7
with the pnedicted secondary structure of the molecul-e
(Bruening, unpublished results). The pnedicted native
mol-ecul-e possesses an overal-1 rod-11ke structure with
four prominent stem-1oop structures and a slngle-strand
5t-proximal region. AIso indicated in Figune 6-7 is the
remankabl-e extent of sequence homology between TobRV
satellite RNA and the virusoid RNAs of VTMoV, SNMV and
SCMoV. The homol-ogous sequences correspond to the
central- conserved regions of the virusoids, and are al-so
positioned in the rod-Iike centre of the TobRV mol-ecuÌe.
Furthermore, the GAAAC sequence which is comnon to the
central- conserved regions of viroids and virusoids(except that of LTSV) is also present 1n the TobRV
Figure 6-7
structure of
Sequence and p noposed secondary
TobRV satel-lite RNA.
This data vüas kindly provided by Dr. George Bruening
( unpubl-ished results ) . Sequences which are
conserved between TobRV satellite, SCMoV RNAs 2 and
2' , SNMV and VTMoV RNA 2 are shown indicated in
green.
TobRV satelllte RNA
I,. tsic-a-lU
uaG
UGUç.UUuUcc
U
Uu'ci. : ü--t'o6'cu
G AÂ50'C
^G'cc'cU.AG'C
u G.CG
¡20u\q:9191ccccGcic I a0
¡c0
c.cG.C ¡51
G.C ¡G.cC'CAUACCCUGUe
20
u
l0
ACGU ACUAGlJ
UGCG UCAUCGcc a
Ita0
l0laG I
G C G U C CU ^
cu9
OGçC GCUAC
cuuc ccÂuGcucaiC AGUIJ
C
G6\ o!
c
uCU
G
IUU' c 5 Ga
GC .CGc
:Y9c,^G
UaU
CÈ
U(,c
s auo0gGca6cGCCÂ.rJ IG.C ¡50
G.C
uuc
G
t,GA
GG
GAUUÀC UC ^GGCt cc u
UUcIa
uuu 6u cuclcu 770
GU
G
6uGua
CGCGa' G
I¡a
u
Uçcuc
cc
^
UAA
E-ttoc
UlAC
u u .l?00-u 'a
c'uu'aG.Uu'^c.c
cAAAC
2t0 ç
B5
satellite RNA, b¡hile tha GAUUUU sequence found in all
virusoids, incl-uding that of LTSV, iS absent '
The presence of sequences and structures
whlch are shared by the TobRV satellite RNA and VTMoV,
SNMV and SCMoV virusoids suggests the nesidence of
common functions and/or signals wlthin lhese RNAs.
These RNAs replicate with viruses of quite different
properties ( nepovirus group versus vTMoV/sobemovirus
group), and this may indicate that the possible
conserved functions and/or signals shared by the RNAs
are invol-ved in interaction with host cel-l components
rather than components of the different viruses. Klefer
et a1 . (1982) concl-uded from evidence outlined above
that TobRV satellite RNA may replicate via circul-ar and
rnultimeric RNA intermediates thnough a roll-ing-circle
type mechanism similar to that proposed for viroids
(Branch e! âf., 1981; Owens and Diener, 1982; Kiefer et
âf., 1982; Bruening et al., 1982) . The work presented
in the final chapter is the nesult of preliminary
attempts to determine whether virusoids also replicate
via a rol_l_ing-circle type mechanism, with the attendant
possibility of involvement of the consenved sequences.
CHAPTER 7
vrRorDS, VIRUSOIDS AND SATELLITES
B6
]NTRODUCTION
Larger than unit-length complementary ( - ) RNA
intermediates have been detected in PSTV- and CEV-
infected plant tissues ( Branch et âf. , 1 981 ; Rohde andilSanger,19B1; Owens and Dienerr l9B2), and appean to
exist malnly 1n extensively double-stranded RNA. 0wens
and Cress ( 1 980 ) and Bnanch et al. ( 1 981 ) have shown
that RNase trealment of doubl-e-strand RNA intermedlates
from PSTV infected pì-ants results in the production of
complemenLary PSTV RNA of unit length or slightly tanger
whil-e 0wens and Diener (1982) demonstrated that,
denaturation of the double-strand RNAs released
monomeric PSTV strands that had been complexed with
multimeric compl-ementary RNAs. In addition, multimeric
series of both ASBV and its complement have been found
in viroid-infected tissue (Bruening et â1., 1982), and
dirneri-c RNAs have been shown f or CCCV. Various workers
have postul-ated rolling-circl-e. type mechanisms f or the
repl-ication of viroids ( Branch et âf. , 1 981 ; Owens and
Dlener, 1982; Brueni-ng et âl ., 1982).
As outlined in the previous chapter, the
replication of the linear satellite RNA of TobRV shares
features common to viroid replication. Compl-ementary
RNA intermediates have been detected in high mol_ecular
weight double-strand RNAs, âhd RNase treatment of these
duplex RNA intermediates reduced the molecules to a size
B7
slightj-y but significantly larger than unit-length ( Sogo
and Schneider, 1982). Double-strand RNAs vüere shown to
produce detectable satellite activlty only if denatured
( Schneider and Thompson, 1 977 ) , and to consist of
multimeric series of apparently concatenate forms of
RNAs of both pol-aritles ( Kiefer et â1., 1982). Thus
Kief er et al- . ( 1982l, have also postul-ated a
rol-ling-circfe type mechanism for the replication of
TobRV satellite RNA. It seemed feasible that virusoids
may al-so replicate via a rol-11n9-circle type mechanj-sm
and the remainder of this chapter 1s devoted to
descniption of the experimental- support for thls notion
and to its possible ramlfications.
ME THODS
A. Isolation of RNA
VTMoV and SNMV $rere kindly provided by Drs.
R.I.B. Francki,
extracted from
vi rus infected
J.Vt. Randles and A. R.
(GouJ-d,
GouId.
1981 ) ,
( Randles
RNAs v\iene
and from
et â1.,
purified
Nicotiana
VITUS
clevelandii
1 981 ) using phenol-SDS extractions as previously
described.
B. Blot hybridization
Nucleic acid samples hlere denatured by
treatment with 1M gl-yoxal and 50% (v/v) dimethyl
BB
sufphoxide ( McMaster and Carmichael, 1977 ) and
electrophoresed on 2.0% agarose slab gels (l5xl 4*.0.15'
cm) in 1OmM sodium phosphate pH 6.5 aL 30 mA. Nucleic
acids were transferred to nitrocel-l-ulose by blotting and
baked in vacuo at B0oC (Thomas, 1 980). Nltnoceflul-ose
sheets h¡ere prehybridized, hybridized and washed
essentialJ-y as described by Thomas (1980 ).
Complementary 32p-o¡la hybridization probe r^ras prepared
using recombinant M13 ss DNA, containing
corresponding to SNMV RNA 2 residues 13'1
Chapter 5), essentiatJ-y as described by
(1982).
RESU LTS
sequences
to 216 (see
Bruening et a1.
A. Analysis of VTMoV and SNMV RNA 2 se uences resent in
vinus and infected tissues
Using recombinant M13 ss DNA containing
cl-oned SNMV RNA 2 sequences, ^ 32P-cDNA bras syntheslsed
and isolated as a probe specific for VTMoV and SNMV RNA
¿ Fi gure
P-probe
7-1 shows the pattern obtained when bhis
hras used to detect VTMoV and SNMV RNA 232
sequences present
infected plants.
mul-timeric series
encapsidated and
dimeric forms of
in RNAs extracted from virions and
It can be seen that for both viruses,
of (+) RNAs are found both
in tlssue extracts. In addition, the
VTMoV and SNMV are detectabl-e by
Figure 7 -1
SNMV RNA 2
Multimeric RNAs containing VTMoV and
sequences.
Nucleic acids r¡rere extracted from either purified
virus (V) or virus infected plant tissue (E) for
both VTMoV and SNMV. The nucleic acids hrere glyoxaJ-
treated, electrophoresed on a 2% agarose gel and
subsequenbly transferred to nitrocellulose. VTMoV
and SNMV RNA 2 sequences were detected using_ a
')a"P- cDNA p robe prepared f rom a cloned SNMV RNA 2
32sequence as described in the text. 3r- P-labelled
Hpa II cut M13 mp7 RF vJas contransferred to provide
si-ze markers.
PLUS OLIGOMERS OF VIRUSOIDS
2% Agarose gel
Glyoxal RNA s
tr. - 1596
{;^-lf - 819
- 454
- 357
VTMOV SNMV
a
MVE V E
B9
toluidine blue staining of
denaturing polyacrYl-amlde
not shown).
viri-on RNAs fractionated bY
gel electrophoresis ( results
DISCUSS]ON
A. Multimers of VTMoV and SNMV RNA 2
Fnom btot hybridization experiments, such as
that shown in Figune 7-1, it is apparent that multlmeric
series of RNAs containing VTMoV and SNMV RNA 2 sequences
are found both packaged in virions and in infected plant
tissues, as j-s the case f or TobRV satel-lite RNA. On
this evidence, it seems IikeIy that the vinusoids of
VTMoV and SNMV replicate via a roll-ing-circfe type
mechanj-sm similar to that- proposed for TobRV satel-lite
RNA (Kiefer et â1., 1982) atthough the existence and
propertles of mul-timeric complementary RNAs and ds RNAs
have yet to be determined.
RoIling-circl-e mechanisms require a circufar
templ-ate to al-l-ow tnanscription of multimeric RNA
intermediates. Therefore, such a model woul-d require
that aL some stage during replication, the linear TobRV
satellite RNA be ligated to produce a circulan template
mol-ecul-e. As one of the f inal steps in replication,
unit-length finear virusoid or satellite RNA must be
produced by either specific transcription or cJ-eavage of
mul-timerlc RNAs . Th e 5' - hydroxyl and 3 t -phosphate
90
groups present on TobRV sateltite RNAs (Kiefer et âf.,
1982; G. Bruening, personal communication) suggest that
these molecufes are produced by specÍfic cleavage rathen
than being primary transcripts. fn the case of
virusoids, the unit-l-ength l-inear mof ecuf es must also be
ligated to produce the final- circulan product. Thus, it
is feasible that the TobRV satellite RNAs are simply
defective 1n l-igation and correspond to l-inear RNA
intermediates in virusoid replication which, in
contrast, a?e capable of cj-rcuf arizatlon.
B. A possible site for RNA pnocessing
The 5t terrninus of TobRV satellite RNA is
adjacent in the
sequences shared
SCMoV. When the sequences of these
aJ-igned
be tween
as in Figure 7-2, extensive
TobRV satellite and VTMoV and SNMV RNA becomes
apparent. Homologous sequences extend from the central-
conserved regions to residues corresponding to the 5l
terminus of TobRV satel-lite RNA (VTMoV and SNMV RNAs 2
residues 49) and include several- residues correspondÍng
to the 3r terminus. Therefore, 1t is proposed that the
TobRV satetlite and virusoid RNAs are pnoduced by
cleavage of multimeric RNA pnecursors at sites
corresponding to between residues 357 and 1 for TobRV
molecule to the centnal- conserved
with the vinusoids of VTMoV, SNMV and
molecufes are
sequence homology
Figure 7-Z A possible site for RNA processing.
A) The proposed structures of TobRV
SNMV and VTMoV RNAs 2 are shown with
sequences conserved between these 3
in colour. These sequences consist
in Figunes 6-6 and 6-T , and include
sequences not shared by SCMoV RNAs 2
satellite RNA,
the nucl-eotide
RNAs indicated
of those shown
addi tional
and 2t .
B) Comparison of the 3r and 5t
conserved reglons of TobRV with
proximal-, and central
the cornesponding
and SCMoV RNAs 2 andregions in SNMV and
2t . Regions of the
and proposed sites
and SCMoV virusoids.
lndicated in col-our.
VTMoV RNA 2
RNAs are shown in linear form,
for RNA'processing of
Conserved nesidues
SNMV, VTMoV
are
A
To bRV
SNMV
u
CGCGCCcla
¡le
U
ugccuocc
gOACgO. uC
{-rU
uG
..1
e^vc¡ !c^c
o9 ^cue
U.cG oC
GC.CGc
I¡ lio
¡ tì¡
G:¡0
ccccoGcG.cc.G^urcclul¡
AAAuc
c
c
u
u
cu
u.ci l!-t'oG'cuc.cG.CG.C
uuc
Gl
c
¡
GA
GG
UA
/ 100g
2oululr
::^c cFõî¡Ic
cct
'g' glo
Ga^1o - t. 'o ^c'cc'cu'a ¡!C G'C r
^u c'c a^ \ c¡99 9Y:t içgY î:Y1fY.+ç
uGCGU
l3!
^Gl99YGue
cUUG OCUAC.CUC
cg^uo G^oUA
IJ
Gv
oç
CL
, î,cccuucucc
CAcAG
UtJ
.U
.c
.c
u AucGcccAGc6cç .G^uuaclc¡l'
¡14
u u u c u c u c AEc I cuu
G UCAUCCcc ^
tlc
gA
U
uluAc
cA
UU. G.Cc'cu'ac.G
cu'c
cU
uc
cA
A
UG
U
cccc vcc
ô0
¡¡0 u
0c
u
uu.¡ ôc-u .
100co'A U CCt/0 ^c t¡uc9u
^^
u
l.g
r00I
c
I
Ig
c
20. 40[u, Icl^ u c u
uo uuoouucoccuqcuc ouo ¡!c c
{þ@$,crc qqc . cac c.^c^cc.ueo-116}
| '^u lü @r340 320
r20
I ^c
r40I
Cq^CCu.CO^G.CUg êu.^oUO sAO00 ^Coocuc c{}ucr. coc
160
c \ohjocu 0
rô0
"^ \,u^g¡. rse^se.cg) u^t0
c
"rrü¡260
cuccc uc0ao q30 0
l'?40
op\ ouo cpþ\cl^ u c
ceu@ rcevcr of| euc
| ^o ' ^.^220
u/"200
gg
oo
co^coo oo^cUC
cuffiõì olõiìcoo
.1:$,"-AOA C ooo{þ ocugoA 0uuc 9^u
I
280
ccc
^c3tt
VTMoV
40
IoocuG^E
C
!CoAO'OoC g OUO AgC C^
Io
lô0
t_c.uclgl. ou^o cA ueAc
cu ¡cue
u cc u uc 0^0 ^c uucg oc 0Accu,coAc,cu0 cu^0
Itr!l a90
2øO
c u ccuucu @ orccu
gOO^ CO ' CUggA
sl
120 140
Io
0ccc
c
c
c
c
c
U
uu cA uc
^U
tr0o0
c g0uo0uu sccv uccou0 o^ooo
cuccc
c
uourcu.{J urc
uc rcruce { ruo
lq" u ^a
u
c0
c
UO
^cooo.[þocrc 0OC¡CÁ€9.^€ACC uACg.A0 C .cc 909^ cg ' cuogÀ ouuq s^u o@^^r
lc UU
I
320I
260
o0
co AE 220
,1.2@365 360
I
300 240
100o
280
a
B
TcbRV scielTite
UACC¡.F
UU 66
A A(¡O
U6
¡Ê¡. 2
AAi*rÞr"¡y'a
CUA
ViMcV ond SIiMV RNA 2
Gu6ulr+
6
SiMcV RlilAs 2 c¡¡d 2'
cAc6st
S;-Prt UÀ L
llo u A t u'yåglj",, ^
G u A t A
I
91
satellite RNA and between nesidues 48 and 49 for VTMoV
and SNMV RNAs 2. Unit length linean VTMoV and SNMV RNAs
2 woul-d then be l-igated to produce the mature circular
forms of the RNAs. Implicit in this proposal j-s the
assumption that the conserved sequences surrounding
these putative sites for RNA pnocessing are in some I^Iay
functional, perhaps in detenmining the specific sites of
cleavage. The conservation of these sequences in RNAs
from different viruses may suggest their interaction
with host, rather than viral, components (TobRV, VTMoV
and SNMV share common host plants, such as Nicotiana
cf evel-andii ).
Interestingly, during sequence determinatj-on
of VTMoV and SNMV RNAs 2, essentiaJ-J-y compl-ete
termlnation of reverse transcription I4Ias observed at
positions corresponding to residues 49 of the RNA
templates. Thls I¡Ias seen whether intact RNA 2 (see
Figure 2-B) or puri-fied finear RNA fragments u¡ere used
as templates, and hras pnesumabl-y due to the presence of
sequences and/or secondary structunes capable of causlng
reverse transcniptase to chain terminate. For example,
an B-base-pair stem 3-base-pair loop structure can be
formed in VTMoV and SNMV RNA 2 aL residues 40 to 58,
however the same structure cannot be formed at the
corresponding sequences of TobRV satellite RNA (or
SCMoV). It is unknown whethen the precì-se coincidence
92
of sites for termination of neverse transcrlption with
predicted pnocessing sites in vTMov and sNMV RNA 2 is a
product of chance of, perhaps indirectl-y, of function.
In contrast, SCMoV RNA 2 and RNA 2t share
Iittle sequence homology with the other RNAs (Flgure
7-2\ outslde the central- conserved region, with the
exception of several residues approximately
cornesponding in location to the 5t terminus of TobRV
satellj_te RNA. Based so1ely on thls limited sequence
and structural- homotogy, Possíb1e processing sites fon
SCMoV RNA 2 and RNA 2t are between residues 62 and 63 in
each mol-ecuf e. The l-essen extent of sequence homology
between the SCMoV virusolds and the other small viral-
RNAs (Figure 7-2) may' be rel-ated to the limited and
excl-usive host nange of scMov, which is not known to
share host pJ-ant species with TobRV, VTMoV or SNMV
( Franckl et âf . , 1 983 ) . Thus functional nucleotide
sequences might vary to acconodate the different
requirements of host components in different species.
C. Viroid, virusoid and satel-Iite RNAs
I t noI^I appears that there exists in pl-ants a
range of replicating RNA species which overal-1 share
many common features. Thus virolds, virusoids and TobRV
satellite RNA a1l consist of ss ú*O speci-es of betweenL-
240 and 4OO residues which, except that of TobRVß
o?
satellite, are rod-like base-paired circufar molecules '
These RNA species do not appear to code for functional
polypeplide translation products, but repÌicäte lhrough
multimeric RNA intermedj-ates whj-ch are probably
transcribed by a roll-ing-circle type mechanism. The
unit-Iength pnogeny species must be produced by specific
transcription or processing events and thenr eXcept in
the case of TobRV satel-Iite, J-igated'
Given these simitarities, the RNAs f all- j-nto
one of two cl-asses. The first contains viroids which
a?e chanactenisticalty naked and capabl-e of independent
replication. The second conslsts of encapsidated RNAs '
Iike those of VTMoV and SNMV which appear to contribute
some f unction to a vinal- Senome I otr l-ike those of TobRV
and LTSV which are satel-1ites. The members of each of
these groups share at l-east some conserved sequences
with others of the same group (see chapter 4 and 6), and
overalf share remarkable conservation of regions central
to their native stnuctures ( with the notabÌe exception
of LTSV RNA 2) . Furthermone the pentanucleotide
sequence GAAAC is present on all sequenced viroids,
virusoids (except LTSV RNA 2) and TobRV satellite RNA,
and is l-ocated within the central conserved regions of,
these molecul-es.
A s ingle questlon looms f nom th j-s tangl-e of
observatons. Do viroids shane common functions, and
94
perhaps origins, with the second group of encapsidated
RNAs? The possible invol-vement of conserved vinusoid
and satellite sequences in interaction with plant host
components v,IaS inferred from data presented j-n Chapters
6 and T, and viroids appear to nely entireJ-y on host
components for reptication. It therefore seems
reasonable to suggest that these biologicalJ-y disparaLe
RNA species may shane some common mechanisms in
neptication whlch involve functionally similar, if not
identical, host components. The two groups of RNA
specles may be derived from a common ancestral- species
or alternatively be products of convergent evol-ution.
These suggestions are of course based on
inference rather than direct evidence as the appnoach
taken in this work aIlows onJ-y a glimpse of the
functions and origins of these mol-ecules as reflected in
their comparative structures. Confirmation on denial- of
these possibilities and ' ultimately, ansvlering of Lhe
three questions originally posed in Chapter'l wlll rely
upon studies of the host and viral compon"ìt" involved
in the repl-ication of these RNAs, Tãther than the RNAs
themselves.
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