Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence.

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0022-538X/87/082642-06$02.00/0

Virus Attenuation

and

Identification of Structural Proteins of

Vaccinia Virus That Are Selectively Modified during

Virus

Persistence

EDUARDO PAEZ,t SHATHA DALLO, AND MARIANO ESTEBAN*

Departments ofBiochemistry andMicrobiologyandImmunology, State UniversityofNew York,Health Science Center atBrooklyn, New York, New York 11203

Received29December1986/Accepted 1May 1987

Toinvestigate thegenetic stability of vacciniavirusDNA,wehave tested whetheralterations occurred in the polypeptide compositionof thiscomplexvirusduring persistentinfections. We found that variants isolatedat variouspassagesinFrienderythroleukemia cells persistently infectedwith vaccinia viruscontained,in addition to an8-megadalton (MDa) deletion onthe left terminusof theviralgenome,majoralterations in the sizes of three structuralproteins with molecularmassesof about39, 21, and 14 kDa. Alterations in isoelectricpoints werealsoobserved inproteins of48, 27,and 14 kDa. The 14-kDaproteinispartof the virusenvelope,and the variants increased thesizeof thisproteinfrom 0.5to3 kDawithincreasingpassage number. Alteration in size of the 14-kDaproteinisadominant trait sinceitappearedin the whole viruspopulationbypassage 48. With more passages, somevariantswerefoundtoincreaseordecreasethesizeofa39-kDacoreprotein byabout 2 kDa and todecrease the sizeofan envelopeprotein of21 kDa byabout 2 kDa. These three proteinswere immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteinswereprevented by continuoustreatmentofthepersistentlyinfected cultures with interferon.However, after interferonwasremoved,proteinmodificationsappearedwithincreasingpassagenumber. Generationof the 8-MDa deletion and alterations inthesize ofthe 14-kDaprotein correlated witha marked decreasein virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introducedin the vaccinia virusgenome that leadtoprotein alterations andtohighly attenuatedviruses.

Itis believed thatpoxviruses are wellconserved through evolution. With theexception of heterogeneityatthe endsof

the viral genome, aremarkable sequence conservation has

been observed withinthecentralregion ofthe genome(fora

review, see references 3 and 10). These previous studies were carried out by restriction enzyme analysis of viral genomes (9)obtained after afewcycles ofvirus multiplica-tion and with serially propagated viruses(10). Becauseofthe

cytocidal nature ofpoxviruses (1), most procedures used

would have selected for the virus population that grew

faster. Our aimwas toestablishpersistent infectionsto test the genetic stability of poxviruses. This method could

pro-vide geneticmarkers thatpredict with certainty the virulence or attenuation of these viruses. We have previously

de-scribedtwopersistently infected vaccinia virus-cell systems

from Friend erythroleukemia (FEL) cells. The first persist-entinfectionwasin theuntreatedcultures (11), whereas the second persistent infection was established in the presence

ofinterferon (IFN) (13).During virus persistence, a

charac-teristic8-megadalton (MDa) deletion is generated at the left terminus of the viral genome (11). By restriction enzyme analysis, no other changes except genetic alterations at the ends wereobserved during virus persistence. Because vac-cinia virus has a large DNA (185 kilobases) which encodes more than 100 structural polypeptides (5), it is likely that

genetic alterations other than deletions are also introduced

into the virus population during virus persistence. In this

investigation we havetested whether protein modifications are introduced in vaccinia virus when it is grown under

*Correspondingauthor.

tPresent address: Centrode Investigaciones Biologicas, 28006 Madrid, Spain.

stringentselective conditionsimposed byapersistent infec-tion in FEL cells and by IFN treatment. We have charac-terized representative virus mutants in the course of a

persistentvirus infection that lasted 2 years.

Structuralproteinmodifications in variants from untreated

persistently infected FEL cells and in variants from IFN-treated persistently infected FEL cells were examined by

one-and two-dimensionalgel electrophoresisof35S-labeled

proteins frompurified virions and by immunoblot analysis.

Unlabeledor[35S]methionine-labeled purifiedvaccinia virus orvariants weredisruptedat100°Cfor 3 min insamplebuffer

(62.5 mMTris hydrochloride [pH 6.8], 4% sodiumdodecyl sulfate [SDS], 10% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue). One-dimensional gel electrophoresis

was run on 12% gels as described elsewhere (12), and two-dimensional gel electrophoresis was carried out as

de-scribed previously (2). Isoelectric focusingwas done in the

first dimension withampholines (pH 5 to 7 and 3 to 10) and in the second dimensionwas doneby SDS-12%

polyacryl-amidegelelectrophoresis (PAGE). Thegels weredriedout and exposed to X-AR5 film for autoradiography. For

im-munoblotanalysis,40,ugofprotein from purified virions was

appliedper well and fractionated with SDS-12% polyacryl-amide gels. Theproteins were transferred to nitrocellulose and immunoblots were developed as described previously (16), except that 5% blotto (nonfat dry milk) was used

instead of fetal calf serum. Monoclonal antibodies against the14-kDa envelope protein of vaccinia virus wereprepared as described previously (16). Polyclonal antiserum was raised in rabbitsimmunizedwith either purified virus

enve-lopesorpurified virus cores.Viralenvelopes wereprepared from5mg ofpurified virus suspended in 1 ml bysonication (500 W for 10 s) in 50 mM Tris hydrochloride (pH 8.5)-10

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mM

MgC12-10

mM dithiothreitol-0.5% Triton X-100. After incubation at

37°C

for 30min,the extract was layered on top of a 36% sucrose cushion in the same buffer, and cores were pelleted by centrifugation at 20,000 x g for 60 min at

4°C.

The upper phase, containing the viral envelopes, was recov-ered and dialyzed extensively against phosphate-buffrecov-ered saline. Cores were resuspended in the same buffer and pelleted through a sucrose cushion. This process was re-peated three times. Cores were finally suspended in phos-phate-buffered saline. Rabbits were immunized by three intramuscular injections of purified viral envelopes or cores (100

,ug

of protein). The first injection was in Freund complete adjuvant, and the other two injections were admin-istered every 15 days but without adjuvant. One week later, rabbits were bled from the ear vein and serum samples were collected. The antibody levels remained high even 6 months after the last inoculation. When the pattern of35S-labeled

virion proteins was analyzed by one-dimensional SDS-PAGE (Fig. 1A), we observed changes in the sizes of proteins with molecular masses of about 39 (clones 65-16 and 101-14), 21 [clone (88)-24, not shown], and 14 [clones 53-8, 65-16, (74)-23, and 101-14] kDa. Two-dimensional gel elec-trophoretic analyses revealed additional information. For simplicity, only the results obtained with the wild-type virus and clone 101-14 are presented. Several proteins were found to change in mutant 101-14 compared with proteins of the

wild-type

virus. These proteins (Fig.1A, denoted by arrows in part WR) have molecular weights of about 64, 48, 39, 27, 24, and 14 kDa.

Next, we tested whether these modified proteins were immunogenic. Purified virus mutants were run on one- and two-dimensional SDS-PAGE gels, blotted on nitrocellulose

paper, and reacted with anti-vaccinia rabbit antiserum, and the antigenic viral proteins were identified by the immuno-peroxidase staining method (16). By inspection of the im-munoblot of proteins run on one-dimensional SDS-PAGE gels (Fig. 1B), it was clear that three proteins of molecular weights of about 39, 21, and 14 kDa changed in size in variants from persistently infected FEL cells. In some variants, the 39-kDa protein either gained (variants 65-16 and 65-17) or lost (variants 101-12 and 101-14) about 2 kDa, the 21-kDa protein lost about 2 kDa [clone (88)-24], and the 14-kDa protein gained from 0.5 to 3 kDa inall variants at or after 48 passages. These protein modifications were not observed in variants isolated from IFN-treated persistently infected FEL cells. Significantly, when IFN treatment was discontinued and virus persistence was maintained for vari-ous passages, variants emerged that had protein modifica-tions similar to those of variants from untreated persistently infected FELcells. Immunoblot analysis of proteins run in two-dimensional gel electrophoresis (Fig. 1B, lower panel) confirmed that the changes occurred in proteins that have an

immunogenic role.

To identify whether mutations occur within the same protein, we used monoclonal antibodies prepared against vaccinia virus (16). One such monoclonal antibody (mAbC3) was found to react against the modified 14-kDa protein, as revealed by immunoblot analysis (Fig. 1C); a breakdown product was also detected (16). The 14-kDa polypeptide is an envelope protein that must play an important role in virus-host cell interactions. It has been demonstrated that this protein causescellfusion and is involved in virus penetration and that monoclonal antibodies reactive against this protein have neutralizing activity (16, 17). In variants from untreated persistently infected FEL cells, the 14-kDa protein gained from 0.5 to 3 kDa with increasing passage numbers. In

contrast, no change oraminor increase in size was observed in variantsfrom IFN-treated persistently infected FEL cells. For example, at passage 48, the 14-kDa protein gained 1.5 kDa in size with variants from untreated persistently

in-fected FEL cells and only about 0.5 kDa in variants from

IFN-treated persistently infected FEL cells. After removal ofIFNandanadditional40passages in the absenceofIFN, variants were generated that had gained 1.5 to 2 kDa insize, whereas if IFN treatment was maintained, no additional changes were observed.

Next we examined thesevariants for a number of proper-ties relevantto virulence. These findings are summarized in Table 1. Wefoundthat variants from untreatedpersistently infected cellsorfrom persistently infected cultures afterIFN

was removed had the same deletion of about 8 MDa atthe left end of the viral genome as previously described (11). This deletion appeared at relatively early passages and was maintained with later passages. Significantly, continuous treatment of the persistently infected cultures with IFN

completely blocked the generation of deletions, even 2 years after the establishment of virus persistence. Another obser-vation was that variants isolated after later passages of untreated persistently infected FEL cells had greatly

re-duced plaque sizes compared with the wild-type virus or

with variants isolated at similar passages but from

IFN-treated persistently infected FEL cells. This reduction in plaque size was greater at later cell passages (clone 101-14) than that at passage 48 described previously (11). However, in IFN-treated cultures, the plaque size was only slighty reduced even at late cell passages. An enhanced cell fusion ability of the virus isolates was observed with increasing passage number, independently of the IFN treatment. For example, mutant 30-10 from untreated cultures and mutant

IFN 87-4 from IFN-treated cultures caused cellstofuse into large polykaryocytes, a cytopathic effect completely dif-ferentfromthatof the wild-type virus, which usually caused cells to round and clump together. However, virus isolates from late cell passages of untreated cultures lost this char-acteristic (clones 101-12 and 65-16). This effect may be due to reversion to the wild-type pheotype, because mutations occur within the same gene or within another gene(s) that blocks this cell fusion capacity. These mutants induced a

typical early cell rounding, with the exception of clonesIFN

87-4, 53-8, and (56)-14, which showed a delay of several hours in the appearance of cell rounding. The degrees of

infectivity and virulence of these variants were tested with culture cells and with experimental animals. Some of these mutants had greatly reduced infectivity and virulence. Sig-nificantly, a good correlation was established between the appearance of deletions at the left terminus of the viral genome and greatly reduced morbidity compared with that

of the wild-type virus. Mutant 14-3 contained an 8-MDa deletion at the left end of the genome, produced a normal plaque size, and had a 2-log reduction invirulence. With the appearance of the small plaque size (mutant 48-7), the virulence decreased by 3 logs,suggesting thattheplaque size may be another genetic markerresponsible for virus

atten-uation.

The particular relevance of this study is that we have identified specific protein modifications in vaccinia variants from untreated persistently infected FEL cells. These mod-ifications do not occur in variants from IFN-treated persis-tently infected FEL cells. Changes in size have been ob-served primarily in structural viralproteins of about 39, 21, and 14 kDa. These are immunogenic proteins (Fig. 1). We have also observed alterations in the isoelectric points of

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-FIG. 1. Alterations in the electrophoretic mobilities of several structural vaccinia virus proteinsduring virus persistence. Conditions for virus persistence in FEL cells, treatment with IFN, virus purification, and virus titration weredone asdescribed previously (11-13). (A) [35S]methionine-labeled purified viruses isolated at different passages from persistently infected cells run in one-dimensional (left) and two-dimensional (right) gel electrophoreses and analyzed by autoradiography. Numbersatthetopindicate variantsasdescribed inTable1. Abbreviations: WR, wild-type virus; SDS, sodium dodecyl sulfate; IEF, isoelectric focusing. (B) Alterations in immunogenic structural proteins of vaccinia virus mutants. Purified viruses run in one-dimensional (top) and two-dimensional(bottom) SDS-PAGE areshown. (C) Immunoblot of virus proteins reacted with monoclonal antibody mAbC3 specific for the 14-kDa envelope protein and visualized bythe immunoperoxidasestaining method. The low-molecular-weight product is thoughttobegeneratedby specific proteolytic cleavage. Numbers atthe top of thegels indicate variants asdescribed in Table1. Arrowspointtopolypeptides (39, 27,24,and 14kDa) whicharealtered in mutantviruses. The molecular masses (kDa) of some structuralpolypeptides of vaccinia virusareindicatedatboth sides.

TABLE 1. Propertiesofspontaneous vaccinia virus mutantsfrompersistently infectedFELcellsa

Reduced

Treatment Virus Deletion Plaque size Amt of cell infectivity LD50

variantb occurrence (mm)' fusiond incultured (PFUY

cells'

None WT No Normal (3) + No 106

5-3 No Normal (3) ++ No 106

14-1 Yes Normal (3) + + No

14-3 Yes Normal (3) + + No 108

30-1 Yes Normal (3) + + + + No 108

30-10 Yes Normal (3) + +++ No

48-7 Yes Small(1) ++ + + Yes 109

48-21 Yes Small(1) ++ + + Yes

101-12 Yes Verysmall (0.5) + Yes

101-14 Yes Verysmall (0.5) + Yes >109

IFN 48-42 No Normal(3) + + + No 107

48-47 No Medium(2) ++ + + No

87-1 No Medium(2) + + + + No

Noneg 53-7 Yes Small(1) + + Yes

53-8 Yes Small(1) + + Yes

65-16 Yes Very small(0.5) + Yes >109

65-17 Yes Very small(0.5) + Yes

IFNremovedh (56)-12 No Small (1) + + Yes i07

(56)-14 Yes Small(1) ++ Yes

(74)-22 Yes Small(1) ++++ Yes

(74)-23 Yes Small(1) ++++ Yes

(88)-24 No Small(1) ++++ Yes

aVariantswereisolated fromuntreatedandIFN-treatedpersistentlyinfectedFELcells.Theoccurrenceof deletionswasdeterminedbySouthernblots,as

describedpreviously (13),andtheplaquesizewasestimated in BSC-40 cells.

bThefirst number indicates the cell passage, and the secondnumberindicatestheclone. Relativetothenormalsize ofWT virus.

dScreenedat24hafter infectionwith 1 PFUofeachvariantpercell. +,clumpsof cells with littlecellfusion; ++,scatteredpolykaryocytes; + ++, many

polykaryocytes;++++,allcells fused intolargepolykaryocytes.

'Viralinfectivitywasmeasuredbytitrationvirusyieldforat24hafterinfectionwith 1 PFUof eachvariant percell;variantsthatproducedless than70% ofthe

wild-type virus yieldwereconsideredtohave reducedinfectivity.Evaluationofvirulencewasestimated inwhitefemaleBALB/cmice(CharlesRiverBreeding

Laboratories, Inc.)6 to 8weeksold afterintraperitonealinoculation(0.2ml)with differentdilutionsinphosphate-bufferedsaline of wildtypeand variants. fVirulencewasscored14daysafter virusinoculation(10micepergroup),and the dose that caused50%mortality (LD_%)wasestimated(21).

gVariants isolated fromindependentlyestablishedculturesof untreatedpersistentlyinfected FELcells.

hCultures were treated with IFN continuously during 14, 30, or45 passages; thereafter, IFN treatment was discontinued and cultures were passaged continuouslywithoutIFNuntil cellpassages56, 74,or88,respectively.

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TABLE 2. Alterations in sizes of structuralproteinsfrom spontaneous vaccinia virusmutants'

Finalsize(kDa)of virionprotein with

Treatment Variant of original size of:

WTvirus

14 kDa 21kDa 39 kDa

None 5-3 14 21 39

14-1 14 21 39

14-3 14 21 39

30-1 14.5 21 39

30-10 14.5 21 39

48-7 15.5 21 39

48-21 15.5 21 39

101-12 15.5 21 37

101-14 15.5 21 37

IFN 48-42 14.5 21 39

48-47 14.5 21 39

87-1 14.5 21 39

87-4 14.5 21 39

None 53-7 16.5 21 39

53-8 16.5 21 39

65-16 17.0 21 41

65-17 17.0 21 41

IFN removed (56)-12 16.5 21 39

(56)-14 16.5 21 39

(74)-22 15.5 21 39

(74)-23 15.5 21 39

(88)-24 15.5 19 39

a Alterations of the 14-, 21-, and 39-kDa virionproteinsweremeasuredby

one-andtwo-dimensional PAGE ofpurifiedvariants with vesicular stomatitis virusand vaccinia virusproteinsasmarkers. Theoriginandtreatmentofthe

variantsare asdescribed in Table1footnotes.

proteins of about 48, 27, and 14 kDa; other alterations included proteins of about 64 and 24 kDa which were

apparentlymissingin some mutants,possiblybecauseoneof

the proteins was altered in size and the mutated protein comigrated with theotherprotein. By examiningthe distri-bution of these proteins between the envelope and core componentsof the virus(16), wehave foundthatproteins of

24, 21, and 14kDaarepartof theenvelope,whereasproteins of39and 27 kDa arepartofthe core structureof vaccinia virus. It is significant that the most frequent protein

alter-ation occurs in the 14-kDa protein. Modifications in this

proteinareintroduced intheviralpopulation atpassage48. Thechangeconsists of anincreasein size from 0.5 to 3 kDa with increasing passage number of untreated persistently

infected FEL cells. In contrast, no change or a minor increase insize was observed in variants from IFN-treated

persistently infected FEL cells. Significantly, when IFN treatmentwasdiscontinuedandviruspersistence was

main-tained for various passages, variants emerged that have

14-kDa-protein modifications similar to those of variants

from untreated persistently infectedFEL cells. Thus, there is a cause-and-effect relationship between IFN treatment andprevention ofprotein modifications. It should be noted

thatthe8-MDadeletionis the first apparent mutational event

introduced in the virus population at relatively early pas-sages.Thereafter, protein modifications follow (Tables 1 and

2). How IFN blocks DNA sequence alterations is not known. This inhibition could result from IFN-mediated

changes in the topology of DNA, in the activity of DNA

replicating enzymes, orin other cellular factors involved in DNA metabolism (7). In fact, various effects of IFN at the DNA level have been described. These various effects

include decreased elongation (6), impaired homologous DNA recombination (14), and inhibition on stabilization or

integration of viral and cellular DNAs into animal cells (4, 15).

Inthisstudy wehave also defined a number ofbiological properties of vaccinia virus that are relevant to virulence. Compared with thewild-type virus or with variants isolated at similar passages but from IFN-treated persistently in-fected FELcells, variants isolated afterlong-termpassages of untreated persistently infected FEL cells have greatly reduced plaque sizes in cells in culture and reduced morbid-ity wheninoculated intraperitoneally into mice. The small-plaque-sizephenotypeand an 8-MDa deletionatthe left end of the viral DNA are two genetic determinants that correlate with a decreased virulence of vacciniavirus (Table 1). We have also observed that some of these variants produce

extensive cell fusion.Analysis of the cytopathic effect at late timespostinfection revealed an enhanced cell fusion ability of the virus isolates withincreasingpassagenumber, despite

the IFN treatment. Cell-cell fusion ability has been associ-ated with the production of nonglycosylated forms of the virushemagglutinin present on thesurface of virions(8, 20).

However, we have observed agoodcorrelation between the enhancedcell-cellfusionability and alterationsin the 14-kDa viralenvelopeprotein (Tables 1and 2). Infact, thisprotein

is involved in virus-induced cell-cell fusion at late times

postinfection, because the formation ofpolykaryocytes can beblockedcompletely by monoclonal antibodiesagainstthe 14-kDaprotein(17). Since some mutants isolatedatlate cell passages have lost theabilitytoformpolykaryocytes despite

altered 14-kDa proteins, it is possible that this viral mem-brane protein forms interactive complexes and that muta-tions in any one component of the complex may alter the structure andfunction ofthe entire complex. This process has been observed inherpesvirus (18, 19).

The significance of mutational changes in the structural

polypeptides of vacciniavirusduring persistent infectionsis that some of these changes might contribute to decreased virus pathogenesis and, in turn, provide specific genetic

markers relevant to virus attenuation. In addition, some of these changes may have contributed to strain variation of

poxvirus through evolution. In this regard, we have noted

thatamutated 41-kDa protein of vaccinia virus comigrates

and cross-reacts with amajor structural protein ofcowpox

virus.Itwillbeof interesttodeterminetheroleofthealtered

vacciniaviruspolypeptidesinvirusattenuationand whether

genetic diversionby members oftheorthopoxvirusgroup is related to any of the mutated proteins described in this report.

Thisinvestigation was supported by PublicHealth Servicegrant CA44262fromtheNational Institutes of Health andbygrantCCA 8510from theUSA-Spain Joint Committee for Scientific and Tech-nologicalCooperation. E.P.was arecipient ofafellowship fromthe ConsejoSuperiordeInvestigaciones Cientificas, Spain.

We thank VictoriaJimenez for her skilledtechnical assistance.

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