INFECTION AND IMMUNITY, Sept. 1976, p. 783-792Copyright © 1976 American Society for Microbiology

Vol. 14, No. 13Printed in U.S.A.

Rhesus Monkey Kidney Cells Persistently Infected withSimian Virus 40: Production of Defective Interfering Virus

and Acquisition of the Transformed PhenotypeLEONARD C. NORKIN

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002

Received for publication 7 May 1976

Monolayer cultures of LLC-MK, rhesus monkey kidney cells became persist-ently infected with simian virus 40 (SV40) when infected at a multiplicity ofinfection of 100 plaque-forming units/cell. A stable carrier state developedcharacterized by extensive viral proliferation without obvious cytopathic effectother than the slow growth of these cultures. By 11 weeks all cells produced theSV40 T antigen. In contrast, less than 5% ofthe cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuouscultivation in antiserum resulted in a temporary cure of uncloned cultures.When virus did eventually reappear in the "cured" cultures the titers remainedlow. The virus produced by the carrier culture was defective at both 31 and 37°C,and it interfered with the growth of standard SV40 during mixed infection ofCV-1 green monkey kidney cells. All of the interfering activity in carrier culturehomogenates could be sedimented by centrifugation at 109,000 x g for 3 h. Thesecultures were completely susceptible to vesicular stomatitis virus. Extensiveviral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrierculture virus. Carrier culture homogenates are only slightly less cytopathic toCV-1 cells than standard SV40. The carrier cultures express several properties ofSV40 transformation.

The interaction of simian virus 40 (SV40)with rhesus monkey kidney cells is unusual inseveral respects. Most striking, perhaps, is theslow killing of these cells by SV40. Infectedrhesus kidney cells are killed much moreslowly than infected green monkey kidney(GMK) cells despite the fact that the 72-h viralyields per infected cell are comparable on eachcell type (16). This is surprising since the pro-duction of deoxyribonucleic acid (DNA) virusesis generally correlated with cell killing. Theinteraction of SV40 with rhesus kidney cells isalso characterized by an initially resistant cellfraction (16). In addition, the plating efficiencyof SV40 is about 25-fold lower on susceptiblerhesus kidney cells than on several lines ofGMK cells (16).

It is shown here that a persistent infection isestablished when rhesus kidney cell culturesare infected with SV40. These cultures arecharacterized by extensive viral proliferationwithout obvious cytopathic effects. Althoughall of the cells eventually produce the SV40 Tantigen, infection is perpetuated in only asmall fraction of susceptible cells. A componentof the virus produced by these carrier culturesis defective and able to interfere with the

growth of standard SV40. In addition, the car-rier cultures express several properties of SV40transformation.

MATERIALS AND METHODS

Cell cultures. The LLC-MK2 line of rhesus kidneycells and the CV-1 line of GMK cells were obtainedfrom the American Type Culture Collection. A-31BALB/3T3 mouse cells were obtained from H. L.Ozer. Cells were cultivated in Dulbecco modifiedEagle medium (DME, GIBCO), containing 10% fetalcalf serum (Flow Laboratories and GIBCO), in ahumidified 5% CO2 atmosphere.

Virus. Small-plaque SV40 (strain 777) was pre-pared for infection as previously described (16). Theheat-resistant strain of vesicular stomatitis virus(Indiana serotype) was kindly provided by Philip I.Marcus.

Antisera and immunofluorescence techniques.Anti-SV40 horse serum with a homologous titer of1:640 versus 500 mean tissue culture infective dosesof SV40 strain 911 was obtained from Flow Labora-tories. Incubation of 107 plaque-forming units(PFU) of SV40 strain 777 per ml with an equal vol-ume of a 50-fold dilution of this antisera for 1 h at37°C results in a 104-fold decrease in titer.

SV40 T or V antigen-producing cells were assayedby indirect immunofluorescence as previously de-scribed (16).

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Plaque assays. SV40 was titered by plaque assay

on CV-1 monolayers as previously described (16).Assays for infectious centers. Infected monolay-

ers were washed five times with Hanks balancedsalt solution (HBSS), and the remaining virus was

neutralized for 1 h at 37°C with SV40 antiserumdiluted 1:50 in HBSS. Monolayers were then washedfive more times in HBSS, suspended with 0.025%trypsin, diluted in DME medium containing 10%fetal calf serum, and plated as infectious centersonto CV-1 monolayers in 35-mm petri dishes fromwhich the overlay medium was removed. The plateswere immediately overlayed with 0.5 ml of DMEmedium containing 0.09% agar (Difco), 10% fetalcalf serum, and 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (Sigma ChemicalCo.). Ten minutes later, 2.5 ml more of the sameoverlay was added to each plate. Assays were incu-bated at 37°C in a humidified 5% CO2 atmosphereand stained on day 7 with the above overlay (minusthe serum) containing 0.009% neutral red.To insure that all extracellular virus was re-

moved by these procedures, A-31 cells were infectedwith SV40 at a multiplicity of infection (MOI) of 100PFU/cell. After an adsorption period of 2 h at 37°C,these samples were treated as described above.

Viable cell count. Attached cells were suspendedwith 0.025% trypsin and added to the originalgrowth medium containing detached cells. Trypanblue (GIBCO) dissolved in HBSS was added to a

final concentration of 0.08%. After 5 min, a total cellcount and a stained cell count were made by using a

hemocytometer.Cell cloning. Cell suspensions were examined mi-

croscopically to confirm the absence of cell clumps.Cells were then plated in Falcon micro test plates at0.1 cells per well. Any wells containing more thanone cell, as revealed by microscopy examination 24 hlater, were eliminated from further consideration.Assay for transformation. Monolayer cultures

were suspended with trypsin, washed, serially di-luted in DME medium containing either 2.5 or 10%fetal calf serum, and plated at several concentra-tions ranging from 1.3 x 105 to 1.6 x 103 cells per 60-mm plastic dish (Nunc) in DME medium containingeither 2.5 or 10% fetal calf serum. Plates were incu-bated at 37°C. The medium was changed on day 6.On day 9, the medium was removed and the cellswere washed with HBSS, fixed with 95% ethanol for10 min, air dried, stained with Giemsa (Fisher),washed with distilled water, and dried.

Viral DNA. Cultures were labeled with [3H]thy-midine (5 ,Ci/ml, 50 ,uCi/mmol, New England Nu-clear Corp.) from 24 to 32 h after infection. Theradioactive medium was then removed and the cellswere washed twice and incubated with fresh me-dium for 2 h to exhaust the intracellular pool ofradioactive thymidine and thymidine phosphates(7). Viral DNA was then extracted from cells by themethod of Hirt (7) and counted in a Packard Tri-Carb liquid scintillation counter.

RESULTS

Establishment of the persistent infection.Subconfluent monolayer cultures of LLC-MK2

cells were infected with SV40 at an input multi-plicity of 100 PFU/cell. About half of the cellswere initially infected as indicated by indirectimmunofluorescent staining for the SV40 T andV antigens at 48 h postinfection. The resistantcell fraction does not result from a heteroge-neous cell population. Instead, resistant cellsoccur at random in the population and resist-ance is transiently expressed (16).The viral yields after the first 72 h of infec-

tion were about 103 PFU per infected cell asindicated by titration of both infected centersand crude cell homogenates. Nevertheless, theinfected rhesus kidney cells were killed slowlyby SV40. By 5 days postinfection, only 17% ofthe initially infected cells were unable to ex-clude trypan blue (16). By 9 days postinfection,only 25% of the cells were unable to exclude thedye. In contrast, infected CV-1 cell cultures arecompletely stainable by 4 days under these con-ditions.As seen in Table 1, 28 days elapsed before the

infected LLC-MK, cultures reached confluencyand were passaged. Although growth of theinfected cultures was markedly inhibited dur-ing this time, there was no apparent cytopath-ology. Both infected and control cultures had asimilar appearance, and the frequency of try-pan blue-stainable cells was always less than10% after the 2nd week. Large amounts of viruswere produced at all times after infection. By 77days after infection, 100% of the cells expressedSV40 genes as indicated by immunofluorescentstaining for the SV40 T antigen (Fig. 1). In

TABLE 1. SV40 production by persistently infectedLLC-MK2 cells

Time Virus yieldCell pas- after in- Treatment at indi- (PFU/cul-sage no." fection cated time ture)b

(days)0 0 Infect, MOI = 100 1 x 1080 7 Wash, FCc 8 x 1070 14 Wash, FC 4 x 1070 21 Wash, FC 6 x 1070 28 Passed, 1/2d 6 x 1071 35 Passed, 1/2 2 x 1082 42 Passed, 1/2 4 x 1083 49 Passed, 1/2 2 x 1084 56 Wash, FC 1 x 1084 63 Passed, 1/2 4 x 1085 70 Passed, 1/2 4 x 1086 77 -

a Cell passage numbers after initial infection of the cul-tures.

b Virus yield per culture grown on 35-mm petri dishes. At7, 14, and 21 days the cells were harvested into the media,frozen and thawed, and sonically treated, and combinedcells plus media were titered. At subsequent times only themedia were titered.

c FC, Fluid changed.d Fractions indicate the fraction of the cell population

used to initiate a subculture.

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I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.i"N_. -.......

FIG. 1. T antigen staining (left) and V antigen staining (right) of carrier culture cells after 11 weeks ofpersistent infection. Both plates were photographed at the same magnification. The perinuclear and cytoplas-mic staining seen in the plate on the right was also observed in uninfected control cells and is nonspecific.

contrast, less than 5% of the cells produced Vantigen (Fig. 1).

Characterization of the persistent infectionas a carrier culture. The low frequency of Vantigen-producing cells in the persistently in-

fected LLC-MK2 cultures strongly suggestedthat these are carrier cultures, as opposed tosteady-state infections in which all cells contin-uously produce virus. This was confirmed bythe recovery of virus-free clonal isolates in thepresence of SV40 antisera. Six clones were ob-tained in this way after 56 days of persistentinfection. All clones subsequently failed to pro-duce virus, even after 45 days of cocultivationwith CV-1 cells. All of the virus-free clonalisolates were as susceptible to reinfection bySV40 as normal LLC-MK2 cells. This was

shown by titration of infectious centers andviral yields (data not shown).Temporary cure of the carrier state with

antiserum. After 101 days of persistent infec-tion (12 passages), samples of the carrier cul-ture were grown continuously for 22 days in thepresence of SV40 antiserum (final titer in me-

dium was 1:13 versus 500 mean tissue cultureinfective doses of SV40) with medium changesevery other day. At the end of this 22-day pe-

riod there were no infectious centers among 105cells of the carrier culture. Furthermore, no

virus was found in cell homogenates preparedby freezing and thawing followed by sonic treat-ment. Parallel samples of the carrier culturethat were maintained in medium containing

matched preserum contained about 10' infec-tious centers among 105 cells.Eleven days after removal of the antiserum,

the treated cultures were still not producingvirus, as indicated by assays for infectious cen-ters and for virus in cell homogenates and ex-tracellular fluids. However, by 35 days after theremoval of the antiserum, the treated culturesproduced 100 PFU in a period of 7 days. Incomparison, the untreated cultures produced107 PFU in the same period. By 49 days afterthe removal of the antiserum, the treated cul-ture produced 105 PFU in 7 days. In compari-son, the untreated culture produced 2 x 107PFU in the same period.Even after 41 days of growth in medium con-

taining SV40 antiserum, virus reappeared inthe carrier cultures after the cessation of treat-ment. In this instance, the virus that reap-peared produced microplaques on monolayersof CV-1 cells. The microplaque-producing viruswas neutralized by SV40 antiserum at the samerate as standard virus.During the time that the treated cultures

were not producing virus they were as suscepti-ble to reinfection by SV40 as normal LLC-MK2cultures as indicated by titration of infectiouscenters and viral yields (data not shown).

All the cells of the cured cultures produced Tantigen. Therefore, T antigen production inthis system is not dependent on the presence ofinfectious particles.

Interference within the carrier culture.

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Carrier cultures have been described in whichcells that are genetically susceptible to the car-

ried virus are made temporarily refractory byinterfering factors within the culture, such as

interferon or defective viruses (i.e., 6). Thesefactors are also capable of interfering with thegrowth of standard virus, thereby altering thecourse of infection.The following observations suggested that in-

terference acts to limit SV40 growth in thepersistently infected LLC-MK2 cultures. Lessthan 5% of the carrier culture cells producevirus as indicated by indirect immunofluores-cent staining for the SV40 V antigen (Fig. 1).This is probably not explained by a selection fornonsusceptible cells since carrier cultures thathave been cured by growth in antiserum are as

susceptible to infection by SV40 as normalLLC-MK2 cultures, with about 50% of the cellsbecoming infected.Comparative titration of infectious centers

and intracellular virus at a number of timesafter the establishment of the persistent infec-tion indicated that there were only of the orderof 10 PFU of SV40, on the average, in virus-producing cells. A similar analysis of acutelyinfected LLC-MK., cells showed that there were

of the order of 102 and 103 PFU per virus-pro-ducing cell at 48 and 72 h, respectively (16).These results denote that virus production incarrier culture cells is slow and/or that virusparticles are released almost as quickly as theyare produced. It is likely that viral growth isslower in carrier cells than in control cells be-cause the 72-h virus yields per producing cellare also 10-fold lower in carrier cultures than incontrol cultures (Table 2).

Results in Table 2 show that interferencefactors inhibit the SV40 yield per producing cellwhen the carrier cultures are superinfected atan MOI of 5 PFU/cell, but not when they are

superinfected at an MOI of 100 PFU/cell. Thisis seen by comparing the 72-h viral yields per

infected carrier culture cell with the yields perinfected normal LLC-MK, cell. Because there isno increase in the number of infectious centerswhen the carrier culture is superinfected at thehigher MOI, all susceptible cells are probablyproducing virus before superinfection. Thenumber of infectious centers produced when thenormal LLC-MK2 cells are infected with SV40reflect the low plating efficiency and resistantcell fraction characteristic of this virus-cell in-teraction (16).

Results in Table 3 show that when a sampleof a homogenate from the carrier culture isadded to a standard virus inoculum, the yield ofinfectious virus is reduced by about 96% on CV-1 cells. The interference factor is not interferonsince it can be completely sedimented by cen-trifugation at 109,000 x g for 3 h. Furthermore,the carrier cultures are fully susceptible to di-rect challenge with vesicular stomatitis virus.Following infection with vesicular stomatitisvirus (MOI = 2), the 16-h vesicular stomatitis

TABLE 2. Susceptibility ofLLC-MK2 carrier culturesto superinfection with standard SV40 after 19 weeks

(19 passages) of persistent infection

72-h viralInfectious yields (PFU/Cells MOIcenters" infectious

center)b

LLC-MK2 Pl' 0 104 3 x 102LLC-MK2 PI 5 9 x 10:1 3 x 102LLC-MK2 PI 100 104 3 x 10:3LLC-MK, 5 6 x 10:1 3 x 10:LLC-MK2 100 5 x 10-1 2 x 10:1

Infectious centers were titered as described inMaterials and Methods. Cells were plated 18 h be-fore infection at 105 cells per culture.

b Cells, plus extracellular fluid from parallel in-fected cultures, were harvested at 72 h, frozen andthawed three times, and further disrupted by sonictreatment. Viral yields were titered by plaque assayon CV-1 monolayers.

' LLC-MK2 PI, Persistently infected cultures.

TABLE 3. Infection ofCV-1 cells with standard SV40 and with virus from LLC-MK2 carrier cultures after 25weeks (2 7 passages) ofpersistent infection

Single infection Mixed infectionRatio mixed/

Yieldb Yield single (WT)Inoculumn (PFU/culture) Inoculum (PFU/culture)

WT 2.9 x 107PI 3.2 x 105 WT + PI 1.3 x 106 0.04PI (pellet) 1.6 x 105 WT + PI (pellet) 1.1 x 106 0.04PI (supernatant) 9.6 x 106 WT + PI (supernatant) 3.2 X 107 1.1

CV-1 cells were infected with WT virus at an MOI of 1 PFU/cell and with carrier culture virus (PI) at anMOI of 2 PFU/cell. A sample of the carrier culture containing cells plus overlay was frozen and thawed threetimes and sedimented at 109,000 x g for 3 h. Infection with the resuspended pellet (PI, pellet) was at an MOIof 2 PFU/cell. Infection with the undiluted supernatant (PI, supernatant) was at an MOI of 0.1 PFU/cell.

b Viral yields of 72 h were titered by plaque assay on CV-1 monolayers.

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virus yields on the carrier cultures and on nor-mal LLC-MK2 cells were each 3 x 107 PFU/mlwhen assayed on LLC-MK2 monolayers.The yield of infectious virus was fairly high

when CV-1 cells were infected with the super-natant prepared from the carrier culture ho-mogenate (Table 3). A likely explanation isthat enough of the interfering factor was sedi-mented to permit the residual nondefective vi-rus in the supernatant to grow to high titer.

Defective-interfering particles of SV40 are

most likely the interfering factor. This is sup-

ported by the results in Table 4, which showthat the viral yield is much lower after infec-tion of CV-1 cells with a carrier culture inocu-lum than after infection with standard SV40 ata comparable MOI.A possible interpretation of the data in Table

4 is that the carrier culture virus may be tem-perature sensitive in part. This would be con-sistent with our finding that the capacity of thecarrier culture lysates to interfere with thegrowth of standard SV40 is lower at 31 than at37°C, provided that viral temperature sensitiv-ity is in part responsible for the interferencephenomenon. When CV-1 cells were mixedlyinfected with carrier culture virus and standardvirus and subsequently incubated at 37'C, thenthe ratio of the yield from the mixed infection tothe yield from a single wild-type infection was

0.04 (Table 3). In comparison, when the infectedcultures were incubated for 6 days at 31°C(after adsorption for 2 h at 37°C), then the ratioofthe yield from the mixed infection to the yieldfrom a single wild-type infection was 0.28 (datanot shown).

Viral DNA synthesis in cells infected withcarrier culture virus. The data in Table 5show that the reduced yield of PFU in CV-1cultures infected with carrier culture virusalone, or in cultures mixedly infected withstandard SV40, is not due to a lack of synthesisof viral DNA. On the contrary, more viral DNAsynthesis occurred between 24 and 32 h in cul-

TABLE 4. Defectiveness ofSV40 from persistentlyinfected LLC-MK2 culturesa

Virus yield (output! Ratio (PI/WT)

Virus MOI input)

310C 370C 310C 370C

PI 1 7 5 0.07 0.005WT 2 102 103a CV-1 cells were infected with the same inocula

as in Table 3. After adsorption for 2 h at 37°C thecultures were incubated at the indicated tempera-tures. Cultures incubated at 31°C were harvested 6days after infection and cultures incubated at 37°Cwere harvested 3 days after infection.

TABLE 5. Viral yields and DNA synthesis in CV-1cultures infected with standard SV40 and with virus

from carrier cultures

Yieldb Viral DNAInoculuma (PFU/cul- Yield (cpm/cul-

ture) (mxdW) ture)c

WT 6.4 x 108 796PI 8.0 x 105 5214WT + PI 2.7 x 106 0.04 4541a CV-1 cells were infected with WT virus at an

MOI of 1 PFU/cell and/or with carrier culture virus(from a 23-week sample, passage 25) at an MOI of 2PFU/cell.

b Viral yields of 72 h were titered by plaque assayon CV-1 monolayers.

c Parallel cultures were labeled and viral DNAwas extracted selectively as described in Materialsand Methods. The counts per minute in the Hirtsupernatant of an uninfected control culture weresubtracted from the experimental data to give thevalues shown.

tures either mixedly infected or infected withcarrier culture virus alone than in cultures in-fected with standard virus alone. A likely ex-planation is that the carrier culture lysatescontain an excess of defective particles that arecapable of DNA replication.One cannot conclude from these experiments

that interference is at a stage subsequent toviral DNA replication because the portion ofthe pulse-labeled viral DNA that is wild typeunder interference conditions is not known. Atany rate, interference by the carrier culturevirus does not result from an overall cessationof viral DNA synthesis.Production of defective SV40 in serial undi-

luted passage on LLC-MK2 and CV-1 cells.Because SV40 infection of LLC-MK2 cells re-sults in persistent infection and production ofdefective virus and because viral defectivenessmay be important in the establishment andmaintenance of persistent infections by virusesthat are normally cytocidal, we compared thecapacities of LLC-MK2 and CV-1 cells to pro-duce defective SV40 during serial undilutedpassages. As seen in Fig. 2, serial undilutedpassages on both cell types result in defectivelysates. This phenomenon does not occur anyearlier on LLC-MK2 cells than on the CV-1cells. Lysates from the fourth undiluted pas-sage on the LLC-MK2 and CV-1 cells caused a95 and 97% reduction, respectively, in the virusyields when added to CV-1 cells co-infected withstandard virus (data not shown).

Cytopathogenicity of the carrier culture vi-rus. Carrier culture virus appears to be onlyslightly less cytopathic to CV-1 cells thanstandard SV40. CV-1 cells were infected at an

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87-

0

L ,.~~~~~~~~~~~~~LC-MK,5L1_

2 3 4 5VIRAL PASSAGES

FIG. 2. Viral yields in serial undiluted passagesof SV40 in LLC-MK2 and CV-1 cells. Cells were

initially infected at an input multiplicity of100 PFU/cell. Cells plus extracellular fluid were harvested 4days after each infection. Successive undiluted pas-

sages were made by infecting cells with 0.05 ml oflysates that had been frozen and thawed three timesand sonically treated before infection. Viral yieldswere titered by plaque assay on CV-1 monolayers.

MOI of 10 PFU/cell with either carrier culturevirus from week 11 of persistent infection or

with standard virus. The fractions of trypanblue-stainable cells at 48 h after infection withstandard virus and carrier culture virus were42 and 21%, respectively. The fractions of try-pan blue-stainable cells 72 h after infectionwith standard virus and carrier culture viruswere 78 and 60%, respectively. Cultures in-fected with either inoculum were completelykilled by 96 h. Even when infected at an MOI of1 PFU/cell, CV-1 cultures were completelykilled by carrier culture virus by day 6.

Neoplastic transformation of the carrierculture. As noted above, all the cells of thecarrier culture acquire the capacity to producethe SV40 T antigen (Fig. 1). At the same time,the carrier cultures express other phenotypicproperties of SV40 transformation. As seen inFig. 3, the carrier cultures achieve a greatercell density than the normal LLC-MK2 cul-tures, indicating that they are less susceptibleto density-dependent growth control. Further-more, the cells of the carrier culture have an

enhanced ability to grow and form clones whenplated at low cell density, perhaps indicating a

partial independence from the distance-depend-ent helper effect (2). In addition, the clonesproduced by the cells of the carrier culture are

denser and more heavily stained than those ofnormal LLC-MK2 cultures (Fig. 3 and 4).The cloning efficiencies of both the carrier

culture and the normal LLC-MK2 culture wereserum dependent. However, the carrier cultureachieved a greater cell density, had a markedlyhigher efficiency of cloning, and produced den-

ser clones in medium containing 2.5% serumthan the normal LLC-MK2 culture did in me-dium containing 10% serum.

Despite the expression of these properties ofthe transformed phenotype, the doubling timeof the carrier culture was about 60 h whentested several times between 28 and 112 days.In contrast, the doubling time of the normalLLC-MK2 cultures was about 30 h. This wasdetermined by counting the number of cellspresent per 60-mm plastic dish at intervalsafter plating at 2 x 101 cells per dish. Cells weresuspended with trypsin and added to the origi-nal growth media containing any detachedcells. They were then counted with a hemocy-tometer.

DISCUSSIONViral infections may result in complete cell

destruction or no noticeable cytopathic effect,with all intermediate outcomes possible. Themost difficult situation to explain is that of apopulation of apparently healthy cells persist-ently infected with a cytocidal virus. The inter-action of SV40 with LLC-MK2 cells is consid-ered in this context in the following discussion.

In a previous study we found that only abouthalf of a population of LLC-MK2 cells is ini-tially susceptible to SV40 infection (16). Studieswith clonal isolates of these cells indicated thatthis did not result from heterogeneity ofthe cellpopulation. In addition, the plating efficiency ofSV40 is about 25-fold lower on susceptible LLC-MK2 cells than on several lines of GMK cells(16). Furthermore, infected LLC-MK2 cells arekilled much more slowly than infected GMKcells, despite the fact that 72-h viral yields perinfected cell are equivalent on each cell type(16).The initially resistant cell fraction, the low

viral plating efficiency, and the slow cell kill-ing might all be important in the establishmentof this persistent infection without obvious cy-topathic effects. These factors might providetime for the production of interference factorsbefore the culture can be completely infectedand possibly destroyed. Although persistent in-fection of fully susceptible and rapidly killedGMK cells is also possible, it is preceded byextensive cell death (12). In that instance per-sistent infection is established by a small frac-tion of cells that unaccountably survive theinitial infection (12). We are presently trying tofind conditions that lead to the simultaneousinfection of all of the LLC-MK2 cells in culturein order to more critically evaluate the role ofthe initially resistant cell fraction.Interference by defective viruses or by inter-

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FIG. 3. Effect of persistent infection on colony formation by LLC-MK2 cells. The plates in the first andsecond columns were seeded with normal LLC-MK2 cells in DME medium supplemented with 10 and 2.5%fetal calfserum, respectively. The plates in the third and fourth columns were seeded with carrier culture cellsin DME medium supplemented with 10 and 2.5% fetal ca7fserum, respectively. The top row ofplates received1.3 x 105 cells per 60-mm petri dish, the second row received 4.3 x 104 cells, the third row received 1.4 x 104cells, the fourth row received 4.7 x 103 cells, and the fifth row received 1.6 x 103 cells. Carrier culture cellswere seeded after 11 weeks ofpersistent infection. All plates were fixed and stained 10 days after plating asdescribed in Materials and Methods.

feron has been implicated in a number of per- scribed above. This is not surprising in view ofsistent infections as the mechanism by which a the fact that similar rhesus kidney cell cul-carrier state is maintained (i.e., 6, 9). Defective tures, unknowingly contaminated with SV40,virus, rather than interferon, is responsible for were used for the preparation of poliovirus andthe interference in the carrier cultures de- adenovirus vaccines (22).

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%.E. ,'4 4,

*d' EDS***h 4.*41 j

*-**','-lb

4 4

04,

* 4.. lb*t

JW4

SW. t * (f_ ,*4

s~~~~~~~A-;'Wh '' ' *46'

i;** * *. 9,.,$?J4 '. 'b;* >*t

S .*U -'.<p''Q . *L *~~.*.^'*

* r4-**

*, *'**

1 * ~~4..9.

'"' S~~~~~1

FIG. 4. Photomicrographs showing the colony morphology ofcarrier culture cells and ofnormal LLC-MK2cells. The carrier culture colony (left) is from the plate in the third column, fifth row, ofFig. 3. The normalLLC-MK2 colony (right) is from the plate in the first column, third row, of Fig. 3. Each sample wasphotographed at the same magnification.

As noted above, a possible interpretation ofthe data in Table 4 is that the carrier culturevirus is in part temperature sensitive. Whileproof must await the characterization ofplaque-purified isolates, it should be noted thatthe interference phenomenon was more pro-nounced at 37 than at 31°C. The suggestion thatthe interference phenomenon may be in partrelated to viral temperature sensitivity is con-sistent with the results of a number of studiesthat show that viral temperature sensitivitymay be a factor in the persistence of normallyvirulent viruses, both in culture and in theintact organism (3-5, 8, 14, 19, 21).The presence of defective viruses per se is

probably not responsible for the slow death ofvirus-producing cells of the carrier culturesince carrier culture lysates containing theseparticles are quickly cytocidal to CV-1 cells ateither high or low MOI. The slow killing ofLLC-MK2 cells by SV40 appears to be an inher-ent feature of the virus-cell interaction in thiscase since cell killing is remarkably slow evenduring initial infection by standard SV40 (16).The defective particles may serve primarily

to limit the number of cells producing nondefec-tive virus and to limit the rate of production ofnondefective virus by virus-producing cells. Ifthe defective particles were to play a role in

limiting the fraction of virus-producing cells,then this would be important in maintainingthe carrier state, since virus-producing cells arenot likely to have long-term viability. That vi-rus-producing cells are not likely to have long-term viability is indicated by the observationthat the carrier cultures can be temporarilycured by cultivation in antiserum. It is likelythat the refractory cell fraction also continuesto play a role in limiting the number of virus-producing cells. It might be noted that at anymoment all ofthe susceptible cells ofthe carrierculture are virus producers, as indicated by as-says for infectious centers before and after su-perinfection of the carrier cultures.We do not know exactly how interference

diminishes so that new susceptible cells canemerge to perpetuate the infection. It is possi-ble that interference continues to be partiallyexpressed in some yielder cells because thesecells appear to produce virus at a slow rate.New producer cells are probably recruited fromthe population ofnonproducing, T antigen-posi-tive, transformed cells since all of the cells ofthe carrier culture produce T antigen after 11weeks.The slow rate of production of PFUs by the

producing cells of the carrier culture is proba-bly not related to the transformed phenotype of

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RHESUS MONKEY KIDNEY CELLS INFECTED BY SV40 791

these cells. This is indicated by the following.When cured carrier cultures are superinfected,the viral yields are equivalent to that obtainedfrom acutely infected control cells, despite thefact that all the cells ofthe cured cultures are Tantigen positive.Because the carrier cultures can be tempor-

arily cured by cultivation in SV40 antiserum,transfer of the virus through the medium isimportant in the maintenance of the carrierstate. The reappearance of virus in the carriercultures several weeks after the removal of an-tiserum probably results from the "sponta-neous" activation oflatent SV40 genomes. SV40genes are present in all cells of the carriercultures as indicated by immunofluorescentstaining for the SV40 T antigen. Despite thespontaneous reappearance ofvirus in cured cul-tures, transfer of virus through the medium isrequired for the continued production of highviral yields.The virus that reappears in the cured carrier

cultures is defective. This is indicated by thevery low titers in these cultures, despite thefact that the cured cultures are as susceptible toinfection by standard SV40 as normal LLC-MK2 cultures. In one instance, the virus thatreappeared produced only microplaques. Thisvirus might be similar to the virus rescued fromSV40-transformed human and hamster cells(10, 11).The persistently infected cultures described

above bear some resemblance to the lesions inthe human slow virus disease progressive mul-tifocal leukoencephalopathy (PML). Two sero-logically distinct agents have been recoveredfrom PML biopsies. One is very similar to labo-ratory strains of SV40 (20, 23), and the other isJC virus, which shares some antigens withSV40 (17, 18). SV40, or JC virus, grows andpersists in PML lesions, with virus in excess of109 virions per g of tissue (15). Furthermore,while the persistently infected LLC-MK2 cul-tures acquire the transformed phenotype, thetypical cytological lesions in the late phase ofPML are characterized by giant astrocytes thatresemble the malignant astrocytes of glioblas-tomas (24). Defective particles, characterizedby short genomes containing reiterated viralDNA sequences, may be associated with PML.These particles were observed in an SV40-likePML isolate that had been passaged threetimes in human fetal brain cells and one timein GMK cells (13). We do not yet know whethersimilar particles are present in our carrier cul-ture lysates. Although there are certainly im-portant differences between persistently in-fected rhesus kidney cells in culture and PMLin the human brain, the similarities suggest

that the carrier system may be a useful modelof this human slow virus disease.Knowledge of the underlying molecular

events in persistent virus infections and slowvirus diseases is still very limited (1). Thesestudies, involving a virus that is being inten-sively investigated at the molecular level,should yield important infornation about theseprocesses.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Serv-

ice research grant 5 R01 CA12948 from the National CancerInstitute, by a grant from the American Cancer Society,Massachusetts Division, Inc., and by a grant from the Re-search Council of the University of Massachusetts, Am-herst.

The excellent technical assistance of Cheryl Goguen isgratefully acknowledged.

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