JOURNAL OF VIROLOGY, Sept. 1982, p. 809-8180022-538X/82/090809-10$02.00/0

Copyright 0 1982, American Society for Microbiology

Vol. 43, No. 3

Mapping the Mutation Site of an Autographa californicaNuclear Polyhedrosis Virus Polyhedron Morphology Mutant

ERIC B. CARSTENSDepartment of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received 22 February 1982/Accepted 27 May 1982

A polyhedron morphology mutant of Autographa californica nuclear polyhe-drosis virus, designated M5, was compared with wild-type virus by genotypicanalysis with EcoRI, BamHI, HindIII, SstI, and SmaI restriction endonucleases.M5 DNA revealed several alterations relative to the wild-type pattern: (i) EcoRIfragment I was 400 base pairs larger; (ii) BamHI fragment F was missing; (iii)HindIll fragment F was 400 base pairs larger; (iv) an extra restriction fragmentwas obtained with both Hindlll and SmaI; and (v) SstI fragment G was 400 basepairs larger. M5 virions contained two size classes of circular DNA, one of 100%of the wild type and one of about 58% of the wild-type molecule. A revertant ofM5, designated M5R, was isolated on the basis of polyhedron morphology. Thegenome of M5R contained the insertion of DNA in EcoRI fragment I and inHindlIl fragment F, but was similar to the wild type in its other restrictionfragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptidesmaller in size than the wild type or M5R.

Autographa californica nuclear polyhedrosisvirus (AcMNPV) has become a model for thestudy of the molecular biology of baculoviruses.AcMNPV DNA is a double-stranded, super-coiled circular structure of approximately 128kilobase pairs (kbp) (8, 11, 14). Restriction endo-nuclease fragment maps of the AcMNPVgenome have been derived (4, 8, 12, 15) whichindicate that the viral genome contains predomi-nantly nonrepeated sequences. Plaque purifica-tion of wild isolates indicates that several geno-typic variants may coexist in these populations(7, 11, 14), suggesting that genetic rearrange-ment may be common in baculoviruses.We were interested in studying the functional

organization of the baculovirus genome. Someof these studies take advantage of specific mu-tants of AcMNPV which are now available. Agreat deal of information from several labs hasbeen generated on the polyhedrin protein syn-thesized in AcMNPV-infected cells. This pro-tein makes up the matrix of the intranuclearcrystal in which the virus particles are foundoccluded and is the major late translation prod-uct in infected cells (3). Polyhedron morphologymutants have been isolated and preliminarilycharacterized by electron microscopy and pro-tein analysis (2). We have begun to analyze thegenome of one of these mutants by physicallymapping the mutation site.

This paper describes the DNA restriction en-donuclease analysis of the morphology mutantdesignated M5 and of a spontaneous revertant of

M5, designated M5R. The M5 genome containsat least two regions affected by mutation. Puri-fied extracellular virions of M5 contain two sizeclasses of circular DNA, one of wild-typegenome length and another smaller DNA mole-cule. The DNA sequences contained withinthese two populations will be described. Theyindicate that a specific region of the MS genomehas been deleted in some of the DNA molecules.The genome of M5R contains only some of thegenetic changes of the parental mutant. Finally,it will be demonstrated that M5-infected cellssynthesize a smaller polyhedrin polypeptidethan wild-type-' or M5R-infected cells.

MATERIALS AND METHODSVirus and cels. AcMNPV and the morphology mu-

tant MS (2) were kindly supplied by Peter Faulkner(Queen's University). After plaque purification of allvirus preparations, working stocks of virus were pre-pared by passing the virus three to four times onSpodoptera frugiperda cell cultures. The infectioustissue culture fluid was used directly as inoculum forthe reported experiments. The cells were propagatedas previously described (3).

purificatio of AcMNPV and AcMNPV DNA. Non-occluded virus was purified from infected cell culturefluid by centrifugation as previously described (3).DNA was purified from the nonoccluded virus aspreviously described (14).

Restiction eadonuceases. BamHI, EcoRI, HindIll,SstI, and SmaI were purchased from either BoehringerMannheim Corp. (Dorval, Quebec) or Bethesda Re-search Laboratories (Rockville, Md.). Protocols sup-

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plied with the restriction enzymes were followed forthe cleavage of viral DNA. Enzyme incubations werecarried out at 37°C for 2 to 3 h with excess enzyme toproduce limit digestion. The reactions were stoppedby the addition of 1/10 volume of a solution (7 M urea,50% sucrose, 0.1 mM EDTA, 0.1% bromophenolblue). In some cases, the fragments were end labeledby "filling in" for 15 min, using 1 U of Klenow DNApolymerase (Boehringer Mannheim) and 0.5 jCi ofeach 32P-nucleotide (Amersham Corp., ArlingtonHeights, Ill.) per ,ug of total DNA before adding thestopping solution.Agarose gel electrophoresis. DNA restriction frag-

ments were routinely separated by electrophoresisthrough 0.8% agarose (type V; Sigma Chemical Co.,St. Louis, Mo.) in 0.1 M Tris-hydrochloride (pH 9.0)-0.077 M H3BO3-2.5 mM EDTA (TEB buffer) at 2 V/cm for 16 h and then stained with 1 ,ug of ethidiumbromide per ml. To purify individual fragments ofDNA, long gel slices containing specific fragmentswere individually placed on the inside wall of a plastictest tube and frozen at -60°C for 2 h. When the tubeswere then thawed, most of the liquid from the gelslices could easily be removed from the bottom of thetube. The DNA-containing buffer was brought to 0.2M sodium acetate and then precipitated with 2 vol-umes of 100%o ethanol. After precipitation at -20°C,the DNA was recovered by centrifugation. Recoveryrates were usually above 60%o. The purified DNA wasthen suspended in TEB and rerun through 0.6% low-melting-temperature agarose (Sea-Plaque agarose;Marine Colloids, Rockland. Maine). The ethidiumbromide-stained fragments were then cut out of the geland stored at -20°C. These fragments were digestedwith a second restriction enzyme by melting the DNAfragment-containing gel slice at 70°C for 3 min andthen incubating liquid samples at 37°C with the appro-priate enzyme buffer and restriction enzyme. To maxi-mize the digestion of the DNA, 0.1% bovine serumalbumin was included in all of the reaction mixtures(9). After 2 h, stopping buffer was added, and thesamples were run on 0.8% agarose gels.Mapping the DNA fragments. Purified whole viral

DNA and purified restriction fragments were cleavedwith various restriction enzymes. The resulting frag-ments were separated by electrophoresis through 0.8%agarose gels and were detected by blotting by theSouthern technique (13), as previously described (14).Preincubation and hybridization of the nitrocellulosefilters (Schleicher & Schuell, Keene, N.J.) were car-ried out by the rapid method of Wahl et al. (17). Thedried nitrocellulose filters were placed in sealableplastic bags and preincubated for 4 h at 42°C in 50%oformamide-5x SSC (SSC is 0.15 M NaCI, 0.015 Msodium citrate)-5x Denhardt reagent (5)-0.05 M sodi-um phosphate (pH 6.5)-300 ,ug of heat-denaturedsalmon sperm DNA per ml. Subsequently, the filterswere incubated at 42°C for 20 h in 50% formamide-5 xSSC-1 x Denhardt reagent-10% sodium dextran sul-fate-100 ,g of heat-denatured salmon sperm DNA per

ml-AcMNPV DNA which was 32p labeled by nicktranslation (14) to a specific activity of 1 x 108 to 2 x10' cpm/,ugg After removing the 32p probe, the filterswere washed with three changes of 2x SSC-0.1%sodium dodecyl sulfate (SDS) at room temperaturefollowed by three changes of 0.1 x SSC-0.1% SDS at50°C. The filters were dried at 80°C and autoradio-

graphed with either Kodak XR-5, Kodak Ortho-G, orKodak XAR-5 film.

Polyacrylamide gel electrophoresis. To analyze totalintracellular polypeptides, infected S. frugiperda cellswere pulse-labeled with 25 ,uCi of [35S]methionine perml at various times after infection. Samples were thenprepared and electrophoresed in polyacrylamide gels,as previously described (3). The following methylated"4C-protein mixture (Amersham) was used as molecu-lar weight markers: myosin (200,000), phosphorylase b(100,000 and 92,500), bovine serum albumin (69,000),ovalbumin (46,000), carbonic anhydrase (30,000), andlysozyme (14,300).

Electron microscopy. Purified virions and purifiedDNA were prepared for visualization by electronmicroscopy as previously described (3, 14).

RESULTS

The AcMNPV polyhedron morphology mu-tant M5 synthesizes single, large, cubic polyhe-dra in the nuclei of infected cells (2). Theseinfections can therefore be easily distinguishedfrom wild-type-infected cells which synthesizemany small polyhedra per infected cell. Sincevirtually no virions are occluded into the M5polyhedron, all of the viral DNA in this reportwas isolated from purified extracellular virus.DNA restriction endonuclease fragment pat-

terns. DNA prepared from purified extracellularwild-type and morphology mutant viruses (M5)was cleaved with the restriction endonucleasesEcoRI, BamHI, HindIII, SmaI, and SstI, andthe resulting fragments were separated on ag-arose gels. The fragment patterns are shown inFig. 1, where individual fragments are letteredsequentially by increased mobility through thegel. DNA fragments which comigrated wereidentified by their increased intensity of stainingwith ethidium bromide and their increased radio-activity as seen on autoradiograms of 32P-end-labeled fragments (data not shown). End label-ing of DNA restriction fragments also aided inidentifying the total number of restriction frag-ments produced by each restriction enzyme.The molecular weights of the fragments wereestimated by comparing their mobility in agarosegels to the HindIII fragments of XDNA and theHaeIII fragments of 4X174 DNA.EcoRI produced 24 fragments (A to X),

BamHI produced 7 fragments (A to G), HindIllproduced 24 fragments (A to X), SmaI produced4 fragments (A to D), SstI produced 9 fragments(A to I). Some of the smallest EcoRI and HindIlIfragments migrated off the gel in Fig. 1.The M5 restriction patterns, although very

similar to those of the wild type, were character-ized by several differences. (i) M5 EcoRI frag-ment I was larger by approximately 400 basepairs (bp) than wild-type EcoRI fragment I. (ii)M5 Hindlll fragment F was larger than wild-type HindIII fragment F also by approximately

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FIG. 1. Cleavage of AcNPV DNA with restriction endonucleases. DNA was isolated from purifiedextracellular virus of (1) wild type, (2) M5, and (3) M5R and digested with the indicated restrictionendonucleases. Samples (1 Rg) were separated on 0.8% agarose gels, as described in the text, and then stainedwith 1 ,ug of ethidium bromide per ml. The various fragments are labeled alphabetically according to decreasingsize. Small EcoRI and HindlIl fragments have migrated off the gels.

400 bp. (iii) There was an additional HindIlIfragment in M5 migrating slightly faster than theAB fragments. (iv) Wild-type BamHI fragment Fwas absent in M5. (v) Cleavage of M5 DNA withSmaI resulted in the appearance of five frag-ments, in contrast to the four fragments pro-duced when the wild type was cleaved with thisenzyme. (vi) M5 SstI fragment G was 400 bplarger than the wild type fragment G.

In addition to these very obvious changes inthe genome of M5, several more subtle alter-ations were observed. Many EcoRI and HindIIIfragments of M5 DNA appeared to be present inless than molar ratios. Specifically, EcoRI frag-ments I, J, K, M, N, 0, T, U, V, and the Hindlllfragments D/E, F, I, J, L, M, N, 0, R, T, U, V,W/X were present in submolar ratios comparedwith the other fragments. M5 SmaI-A and SmaI-D also appeared to be present in submolar ratioswhen compared with fragments B and C as wellas the extra SmaI fragment Al migrating be-tween A and B. In addition, M5 SmaI fragmentD appeared to migrate slightly slower than didwild-type SmaI fragment D.

Obviously, many changes from the wild typehave occurred in the genome of M5. Its veryunusual phenotype and genotype implied that itmight be a very useful mutant to aid in the studyof the organization of the AcMNPV genome.Mapping the mutation sites. The restriction

fragment patterns of wild-type DNA closely

resembled those of the AcMNPV E2 DNA (12),for which physical maps have been derived. Wehave recently produced similar maps for ourwild-type strain of AcMNPV (4). The physicalmapping of the M5 mutation site was investigat-ed to locate the region(s) on the genome poten-tially affecting the synthesis and morphogenesisof polyhedrin. As can be seen in Fig. 1, M5BamHI fragment B appeared to be slightly largerthan the wild type or M5R BamHI B fragments,implying that the BamHI site between BamHI-Fand BamHI-B had been lost in M5 (see Fig. 7).This was confirmed by purifying the EcoRI Ifragments of the wild type and M5 and redigest-ing them with another restriction enzyme. Thepurified EcoRI fragments were cut with eitherBamHI or SmaI, then blotted and probed with32P-labeled wild-type DNA. EcoRI fragment Iwas contaminated with other EcoRI fragments,particularly Eco-B, -C, and -D. However, thisdid not interfere with the subsequent analysis.When wild-type EcoRI fragment I was digestedwith BamHI, BamHI fragment F (labeled c) wasgenerated along with fragments correspondingto part ofBamHI fragment C (labeled d) and partof BamHI fragment B (labeled b) containedwithin EcoRI fragment I. However, with M5EcoRI fragment I digested with BamHI, BamHIfragment F and partial BamHI fragment B weremissing. Instead, there was only a larger frag-ment (Fig. 2, lane 6) which corresponded to a

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1 2 34 56 78 9

A-

B.-_ _

a

b o

D- _

E-- _

F- ,. C

dG- d

FIG. 2. Cleavage of purified EcoRI fralBamHI and SmaI. Analysis was by thmethod. EcoRI fragment I of the wild t!were purified and digested with eitherSmaI. The resulting fragments were s5agarose gels, blotted onto nitrocelluloseprobed with 32P-labeled wild-type DNA, zin the text. Autoradiographs were obtaineOrtho-G film. (1) 0.25 jig of wild-type DN;BamHI; (2) 0.025 Fg of wild-type DNBamHI; (3) purified wild-type EcoRI-I; (4)EcoRI-I, (5) purified wild-type EcoRI-:BamHI; (6) purified MS EcoRI-I, cut withpurified wild-type EcoRI-I, cut with SmiaIMS EcoRI-I, cut with SmaI; (9) 0.025 ptgDNA, cut with EcoRI; (10) 0.25 ,xg of wildcut with EcoRl. (a) Partial product,BamHI-F; (b) part ofBamHI-B; (c) BamHof BamHI-C.

partial digest product BamHI-F plBamHI-B seen in the wild type (labthe case of M5, BamHI-F plus Ban400 bp larger than the correspondingfragment. These results indicated thaiBamHI site at 3.0 between BamBamHI-B which was missing in MS. Ithe increase in size of MS EcoRI I

corresponded to the increase in siBamHI-F plus a partial BamiHI-B,that the insertion was located betwe4.5 map units.

lO Information could also be deduced from theSmaI digestions of EcoRI fragment I: (i) therewas no cleavage with SmaI of EcoRI fragment Ifrom either the wild type or M5; (ii) there wascleavage of the contaminating EcoRI-C andEcoRI-D in both the wild type and M5, but the

_-A generated fragment patterns were similar for the_I_6C two viruses, implying that the extra SmaI site in-E M5 was not located within either EcoRI-C orFGH EcoRI-D. These results satisfactorily explained

5^ - ! the disappearance ofBamHI fragment F and theincrease in size of EcoRI fragment I in M5.

- K The results of digesting M5 DNA with SstIindicated that a second mutation site was locat-

LM ed within SstI fragment G (Fig. 1). This fragmentshowed an increase in size of about 400 bp. Allof the other restriction enzymes used in this

-N study produced either very large fragments fromthis region, or produced several fragments verysimilar in size so that it was not possible to see

-PQ the effect of this second insertion with theseenzymes. The only exception was SmaI, whereSmaI fragment D of M5 was slightly larger than

S the wild-type fragment (Fig. 1).These results revealed that there were two

widely separated insertions of about 400 bp each-UV in the M5 genome, but they did not explain other

results, including the presence of extra SmaIand HindIII fragments.

gment I with Digestion of purified SmaI fragments. To deter-ie ISouther mine the origin of the extra DNA apparentlyypeSandheM present in M5, all of the SmaI fragments fromBamHl or the wild type and M5 were isolated by two

eparated on consecutive electrophoresis purification steps.filters, and All of the purified fragments appeared as single

as described bands on blots of uncut material, indicating thatd on Kodak they were not cross-contaminated with the other{A, cut with fragments. In addition, M5 SmaI fragment Al[A cut with did hybridize to the wild-type DNA probe, indi-purified M5 cating that it did contain at least some viral DNABarnH. (t7) sequences. These purified fragments were then(8) purified cut with either EcoRI or HindIII, the resulting

of wild-type fragments were blotted onto nitrocellulose fil-l-type DNA, ters, and the filters were probed with 32P-labeledgamHI-B + wild-type DNA.I1-F; (d) part Wild-type and M5 SmaI fragments B, C, and

D produced all of the expected EcoRI andHindlIl fragments (data not shown). These di-

us part of gestions also revealed the presence of a second,ieled a). In very small DNA fragment, EcoRI-X, identical iniHI-B was size to EcoRI fragment W. These two fragmentswild-type have not been previously mapped, but one maps

t it was the within SmaI fragment C, probably near 60.0,tHI-F and whereas the other maps somewhere within SmaIn addition, fragment A, probably near EcoRI fragment S atfragment I 87.8 (4). The insert in M5 EcoRI fragment I wasize of M5 observed in digestions of SmaI fragment A (Fig.indicating 3), and the second insert within EcoRI fragmenten 0.0 and C was observed in digestions of SmaI fragment

D (data not shown).

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AcMNPV MORPHOLOGY MUTANT DNA 813

3_ ISa a Go -..-^.,

T, K, J, and A were all left intact, indicating thatnone of these fragments contained an extraSmaI restriction site.

Since fragments U, V, and F were also under-UP " <- represented in EcoRI digests of whole M5 DNA,

these results suggested that a continuous stretch;--- of DNA containing the EcoRI fragments part of

C, U, V, F, N, M,T, K,J, A, 0, R, andpartofIhad been deleted in some of the M5 genomes.This deletion would remove the SmaI site at 39.1and create a new SmaI fragment (SmaI-Al),consisting of sequences from the 84.1 to the 0.0to 3.0 region of SmaI-A joined to sequencesfrom the 43.4 to 49.4 region of SmaI fragment D.This mutated DNA molecule would also containan aberrant HindIII fragment (HindIII-Bl), con-sisting of sequences from 96.3 to 0.0 to 3.0 ofHindlIl fragment F joined to sequences from

1 2 3

FIG. 3. Cleavage of purified SamI fragments withEcoRI. Analysis was by the Southern method. TheSmaI fragments of the wild type, M5, and M5R werepurified and digested with EcoRI. The resulting frag-ments were separated on agarose gels, blotted ontonitrocellulose filters, and probed with 32P-labeled wild-type DNA, as described in the text. (1) Wild type; (2)M5; (3) M5R; (4) Sma fragment Al of M5. (A) purifiedSma-A and Sma-Al digested with EcoRI; (B) shorterexposure of (A); (M) 0.05 ,ug of wild-type DNA cutwith EcoRI. Terminal fragments are indicated by <.

When SmaI fragment Al from M5 was cutwith EcoRI, it was found to contain a few of thesame EcoRI fragments found only in SmaI frag-ment A. Specifically, those EcoRI fragmentsmapping at the left-hand end ofSmaI fragment A(84.1 to 100) were seen, the EcoRI part of H, S,X, P, and B. However, all of the EcoRI frag-ments mapping to the right of EcoRI fragment B(0.0 to 38.1) were not observed. A single frag-ment of 10.1 kb (labeled IC) appeared to formthe right end of this Al fragment. The EcoRIfragments which were missing from the right endof the SmaI fragment A region were exactlythose which appeared to be underrepresented inthe EcoRI digest of whole M5 DNA.Double digests of total M5 DNA with SmaI

and EcoRI produced very similar patterns tosimilarly digested wild-type DNA, exceptingone extra fragment of 10.1 kb in M5 (Fig. 4). Inthese digests, M5 EcoRl fragments U, V, N, M,

FIG. 4. Cleavage of AcNPV DNA with EcoRI andSmaI. DNA purified from extracellular virus wasdoubly digested with restriction endonucleases EcoRIand SmaI and analyzed as described in the legend toFig. 1. (1) Wild type, (2) M5, and (3) M5R. The EcoRIfragments not cut by SmaI have been labeled alphabet-ically, whereas the extra M5 fragment has been labeled10.1.

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wt7 8 9

M56 7 8

- B

-

.moa

KI _"NS L-

4.,

-TU-V/wX

FIG. 5. Neutral sucrose gradient analysis of viralDNA. Purified virions were treated with 0.5 ,ug ofproteinase K-0.1% SDS-0.1% sarcosine for 1 h at37°C. The total extract was then layered on top oflinear 5 to 20% sucrose gradients in 10 mM Tris-hydrochloride (pH 7.5)-i mM EDTA-0.1 M NaCl andcentrifuged for 2.5 h at 45,000 rpm in an SW60 rotor.The gradients were then fractionated, and each frac-tion was ethanol precipitated. A sample of each pre-cipitate was digested with HindIll, electrophoresed,blotted, and then probed with 32P-labeled wild-typeDNA. Autoradiograms of the wild-type fractions 7, 8,and 9 and the M5 fractions 6, 7, and 8 are shown. Dotsto the right of the M5 fraction 8 track indicate theHindlll fragments which are missing in this fraction.

43.4 to 53.3 of HindIII fragment A. Similar typesof hybrid fragments would, of course, be seenwith all restriction enzymes. However, the mo-

bilities of these fragments were similar to those ofnormal fragments, and their appearance was

masked in the digestions of total MS DNA withthese restriction enzymes.

Separation of two sizes of M5 DNA. The re-

striction enzyme analysis ofDNA prepared frompurified extracellular M5 virions suggested thatthey contained two populations of DNA. At-tempts were made to separate these two DNApopulations. Purified wild-type and M5 virusparticles were treated with proteinase K andSDS. The total extracts were then sedimentedthrough 5 to 20% neutral sucrose gradients.Each fraction was precipitated with ethanol;

then a portion of each precipitated fraction wasdigested with HindlIl. The resulting restrictionfragments were separated on agarose gels, blot-ted onto nitrocellulose paper, and then probedwith 32P-labeled wild-type DNA. Figure 5 showsthe results obtained with the peak fractions ofwild-type and M5 DNA. The peak of wild-typeDNA was found in fraction 7, exhibiting a nor-mal HindIll fragment pattern. The peak of M5DNA, found in fraction 6, also exhibited anormal MS HindIII fragment pattern. However,MS DNA in fractions 7 and 8, sedimentingslower than normal, appeared to be missingcertain HindlIl fragments. Specifically, HindIIIfragments A, I, U, 0, E, R, M, L, J, X, D, N, T,V, and F were missing. These fragments comefrom a continuous stretch of DNA mapping from96.3 through 0.0 to 53.3. This DNA populationwas approximately 60% of the size of the wild-type DNA.The presence of two size classes of circular

M5 DNA, one of 100% and one of about 58% ofthe wild-type length, was confirmed by electronmicroscopy (Fig. 6).

Revertant of M5. A single spontaneous rever-tant of M5, designated M5R, was isolated as aplaque from the stock of MS on the basis ofpolyhedron morphology. Instead of a single,large polyhedron, MSR produced polyhedraclosely resembling the wild-type phenotype. Thefrequency of reversion is not known. The fre-quency must be very low since only one plaqueshowing wild-type polyhedra has been observedduring the experiments with MS over the past 3years. Based on endpoint 50% tissue cultureinfective dose titrations, the reversion frequencywas below the detectable limit of 3.3 x 10-5 (2).DNA was purified from M5R extracellular

virus and analyzed with a series of restrictionenzymes (Fig. 1). Some of the fragment patternsofMSR were similar to that of MS. For example,EcoRI fragment I and HindIII fragment F werethe same in MS and M5R. However, the extraHindIII-Bl and SmaI-Al of M5 were absent.The BamHI and SmaI fragment patterns ofMSRwere similar to the wild-type pattern. Apparent-ly, M5R had lost the small insertion near 45.6 to48.6 and had regained the BamHI site at 3.0.These data suggested that the mutation caus-

ing the increase in length of EcoRI fragment Iand the mutation causing the loss of the BamHIsite in MS could be differentiated and wereprobably affecting separate sequences withinEcoRI fragment I.

Polypeptides in cells infected with wild type,M5, and M5R. The polyhedrin polypeptide canbe detected in wild-type-infected cells by 10 to12 h postinfection (3), and its synthesis contin-ues at a very high rate until very late times afterinfection. The synthesis of this polypeptide in

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AcMNPV MORPHOLOGY MUTANT DNA 815

dP/

& ^ , S ' . U ..

FIG. 6. Electron micrograph of purified M5 DNA. DNA was purified from extracellular M5 virions andspread for examination in the electron microscope. The circular DNA molecules of different lengths are shown.Measurements of 20 such molecules indicated that the smaller circles were 58% of the length of the larger circles.

M5- and MSR-infected cells was studied bypulse-labeling infected cells for 1 h at varioustimes after infection and then analyzing thewhole cell extracts on SDS-polyacrylamide gels(Fig. 8).The polypeptide synthesis patterns of the wild

type, M5, and M5R were all very similar. Theonly obvious difference among them was thealtered mobility of the M5 polyhedrin polypep-tide (2). The polyhedrin polypeptide of both thewild type and MSR had a molecular weight of34,000, whereas that of M5 had a molecularweight of 33,000.

DISCUSSIONA morphology mutant of AcMNPV, designat-

ed M5 (2), which synthesizes a single, large,cubic crystal in infected cells, was investigatedby restriction endonuclease analysis of the puri-fied viral DNA. A complicated set of alterationshas occurred in the M5 genome, resulting inDNA fragment patterns which, although verysimilar to the wild-type patterns, can easily bedistinguished from them. These alterations havebeen physically mapped on the M5 genome.

Digesting purified EcoRI fragment I withBamHI indicated that the BamHI site at 3.0 wasaltered by mutation. In addition, the data re-vealed that an insertion of DNA of about 400base pairs had occurred within EcoRI fragment Ibetween 0.0 and 5.9 on the map. This insert was

also observed in HindlIl fragment F. Since theright end of Hindlll fragment F maps at 3.3 (4),this places the insertion site between 0.0 and 3.3.However, this site did not correlate with theincrease in size of SmaI fragment D of M5,which maps between 38.1 and 49.4. Digestion ofM5 DNA with SstI confirmed that there was aninsertion in SstI fragment G, again of approxi-mately 400 base pairs, mapping between 45.6and 48.6.The presence of these two insertion sites in

the M5 genome did not explain the presence ofsubmolar amounts of some DNA restrictionfragments, easily seen with EcoRI and HindIII,or the presence of extra DNA restriction frag-ments seen with HindIII and SmaI. By correlat-ing the intensity of ethidium bromide staining tothe relative quantity of a particular DNA frag-ment, it was determined that a continuous se-quence of DNA, including EcoRI fragments N,M, T, K, J, A, 0, R, and I was present in asubmolar ratio compared with sequences repre-sented by the other EcoRI fragments. The sameregion of the genome was found to be underrep-resented, as well, in HindlIl fragments I, U, 0,E, R, M, L, J, X, D, N, T, V, and F. An obviousexplanation of these results would be that thestock of M5 contained two or more variants. Ithas already been shown that uncloned polyhe-drosis virus preparations may contain severaldifferent genotypic variants which can be isolat-ed by plaque purification (7, 11, 14). However,

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I

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AB E C F GH D AB AB

139 225 39.6 45.6 48.6 61.862.4 89.9

A D C B AI I I I

38.1 49.4 64.8 83.7

F C A EGD B

3.0 4.5 11.3 78.9 82.3 88.081.5

D F H E LM A B G I J K CI I I I 11 I I I III1.9 13.1 19.1 24.1 32.2 34.8 57.6 75.7 81.6 864 W.7

33.9 89.0

D G OI J K F E H A C L M B NI II III I III II

8.0 13.4 8&5 21.2 23.5 30.1 38.2 42.2 62.3 75.7 779 97.0 99.014.7 80.1

F VTN D X J LMR E OU I a C WH S A K QP G FIII I 11 I I II I if I 11 II I i I I I

3.3 4.8 142 18.4 22.2 N33 33.8 377 53.3 62.0 66.9 85.1 8. 96840 62 14.7 20.4 23.6 33.0 62.6 68.2 872 90.4

Sst

Sma I

Bom Hi

Xho I

Pst I

Hind III

I ROEco RII 8I

70

A J K T M N F VU CI I II I I III193 25.0 292 33.1 3.0 41.9 43.4

30.2 426

G W D Q L EI I . I I I

52.9 59.7 68.3 72.860.1 69.8

H SXPI LI l

798 86.6 88.1877 89.6

B

100.0

% I I I I I I I I IGenome O t0 20 30 40 50 60 70 80 90 100

I I I I I I I I I I I I I IKbp o 10 20 30 40 50 60 70 80 90 100 110 120 130

AcNPV , HR3FIG. 7. Physical map of AcNPV HR3 DNA. The diagram shows the map positions of various restriction

endonuclease fragments of HR3 DNA, the parental wild-type strain of M5 and M5R (4).

extensive plaque purifications of M5 have failedto remove the submolar bands from the M5DNA preparations.Another explanation would be that more than

one nucleocapsid was enveloped as the virusparticles budded out through the cytoplasmicmembrane, generating a heterogeneous popula-tion of extracellular virions, some of which weresingly enveloped, whereas others were multiplyenveloped. The multiply enveloped particlescould contain two or more virions with differinggenomes. However, electron microscopic ex-amination of M5 extracellular virus preparationsrevealed only singly enveloped nucleocapsids(data not shown).A third explanation would be specific hetero-

geneity in the population of DNA moleculesisolated from purified M5 virions. The presenceof extra DNA fragments generated by cuttingM5 DNA with SmaI or HindlIl suggested thatperhaps the M5 genome was much larger thanthe wild type. However, because the DNA frag-ment patterns generated by cutting M5 DNAwith EcoRI or SstI were extremely similar quali-tatively to the wild type, it was likely that theextra SmaI and HindIlI fragments resulted fromthe deletion of a specific region of the M5genome in some DNA molecules. This deletionwould result in two phenomena. The DNA re-striction fragments in the genome within thedeletion region would be present in relatively

fewer copy numbers than DNA restriction frag-ments outside the deletion region since only aportion of the genomes would carry a completeset of DNA sequences. Secondly, sequenceswhich overlapped the two ends of the deletionwould be fused, generating new restriction frag-ments not seen in normal length DNA. Thus,there could be two types of DNA molecules inM5; one would consist ofgenome-length circularDNA, the second would consist of a shorter,circular DNA, having a large deletion of viralsequences, spanning the region between the twopreviously mapped insertion sites at 0.0 to 3.0and 46.5 to 48.6.Evidence for this model was derived by cleav-

ing individual SmaI fragments purified fromwild-type and M5 DNA with either EcoRI orHindIII. SmaI fragments A, B, C, and D of thewild type generated the expected EcoRI frag-ments and also indicated that EcoRI-W mappedwithin SmaI fragment C, whereas a previouslyundetected EcoRI fragment X mapped withinSmaI fragment A. These fragments have nowbeen precisely mapped (4). SmaI fragments A,B, C, and D of M5 generated the same EcoRIfragments as did the wild type, except for theincrease in length of EcoRI-I and EcoRI-C.The extra M5 SmaI fragment, Al, generated

fragments the size of the EcoRI part of H, S, X,P, B, and a new fragment of about 10.1 kb. TheEcoRI part of fragment H, S, X, P, and B forms

J. VIROL.816 CARSTENS

AcMNPV MORPHOLOGY MUTANT DNA 817

WT M5 WT M5R

4 8 12 16 202428322 ""I 7r.--. . .. ,'4.,

E., Mam

I1 14 8 12 16 20 24 28 32 24 4 8 12 16 20 24 28 321

-92 .8;.- L .

4Uia0t; w0ii X4

e;-46o Mg u _S^34

*30.

x :.--S

-14.3-

FIG. 8. Autoradiogram of SDS-polyacrylamide gel electrophoresis of infected cell extracts. S. frugiperdacells, infected with the wild type (WT), M5, and M5R were pulse-labeled for 1 h with [32S]methionine at theindicated times after infection. The whole cell extracts were analyzed by gel electrophoresis. The polyhedrinpolypeptide was apparent by 16 h postinfection and continued to be synthesized up to 32 h postinfection. The M5polyhedrin polypeptide had an apparent molecular weight of 33,000, whereas the wild-type and M5R polyhedrinpolypeptides had molecular weights of 34,000.

a continuous stretch ofDNA from 84.1 to 100.0/0.0 of SmaI fragment A. Since EcoRI-I andBamHI-C purified from M5 did not contain aSmaI site, the right-hand end of the M5 SmaIfragment Al must be located farther to the rightthan 11.3 on the physical map. There are manyEcoRI sites to the right of the EcoRI site at 0.0.However, only one other EcoRI fragment wasseen in SmaI fragment Al, the 10.1-kb fragment.These results suggested that a portion of thesequences, most likely including the SmaI site at38.1 and the EcoRI site at 5.9, had been deletedin M5 DNA to produce the Al fragment. Sincethe 10.1-kb fragment was the only new fragmentseen in SmaI plus EcoRI double digests of M5DNA, the extra SmaI fragment was not generat-ed by a new SmaI recognition site, but rather bya rearrangement in the viral DNA, resulting insequences contained within the region of 0.0 to3.0 being joined to sequences within the 45.6 to48.6 region.Two different size classes of M5 virion DNA

could be separated by neutral sucrose gradientcentrifugation. One population of M5 DNA sedi-mented like wild-type DNA, whereas a secondsmaller population sedimented slower than wild-type DNA. When the HindIII restriction frag-ments of these two DNA populations were sepa-rated on agarose gels, it was apparent that the

smaller DNA was generated by deleting astretch of DNA from 0.0 to 3.0 to 45.6 to 48.6.This deletion resulted in a genome which wasapproximately 58% of the size of the wild-typegenome. These results were confirmed by elec-tron microscopy of purified M5 virion DNAwhere two size classes of circular DNA wereseen.

This kind of genetic rearrangement is similarto the formation of defective particles observedwith other DNA viruses (1, 10). However, in thecase of M5, the mutant was not derived by serialpassage but rather by mutagenization of thewild-type stock. Nitrosoguanidine seems to in-duce mutations at the growing fork of replicatingDNA, resulting in gaps which may be filled byerror-prone repair (6). Of course, it is possiblethat the mutation did occur before mutagenesisalthough we have never seen this phenotype innonmutagenized virus stocks. It is possible thatthe synthesis of two closed circular DNA mole-cules of different sizes is a result of aberrant M5DNA replication, which generates these twoforms during every round of DNA replication.This is supported by evidence which indicatesthat the DNA restriction fragment patterns ofserially passaged M5 (up to 16 passages to date)remain qualitatively and quantitatively similar tothe fragment patterns of the original plaqued

VOL. 43, 1982

818 CARSTENS

stock. The small insertions may also function astransposable elements and, in this way, generatethe two size classe7s of circular DNA at a highfrequency. Virtually nothing is known about themechanism of DNA replication in baculovirus,but M5 may prove to be useful in studying thisaspect of AcMNPV.The revertant of M5, M5R, appears to have

resulted from a very rare mutational eventwhere an alteration in the M5 genome has gener-ated a new genome capable of coding for a full-length polyhedrin polypeptide. This wild-type-length polypeptide is able to assemble intostructures phenotypically identical to the wild-type polyhedron, and in this case, the normalpolyhedron of M5R acted as a suitable visualmarker. The fact that the revertant genomeretains the 400-bp DNA insertion in the 0.0 to3.0 region indicates that M5R is a true revertant.The M5R genome differs in two respects from

M5. First, it has lost the 400-bp DNA insert inthe 45.6 to 48.6 region. Secondly, it has regainedthe BamHI site at 3.0. Either of these regionsmay be critical for the phenotypic expression ofpolyhedra in infected cells, but presumablythere is only one site corresponding to thepolyhedrin gene which could affect the size ofthe polyhedrin polypeptide. It is possible thatthe polyhedrin mRNA results from a splicingevent where the region between 3.0 and 45.6represents a very large intron. However, recentdata indicate that the polyhedrin gene mapswithin BamHI fragment F (16; Rohel, Cochran,and Faulkner, personal communication). Thesedata suggest that the alterations in the 0.0 to 3.0region of the M5 genome affect the size of thepolypeptide translated from this region. Howev-er, the mutation affecting the synthesis of poly-hedrin may not be related to the phenotypicexpression of the polyhedron morphology sinceat least two distinct regions of M5 are mutated.Other regions of the M5 genome possibly affect-ing polyhedron morphology may also be mutat-ed, but these were not detected by the presentrestriction endonuclease analysis. Experimentsare currently under way to determine the originof the inserted sequences in M5 and to study thetranscription products of these mutated genomesequences.

ACKNOWLEDGMENTSI thank Nancy Butterill and Margaret Hough for technical

assistance, Dorothy Agnew for preparation of media, and P.Faulkner for gifts of cells and viruses.

This work was supported by a grant from the MedicalResearch Council of Canada.

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Evolutionary variants of simian virus 40: characterizationof cloned complementing variants. Virology 66:36-52.

2. Brown, M., P. Faulkner, M. A. Cochran, and K. L.Chung. 1980. Characterization of two morphology mu-tants of Autographa californica nuclear polyhedrosis vi-rus with large cuboidal inclusion bodies. J. Gen. Virol.50:309-316.

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10. Rixon, F. J., and T. Ben-Porat. 1979. Structural evolutionof the DNA of pseudorabies-defective viral particles.Virology 97:151-163.

11. Smith, G. E., and M. D. Summers. 1978. Analysis ofbaculovirus genomes with restriction endonucleases. Vi-rology 89:517-529.

12. Smith, G. E., and M. D. Summers. 1979. Restriction mapsof five Autographa californica MNPV variants, Tricho-plusia ni MNPV, and Galleria mellonella MNPV DNAswith endonucleases SmaI, KpnI, BamHI, SacI, XhoI, andEcoRI. J. Virol. 30:828-838.

13. Southern, E. M. 1975. Detection of specific sequencesamong DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98:503-517.

14. Tjia, S., E. B. Carstens, and W. Doerfier. 1979. Infectionof Spodoptera frugiperda cells with Autographa califor-nica nuclear polyhedrosis virus. II. The viral DNA andthe kinetics of its replication. Virology 99:399-409.

15. VIak, J. M. 1980. Mapping of BamHI and SmaI DNArestriction sites on the genome of the nuclear polyhedrosisvirus of the alfalfa looper, Autographa californica. J.Invert. Pathol. 36:409-414.

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J. VIROL.