3cl - journal of virologyjvi.asm.org/content/suppl/2013/05/06/jvi.00416-13.dcsupplemental/... ·...

Hel

CavVNDiVHanaVMénoVNséVMoumoV

4207 4280ICSYKSQLQN FISLQQQKIL SDNVNLSTID SAQGDEFDIV ILCLSQINNF TLNPNRFNVA ISRAKSVLFI TVPP.......... .......... .E........ .......... .......... .......... .......... .............. .......... .......... .......... .......... .......... .......... .............H .AT..N...I PEF.K..... .......... .......... .......... .......... .............. .A....L.VI PEY....... .......... .......... .......... .......... .............. .T.......I P.F.K..... .......... .......... .......... V......... ....

VIV


4117YHFRCHPTIF QYFKDLYYAD KDMECATSIA DRIIRPLNPI NTVQVSEPTF RNQGVILNQD EADKVLEILV LVNQTLALHS SYEYQPTIAI.......S.. .......... .......... .......... .......... .......... .......... .......... .................M.. .......... .H........ .......... .....G.... .......... .......... .......... T.........D......H.. .....Q.... ...V...KAE ..L....... ...H.GP..Y .......... .T....D..I ..T...T..A .Q....SL..H......S.. .F........ ...V...AAS .......H.. ..I..EG.SY ......R.EN .TE...D... ..S.V.S..A .FN..............L.. .F..N..... ...V.S.QAI .......H.. ...PIQG..Y ......R.EH .TE...D... ....I.S..T .FDFK.....

IV


4027RICVTTIQSF STVQHVKDID LVILDEFSLT SDNYLLTGLA HLKPSTRVLF SGDPRQLSGV DEVRKPLQSR FHTLINYYTE TYPREVHVLK.......... .......... .......... .......... .......... .......... ..I....... .......... .................... .A....V-V. V......... ......S... ..RL...... .......... .VI..L.H.. .......... ...L..L..T.......... ......R... .......... .........S .......... .......... ..T.....P. ......F... ...N......C......... P.....-.V. .......... .........S ...L...... ....K..... ..I....LP. ........A. ...K.LL...C......... P.....-.V. .......... .........S ...L...... ...T...... ..I..T..P. ........A. ...N..L...

II III


3937RFKIMFGGPG TGKSHTLSIL INHLHEKGLR ILVYTPSHQS ANALLYKIAN LIKRRTIQNP GLVRIITDGM KEEIKPHPYI TYRTNMLDKD.......... .......... .......... .......... .......... .......... .......... .......... .................... .......AT. .....D..F. V......... .......... MM...G.... .......... .D.L...... ....H.I.T........... .......... V.....Q.FK ..I....... .....F.... IM...H...S .......E.. .D......F. ...AS..E............ ....Y..... ......S.FK ..I....... .......... VMRK.N.... R......E.. .D.L...... .P..H...Q........... .......... V....AS.FK M.I....... .........G .M...S.H.. .......... .D.L...... .P..H.....

I

ZCavVNDiVHanaVMénoVNséVMoumoV

3601NKGRLITYNC YVCGENAYLT CATCERAFCN SADTNHGSHM EQHLQYSGHT CLYLNCKTVK CHHCFTTDIN LLYTTGRD-H YCEAHKPKNA...K...... .......... .......... .......... .......... .......... .Q....M... ........-. ...S............S... ....D..... .......... NT.......I .......... .......... .Q..Y.S... .......E-. F..I.........Q...... ........I. .....KS... .K..I....I .........D ........I. .R...AI... K...S.K.MY F..T....H....K...... .......... .......... .S.SI....L .......E.N ......R... .S..Y..... V...S.KN-V F............K.VA... .......... .......... .S.PL..... .........D ..F....... .R..Y.Y... V.......-V F..Q.R....

3689* * * * * * * * * * *++++ +

3CL

CavVNDiVHanaVMénoVNséV

1565DTTTGKSNIW TSYKLQHPSE IMITLNNEIN LPNPANYDFE TTKVVYQHPL RNVCATLETL QHLTNKTNAK LPYDSRLLSD FNITAEQYNQ.......... .......... .......... ....T..... .......... .......... ........V. ....P..... ........A..IK....... .......... .......... ....E..... .SS....... ..IS...... .Y........ IA........ ........A.NC...TP... ....I..... .....D.G.. ..Q.ED...Y .A.IC..... .D.R...Q.. .Y...QG.T. ....PQ..A. ...........KLQ...... ....M..... .......... ..E.VD...N .N..I.H... ...M...... .Y...S...R M...K..... ........E.


1385

NKSAASNPSI SHIVLEMPVA INPLIKYTTR TSVSSLRGAV VNGYIYIQRH LFGSKKQEFE ACYNNGKGLL NCKNLERSKY DIDSAELIGT.......... ......L... .........K .......... .......... .......... .......... .....D.... .................... A.N...L..T .......... .......... .......... ...N...V.. .......... .....D.... ..............L..... PY.A.PL..S .....T.DS- --......S. .......L.. ....NQKD.D SH.A.....N K......... ...N................ P.K..GL..T .....T.MSN KYA.....S. .......... ....N.ND.L V........E K......... ..........

! !

pro


1655 1688YGYYIDYNNF VNNFNRYTTT TIGTKSFETC IKYG...N...... I......... .......... ....H...V..... .....H.... .......... ....H......K.. IQ..IK..N. L.NSR...MS ......L.V...Q. .K...Q.... L....T..S. ....


1475LIRIPLHDKH SIPHISIHPD PLSYNGPVTL YLSRYDTELN KDVLCVHTGF MSEGHHDIKT VFGDCGGMLF DPKGRLLGLH CAGSDDVVFM.........Q .......... .......... .......... .......... .......... .......... .......... ................V..S .V.N.N...A ..T....... .......... .......... I......... .......... .......... ................V.PK .V.DVK...K ....T..... ......S.TA .......... IA......R. .......... ..R.Q..... ....A..S.L..K...V.RN .VVD.EL..E ..T....... ......S.QG .......... ........R. .......... .T........ ....E.....

& ! &

OMTAALAKAPLDW DHLTLEIPGY NTRKQHSS-H MTTKALGILH ILQDSMLYTN RKTLNPNLPV ILPGSASYLG DTVLANEMSK TLKQTKFVHI...S...HN. .......... ........-. .......... .......... .......... .......... ........A. .......I..ST.S...PT. N.....L..C .--RK.V.-. T....S...Y .......... .......... .I..A..QF. ....T..I.. N..R...INVSN.Q...SN. N.TQ.L.... E...ISPPG. .LN......S .........H .T...K...I .H..A.GFN. ..I....FR. Y...S.IIN..H.NL..HT. ..Q....... S..INNPC-. .Q...I.V.R .....I...P .....AKY.. .......NY. E.I....FRR H...S..I...H.N...Q.. ..Q....... T..TNNPC-. .Q.....V.. ..K......S .L...AAR.I .......HY. E......FK. Y..LS..I..


4848

X I

#

DPRLKIDNNT THHRKTLMEM LDIGYTTELI ISDIHDNNN- PWIPELMEYT LKYLIDTGTL IMKITSRGAT EAVLQQLEHM AKNFTYVRVC.......... .......... .......... .......K.- .......T.. ....V..... .......... .D......DL S..............L..HN ..Y..P.K.. .......... .........N T..H..I... ....IE.... .......A.. .DT.EY...L S..............R-.DD ..YKLL.KD. .PK.FN.... .....--SDV E.....I... N...QQS... .......M.. .KAI....TL SE.............L.... ..FK...KD. .P...P.... .....N.DDP T..T..ID.. H...L..... .......... STA.E...NL SRI................. ..FK...N.. ..V..S.... .......T.K N..A..I..S T...LES... .......... .EA.AL.QAY S.......T.


4937

IV VI

# #

NLNAVTFSSE LWIVFANKRK PPVQGWTSHE LRAELRKHWY SMTRSIIQPL MRARQSVFRY SPK.......... .......... .......... .......... .......... ..S....... .........P..V ......D... ...L...... ..N....... ..A.N..... T...PCI... ......V..C... .......N.. ...N.....D ..N....... ...HN.LH.T I.S.LD.... ....V....C... ......D... .T.K.....D V.S....... ...QN.KN.I L.Y.LDI... ....I....C... .......... ...N....YD ..T...M... ...QN.RTTV L.P.IGL.K. ...


5026 5088

VIII

#

NMTSFVLSHLEKL AYEPQANLKA FTSMDYRLKN FNPEMCKLRR ELQKVWYEKY IDTK--KTHC NMGCGKEPLQ QALHNIDVLQ GKSNPQNNMN........N. .......... .......... .......... .......SQ. TN.N--.... .......... .......... ..................N. ..D....... .......... .......... ....T..QQ. .A.N--.... .........K H......I.. ..T........V.QPQ..SI ..DN-...N. ........R. .D........ D...H..A.. SE.S--I... ......Q..K .......IN. ..N........L.QP.F... ...QG...R. .L......R. ..I....... ...IE...R. TSINTSR... .......K.S N......IK. ..-.LH.D...L.Q.EY.N. ...Q....S. .L......R. .......... ...DA..TQ. LTASKP.I.. ......QL.K R......IKL .N-K.Y....


4614

I

THTCDSEEHI YFDSHWYKDG GFKKPSYIFS DINKEHYYKL GTTGLCLYLN SKYAKYVHEY RTVSGNDVFK S-LYSPYCDL GRKPHQAVIE.......... ........A. ..T....... .......... .......... .......... .......... .-........ .......E.......A.... .......... ..S....... ........N. .....V.... ......I... .K........ .-..N.F... ..E....A....L..AQ... ........T. ..S....... .......... .A........ ..H...L.AF QPI....... TEF.NSQ.TT N.T..HLS....I.S.D... ........IN ..S....... .M......N. .......... ......C.V. QE.T.T.... .-...SS.E. EL...K.I.T....NA.... ........EN ..Q....... .M......N. .S........ ..H...C.K. .EA..D.... .-...LE.DK DQK..THA.Q


4702 *

PSCSIPDCII TS-NIGERFQ TLVCNVHQDQ MELISKIAQA TKYGYQFIYT GKILLNNH.......... ..-....... .......K.. .DI....S.. .......... ..T............... ..-..D.K.. .....I.K.. ..I....S.. .......V.. ..T.....DNT.M.A... Q.-SSN.D.H .FI.DG.Y.. .QI..D.SK. ........K. .PTT.S..DCT.T....N S.-TL.SQ.. .I..SL.YG. .DI....S.. .Q...R.V.. .S-E...KDCT.T..... ..NTSD.N.. V...SK.Y.. .DI....S.. .....R.... KANE....


4791 4847* * *

ExoN

GIRNDTTVDL RPLLNFCVDN MHVKPVIVTW SGASDHCFLR AHTLYPDIST VCNITTRCTS QPIYASPQGR HTYYLCQYHA HQLKDHINIT.......... K......... .......... .........K ........A. .....I.... .......... .......... ......V.....S...I... K......... V......... ..V......K .Y........ .......... .......... .......... ............K.NV...I KH.....I.. .......I.. A.DN.....K .N....PAAQ I....P.... T.....A.TC .......F.. QTF.EQLT....SDN.I..V KHM....I.. V......... A.T....... .N.....V.Q T...AS..L. .......... .......... .KF.EQL........I.... KHM....I.. T......... A..T.....K .N......TQ I......... ......R... NI........ ..H.EQL...


4447* * * *

I

DCICFDAEFL NPRDNLQEPV MLSYGFSSKY GKRRIAGIPV RYIKDKFNRI IPHKYNYKDN NKPLTSTYSC DWMKKQHPDQ YKHLLTSVLQ.......... .....V.... .......... .......... .......... V.Q....... ........T. ...R....E. ........M..Y........ .....V..H. .......... .......T.. ........K. V.I..P...K H.......V. E.....K.E. .QY..N.IT..Y.F..S... ....DPR... ......T.A. .N...S.T.M ..VYS...QL T.V....... ........IA T.L.AKA.E. .NY.RDTIT..Y.Y..S... ..K..PP... .........H .....S.T.M .....NY... T.I...F..K D.......VS S....DL.T. F...NNA.CK.YVY..T... ..I..R..C. .V.......N .N.....T.M ..V....DKL V...F.F..K ........IT G.L.A.L.Q. F.Q.N.A..N


4357 & &

HFVNLEIIDL KVDRNQYTNE RTLRVYHNDY LKLTLDLDN- VASNSLTDCH TRYCRTIHAP PTPHDPLDDA IMTQCIYQ.......... ........D. .......... .........- .......... ......V... A......... .................. ..EY..F.S. .I......N. .........- .......... K.....V..S I......... .................. .AIN.KQ... K...LT..NE ....IE.SQD IK....N... AY..N.T.R. I......... L.....FK.......... S..K.KH.D. .N...T..NE ...K.EISKS I...K.Q... DF..N...T. I......... ......F........L.. .AEF.VH.G. K.IS.T..EE ...KFEIHED ...CK...V. DF..N.V.T. I......... ...R.VF.


4537 4613

II III

& & § §

Figure S1: Multiple sequence alignments of putative mesonivirus replicative

domains. Partial pp1a/pp1ab sequences were aligned using ClustalX 2.0 (6) as

implemented in Geneious (5). Amino acid numbers relate to positions in CavV

pp1a/pp1ab. Conserved sequence motifs in the helicase (Hel) (3, 4), 3'-to-5'

exoribonuclease (ExoN) (7, 10), guanine-N7 methyltransferase (1, 8) and ribose-2'-O

methyltransferase domains (2, 10) are indicated by black bars and roman numbers.

Other conserved residues are indicated as follows: !, Cys, His and Asp residues

predicted to form the catalytic triad of the mesonivirus 3CLpro (12, 13); &, residues

predicted to participate in the S1 specificity pocket of the mesonivirus 3CLpro (13); *,

conserved Cys/His residues in the helicase-, ExoN- and NMTase-associated zinc-

binding domains (8-11, 13) that are predicted to be involved in zinc ion coordination;

+, other conserved Cys and His residues in the mesonivirus helicase-associated

zinc-binding domain; &, conserved acidic residues (Asp, Glu) predicted to be

involved in metal ion coordination by nidoviral (and cellular) exoribonucleases of the

DEDD superfamily (7, 10); #, conserved residues predicted to form the catalytic

tetrad of mesonivirus (and other viral and cellular) ribose-2'-O methyltransferases (2,

10).

gp64 ? gp116

229TILS 1128LAPR

N

1MNRR

C

sgsg

Protein

mRNA2 mRNA3

Genome 2 3 5’ An-3’

5’5’ An-3’

An-3’

Okavirus (YHV)

Alphamesonivirus (CavV)

2b 2a 5’ An-3’

5’ An-3’

? S

224STRID

S1 S2

224STRID 431WDSS

N

1PATVS

B

sg

Genome

Protein

mRNA2

N Arterivirus 5’ E 1a 1b

GP5

M

GP3

GP4 GP2

An-3’

N Okavirus An-3’1a

1b 5’

gp116 3

? gp64

4?

N Bafinivirus An-3’1a 1b M

5’ S

1b

N Torovirus An-3’1a

1b M 5’

HE S

1b

N Alphacoronavirus 5’ 1a 1b

An-3’ M S E

2b Alphamesonivirus An-3’1a

1b 5’ 2a

3a 3b

4? N

S1

M?

S2 ?

A

Figure S2: Nidovirus genome organization. (A) Schematic view of the genome

organization of viruses representing all currently established nidovirus (sub)families.

Mesonivirus (B) and ronivirus (C) genome organization in the glycoprotein and

nucleocapsid protein coding regions. Open reading frames (ORFs) are shown by

boxes, sgRNAs are indiated by lines, and proteins are shown by rounded boxes.

Leader sequences are symbolized by red and 5’ cap structrures by black boxes. N-

terminal protein sequences and genome positions are indicated. 3' poly (A) tails are

indicted by An. The following genomes are shown: Human coronavirus NL63 (HCoV-

NL63, NC_005831, genus Alphacoronavirus, subfamily Coronavirinae, family

Coronaviridae); Bovine torovirus (BToV) strain Breda (NC_007447, genus Torovirus,

subfamily Torovirinae, family Coronaviridae), White bream virus (WBV, NC_008516,

genus Bafinivirus, subfamily Torovirinae, family Coronaviridae), Equine arteritis virus

(EAV) strain Bucyrus (NC_002532, genus Arterivirus, family Arteriviridae), Cavally

virus (NC_015668, genus Alphamesonivirus, family Mesoniviridae), and Gill-

associated virus (GAV, NC_010306, genus Okavirus, family Roniviridae). Proteins

and corresponding ORFs encoding these proteins are indicated as follows: E,

envelope protein; gp and GP, glycoprotein; HE, hemagglutinin-esterase protein; M,

membrane protein, N, nucleocapsid protein; S, spike protein.

Table S1: Primers used to generate DIG labeled northern blot probes.

Virus Primer name Primer sequence Probe length

CavV CavV-5’-2-F

CavV-5’-106-R

5’-CTAATGAAAATTTTGTTTTCTCAC

5’-ACTGATGTGGCTAGGTTTGGTGAT

105 nt

CavV-3’-19551-F

CavV-3’-20092-R

5’-AAGTTCGACCACTGAATGAGAC

5’-CGCCATACTACTAAGCCCTAAC

542 nt

HanaV HanaV-5’-1-F

HanaV-5’-108-R

5’-ACTAAAGAAAATTCAGTT

5’-GCAACTGATGTGGCTAGGTTTGGTG

108 nt

HanaV-3’-19655-F

HanaV-3’-20062-R

5’-TGCATTAATCTACCACTATAATCTCGCC

5’-TGCCAAGCTCCATACTACTAAGCC

408 nt

MénoV MénoV-5’-1-F

MénoV-5’-121-R

5’-ACTTTGATATCTTTTGATAATCGCC

5’-CAGAGGGTCTCAAACAAACAAAGTTGC

121 nt

MénoV-3’-19717-F

MénoV-3’-19971-R

5’-CACTTTCCGAGGCACGACACAACC

5’-TGCCAATCTGATAACTACTGAGCCCT

255 nt

NséV NséV-5’-1-F

NséV-5’-111-R

5’-ACTAAAGAATAATTTGTATTCAACC

5’-AGCAATAGACGTGGCTAGTTAAATTTCTAGAG

111 nt

NséV-3’-19790-F

NséV-3’-20060-R

5’-TGCAGCATGAACTCGACCACCA

5’-GCGCCAAACTACTAAGTCCTAACCG

271 nt

References

1. Chen Y, Cai H, Pan J, Xiang N, Tien P, Ahola T, and Guo D. 2009. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. PNAS 106:3484-3489.

2. Decroly E, Imbert I, Coutard B, Bouvet M, Selisko B, Alvarez K, Gorbalenya AE, Snijder EJ, and Canard B. 2008. Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. J Virol 82:8071-8084.

3. Gorbalenya AE, and Koonin EV. 1993. Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3:419-429.

4. Ivanov KA, Thiel V, Dobbe JC, van der Meer Y, Snijder EJ, and Ziebuhr J. 2004. Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J Virol 78:5619-5632.

5. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, and Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647-1649.

6. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, and Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948.

7. Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, Canard B, and Ziebuhr J. 2006. Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. PNAS 103:5108-5113.

8. Nga PT, Parquet Mdel C, Lauber C, Parida M, Nabeshima T, Yu F, Thuy NT, Inoue S, Ito T, Okamoto K, Ichinose A, Snijder EJ, Morita K, and Gorbalenya AE. 2011. Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes. PLoS Path 7:e1002215.

9. Seybert A, Posthuma CC, van Dinten LC, Snijder EJ, Gorbalenya AE, and Ziebuhr J. 2005. A complex zinc finger controls the enzymatic activities of nidovirus helicases. J Virol 79:696-704.

10. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, and Gorbalenya AE. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331:991-1004.

11. van Dinten LC, van Tol H, Gorbalenya AE, and Snijder EJ. 2000. The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis. J Virol 74:5213-5223.

12. Ziebuhr J, Snijder EJ, and Gorbalenya AE. 2000. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81:853-879.

13. Zirkel F, Kurth A, Quan PL, Briese T, Ellerbrok H, Pauli G, Leendertz FH, Lipkin WI, Ziebuhr J, Drosten C, and Junglen S. 2011. An insect nidovirus emerging from a primary tropical rainforest. mBio 2:e00077-00011.

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