Persistent infections of green monkey kidney cells initiated with temperature-sensitive mutants of simian virus 40

VIROLOGY

107,

375-388

(1980)

Persistent Infections of Green Temperature-Sensitive

Monkey Mutants

LEONARD Department

qf’Microbio/ogy.

Cnirersity Accepted

Kidney Cells Initiated of Simian Virus 40

with

C. NORKIN qf’Massachusetts, July

Amherst.

Massachusetts

01003

PI, 1980

An early SV40 temperature-sensitive (ts) mutant, tsA.58, and a late mutant, tsBl1, each expressed homotypic interference in CV-1 cells coinfected with WT. Interference required the functional activities of the mutant genomes and resulted from a competitive interaction between the mutants and WT. Another late mutant, tsDlO1 did not display interference activity. Nevertheless, each of the ts mutants promoted the survival of CV-1 cells coinfected with WT at 37” and, consequently, facilitated the establishment ofpersistent infections. Each of the mutants was also more able than WT to establish persistent infections under conditions of single infection. Only a few percent of the cells in these persistent infections produced the SV40 T or V antigens and clonal isolates from these systems contained both antigen-producing and nonproducing cells. These systems were resistant to superinfection with WT SV40 but were completely susceptible to vesicular stomatitis virus. There was a rapid selection forts+ virus in the persistent infections initiated by mixed infection with the ts mutants and WT. The proportion of ts- virus also increased, although somewhat more slowly, in the systems initiated by infection with the mutants alone. Defective interfering (DI) particles vvere detected in some stocks by yield reduction assays. Many viral genomes in stocks displaying DI particle activity contained multiple BglI cleavage sites, indicating that they possess reiterations of the origin for DNA replication.

in the establishment and mainte 3 of persistent infections of African green monkey kidney (GMK) cells with SV40. While persistent SV40 infections of rhesus cells are readily established with barely any cytopathology (Norkin, 19’761, persistent infections of GMK cells are established only after the nearly complete destruction of the cell cultures (Margalith et al., 1969; Norkin, 197713). In the studies reported here we have examined three well-characterized ts mutants of SV40 (tsA58, tsBl1, and tsDlOl), representing three different mutant groups, for their capacities to modulate the acute infection in GMK cells coinfected with WT and to facilitate the establishment of persistent infections of those cells. Persistent infections of the CV-1 line of GMK cells were more readily established at 37” following mixed infection with each of the mutants plus WT than following infection with WT alone. Each of the mutants was also more able than WT to establish persistent infections under

INTRODUCTION

There has been considerable evidence to suggest that temperature-sensitive (ts) viral mutants might function in the establishment and/or maintenance of several RNA virus persistent infections, both in cell culture and in the whole animal (reviewed by Preble and Youngner, 1975). ts mutants might be expected to also play a role in persistent infections with DNA viruses. Thus, we have been interested in the possible role and action of ts mutants in persistent infections with simian virus 40 (SV40), particularly since late virus stocks from an SV40/rhesus monkey kidney cell carrier system display an impaired capacity to replicate at 41” (Norkin, 19’79a). Viral temperature sensitivity is not necessary to maintain the SV40lrhesus cell system because that persistent infection is stable following transfer to 33” (Norkin, 1979a). Nevertheless, it was of interest to determine what role ts mutants might play 375

00426822/80/160375-14$02.00/O Copyright 41i rights

0 1980 by Academc Press. Inc. of reprnductm in any form reserved.

376

LPNNARL)

(:. NORKIS

Zndirect immunojluorescence. SV40 T or V antigen-producing cells were assayed by indirect fluorescent antibody staining as previously described (Norkin and Ouellette, 1976). MATERIALS AND METHODS Viral DNA preparation. Viral DNA was selectively extracted from cells by the Cell culture. The CV-1 line of normal method of Hirt (1967) and concentrated with GMK cells and the LIC-MK, line of rhesus polyvinylpyrrolidone (PVP-40, SIGMA). monkey kidney cells were obtained from the Form I DNA was isolated by equilibrium American Type Culture Collection, Rockcentrifugation in CsCXethidium bromide, ville, Maryland. Cells were cultivated in followed by dialysis against 10 mJ4 Tris (pH Dulbecco modified Eagle medium (Grand 7.5), 1.0 m&f EDTA. Island Biological Co.) supplemented with Restriction endonuclease digestion ctnal10% fetal calf serum in a humidified 5% CO, ysis. Hind111 from Haemophilus injluenxa atmoshere. Cultures were free of mycoand EcoRI from Escherichia coli were obplasma as indicated by phase contrast tained from Boehringer Mannheim. BglI microscopy of Orcein-stained cultures from Bacillus globigii was obtained from (Fogh, 1973). Miles Laboratories. The reaction mixtures Virus. Wild-type SV40 (strain 777) was for either Hind111 or EcoRI contained 6 prepared for infection as previously demJ4 Tris-HCl (pH 7.5), 50 mil4 NaCl, 8 nuJ4 scribed (Norkin and Ouellette, 1976). SV40 MgC&, 6 nuJ4 P-mercaptoethanol, 5 pg ts mutants tsA58, tsBll, and tsDlO1 were BSA, 1 pg of DNA, and 3-6 units of enzyme kindly provided by Peter Tegtmeyer and in a total volume of 0.05 ml. The reaction Harvey Ozer. Stocks were prepared by inmixture for BglI contained 12 mM Trisfecting CV-1 cells at 33” at inputs of 0.1 HCl (pH 7.5), 6 mM MgC12, 6 mM P-mercapPFU/cell. Infections were harvested after toethanol, 1 pg of DNA, and about 3 units of one cycle of viral growth at 33” (5 days). The enzyme in a total volume of 0.05 ml. Incubaheat-resistant strain of vesicular stomatitis tions were carried out for 1 hr at 37”. One virus (VSV, Indiana serotype) was kindly unit of enzyme is defined as that amount provided by Philip Marcus. required to completely digest 1 pg of A DNA Interference assay. Interfering particle in 1 hr under the above conditions. The reactiters in ts mutant stocks and in stocks from tions were stopped by the addition of 10~1 of persistent infections were estimated from 5% SDS, 25% glycerol, and 0.1% bromyield reduction curves obtained by coinfect- phenol blue followed by heating at 65” for 10 ing CV-1 cells with a constant low multiplicmin. Polyacrylamide gel electrophoresis ity (0.1 PFU/cell) of WT and two-fold serial was carried out at a constant voltage of 100 dilutions of the test stocks (Bellett and V for 2.5 hr in a verticle 4% polyacrylamide Cooper, 1959; Marcus and Sekellick, 1974). slab gel (16 by 14 by 0.1 cm) in a buffer conAssuming that a single interfering particle taining 40 mil4 Tris base (pH 7.8), 20 mM is capable of inducing interference and that sodium acetate, and 2 mM EDTA. Agarose interference is an all-or-none phenomenon gel electrophoresis was carried out at a conon a per cell basis, it follows (according to stant voltage of 75 V for 5 hr in a horizontal the Poisson distribution) that each cell is 1.4% agarose slab gel (22 by 13 by 0.2 cm) in infected with one interfering particle on the a buffer containing 50 m&Z Tris base (pH average when the single infection WT virus 8.05), 20 mM sodium acetate, 2 mM EDTA, yield is reduced by 63%. Because it is incor- and 18 m&Y NaCl. Gels were stained by imrect to assume all-or-none interference in mersion in 500 ml of water containing 0.5 these assays (below and Norkin, 1979a, b), pg/ml of ethidium bromide for 25 min the actual interfering particle titers are and were illuminated with a model C-61 probably somewhat higher than the re- short wave uv lamp (Ultraviolet Products, ported estimates. Inc., San Gabriel, California) and photoconditions of single infection. teristics of these persistent also described.

Other characinfections are

PERSISTENT

INFECTION

graphed through’an A23 filter (Kodak) with Polaroid type 47 film. RESULTS

Homotyp,ic

Interference

by SV40 ts Mutants

Monolayer cultures of CV-1 cells were either singly infected with WT or coinfected with WT and each of the ts mutants. Results in Table 1 show that tsA58 and tsB11 are each able to suppress WT growth at 41”. However, the interference activity of tsB11, but not tsA58, is very much diminished at 33”. Although it is not clear from Table 1, tsBl1 is indeed able to interfere with WT at 33”, as shown by the yield reduction curves in Fig. 1. tsD101 did not display significant interference activity at either temperature. The infectious ts mutants were responsible for the interference activity in the tsA58 and tsBll stocks as indicated by the following. Those stocks were free of defective interfering (DI) particles, as indicated by Hind111 restriction analysis of their DNA (not shown). In addition, interference activity was inactivated by uv irradiation, indicating that it was not due to interferon, but TABLE

1

HOMOTYPIC INTERFERENCE ts MUTANTS OF SV40

Virus yield (PFUlml) 33

Inoculum” WT tsA58 t WT tsBl1 + WT tsDlO1 t W’T

4 3 2 2

x x x x

41” 106 lo” 106 106

2 x 106 5 x 104 105 2 x 10”

BY

Ratio of virus yields (ts + wT)/ mT -33” 41”

0.08 0.5 0.5

0.03 0.05 1

” CV-1 cells infected with WT at an input of 0.1 PFUicell and with the ts mutants at about 4 PFUicell as indicated. All inocula were adsorbed for 2 hr at 33”. The cultures were subsequently incubated at the indicated temperatures. Lysates of infections at 41 and 33” were harvested on Days 3 and 5, respectively, and were titered by plaque assay on CV-1 monolayers at 37”. None of the mutant stocks produced plaques under the conditions of assay.

WITH

SV40

ts MUTANTS

377

rather that it required the functional activities of the mutant viral genomes. Also, a 63% reduction of the WT viral yields occurred in yield reduction experiments (e.g., Fig. 1) when the input multiplicities of the ts mutants were about 1 PFU/cell, as predicted by the Poisson distribution (Bellett and Cooper, 1959). The negative exponential relationship between the dose of tsBl1 and the WT yields (Fig. 1) implies that a single tsB11 virion is capable of interference. Similar results were obtained with tsA58 (not shown). Interference results from a competitive interaction between the ts mutants and WT. This is shown by the effect on the WT yields of varying the input multiplicity of WT under conditions of coinfection with tsBl1 at a constant input multiplicity. For example, when CV-1 cells were coinfected with tsBl1 at an m.o.i. of 4 PFU/cell and with WT at inputs of 0.25,0.5, and 2 PFUicell, the respective 72-hr WT yields were 5 x lo”, lo”, and 4 x 1Oj PFUiml. The single infection WTyieldsrangedfrom 1.5 x lo6 to2 x lo6 PFU/ml. Interference is not expressed in an all-ornone manner on a per cell basis, as the following results show. Despite the fact that tsBl1 is unable to produce structures that react with antisera prepared against virions (Tegtmeyer et al., 1974), a similar fraction of V antigen-producing cells was observed in CV-1 cultures coinfected with tsBl1 and WT as in cultures infected with WT alone. For example, when CV-1 cells were infected at 41” with WT at an input of about 1 PFU cell, 30% (1541517) of the cells produced V antigen at 48 hr. Coinfection with WT and tsBll at respective inputs of 1 and 10 PFUi cell resulted in a similar fraction of V antigen-producing cells (1861509). Nevertheless, under the conditions of mixed infection with tsBl1 there was a loo-fold decrease in the WT yield. Persistent Infections of CV-1 Cells Established by Coiqfection with ts Mutants Plus WT

Monolayer cultures of CV-1 cells were infected with WT (0.1 PFU/cell) either alone

LEONARD

378

0

I I25

I 25

C. NORKIN

I

INPUT

I 50

OF tsB11

I

I 75

I

I 10.0

(PFU/cell)

FIG. 1. Effect on the WT yield of varying the input multiplicity of t&f I in coinfected cultures. (X-1 cells were coinfected with WT virus at a m.o.i. of 0.1 PFUicell and with twofold serial dilutions of tsBl1. Adsorption was for 2 hr at 33” and cultures were subsequently incubated at 33” (0) or 41” (0). Infections at 41 and 33” were harvested at days 3 and 5 postinfection, respectively. Wild-type virus yields were titered by plaque assay on CV-1 monolayers at 37”.

or in combination with the ts mutants at inputs of l-5 PFU/cell. Cultures were subsequently incubated at 37”. At weekly intervals all cultures were either washed and fluid changed or were passaged. Weekly viral yields are shown in Fig. 2. Cultures infected only with WT developed extensive cytopathic effects (CPE) during the first week. Nearly all cells were involved and by the end of week 2 no attached cells were evident. One clone did emerge during the third week, corresponding to 0.0005% survival of the initial cells (Table 2). This clone continually displayed CPE and it was not until Week ‘7 that it could be passaged. The culture continued to display CPE until Week 19 when the experiment was terminated. Only about half of the cells in cultures infected with tsA58 + Wt displayed CPE by the end of Week 1. Between Week 2 and

Week 19 (when the experiment was terminated) that infection was free of CPE. Many cells had detached by the end of Week 1 in cultures infected with tsBl1 + WT, although many healthy looking attached cells were present as well. CPE continued to develop in this infection such that by the end of Week 2 relatively few cells remained attached. By the end of Week 3 a total of 17 clones were present in the culture, corresponding to 0.01% survival of the initial cells. Thereafter, this infection was free of CPE until Week 9 when extensive crisis developed. The infection was again free of CPE by Week 11 and remained so for the duration of the experiment. Extensive CPE developed by the end of Week 1 in the cultures infected with tsDlO1 + WT and no attached cells were seen by Week 2. However, by the third week there were a total of six clones in this infection,

PERSISTENT 9

,

I

I

I

-tsA58+WT c--a

ts

Bil

INFECTION

+ WT

I

I

b.4

ts DlOl

---.

WT

WITH SV40 ts MUTANTS I

I

I

1

I

I

I

+ WT

8-O

FIG. 2. Viral yields in persistent infections of CV-1 cells initiated by coinfection with ts mutants and WT SV40. CV-1 cells were infected with tsA58 + WT, tsBl1 + WT, tsDlO1 + WT, and WT at respective input multiplicities of approximately 3,1, and 5 PFU/cell of the ts mutants and 0.1 PFU/cell of WT. Cultures were subsequently incubated at 37”. Viral yields were harvested at weekly intervals and were titered by plaque assay on CV-1 monolayers at 33”. Extensive CPE developed between Weeks 1 and2 in the cultures infected with tsBl1 + WT and tsDlO1 + WT (see text). These cultures had stabilized by Week 3. The WT persistent infection is from a single clone (out of 2 x lo5 cells) which survived the initial infection. It was first passaged at Week 7.

corresponding to 0.003% survival of the initially infected cells. This culture was free of CPE from Week 4 until Week 14 when some detached cells were observed over healthy looking monolayers. The culture was completely recovered by Week 17. It is evident from these results that the ts mutants, representing all three mutant groups, promote cell survival during the initial acute infection (Table 2) and, thereby, facilitate the establishment of persistent infections. The persistent infections initiated by coinfection with WT and either tsA58, tsBl1, or tsDlO1 (ts + WT PIs) will hereafter be referred to as tsA58 +WT PI, tsBl1 + WT PI, and tsD101 + WT PI, respectively. Persistent Infections of CV-1 Cells Established by Single Infection with ts Mutants

Because the ts mutants have the capacity to aid in the establishment of persistent infections of CV-1 cultures coinfected with WT, it was of interest to determine whether

the individual ts mutants could establish persistent infections under conditions of single infection. Thus, monolayer cultures of CV-1 cells were singly infected at 37” with WT, tsA58, tsBl1, or tsD101 at inputs of TABLE

2

CAPACITYOF ts MUTANTSTO PROMOTE CELL SURVIVAL Inoculum” WT tsA58 + WT tsBl1 + WT tsDlO1 + WI-

Percentage cell survivaP 0.0005 NC’

0.01 0.003

WT

0.0

tsA58 tsBl1 tsDlO1

0.02 NC’

0.01

u CV-1 ceils were infected with WT at 0.1 PFU/cell and/or with the ts mutants at l-5 PFUicell as indicated. * Surviving cell fractions were determined from the number of clones detectable during week 3. c No crisis in these infections.

LEONARD

380

2 0

I 5

I IO

I 5

.I 122

TIME

C. NORKIN

AFTER

I IO

INFECTION

LI ‘22

j

I 5

I IO

.Ii ’ 22

(Weeks)

FIG. 3. Viral yields in persistent infections of CV-1 cells initiated by single infection with ts mutants of SV40. CV-1 cells were infected with WT, tsA.58, tsB11, or tsDlO1 at respective input multiplicities of approximately 1,3, 1, and 5 PFUicell. Cultures were subsequently incubated at 37”. Virus stocks were harvested at weekly intervals between Weeks 1 and 12 and at Week 22 and were titered by plaque assay on CV-1 monolayers at 33 and 37”. (A) WT-infected culture titered at 33” (0) and at 37”(H); tsA58 PI stocks titered at 33” (0) and at 37” (0). (B) tsBl1 PI stocks titered at 33”(O) and at 37” (0). (C) tsDlO1 PI stocks titered at 33” (0) and at 37” (0). The WT-infected culture underwent extensive CPE and no attached cells were present by the end of Week 2. The arrows indicate that the 37” titers of the preceding samples were less than 400 PFU/ml, the minimal level of detectability in the plaque assay.

between 1 and 5 PFU/cell. Cultures were either washed and fluid changed or passaged at weekly intervals. Weekly viral yields are shown in Fig. 3. The culture infected only with WT developed extensive CPE during Week 1. No clones emerged during subsequent weeks (Table 2). Extensive CPE also developed in the tsA58-infected culture during Week 1. However, 41 clones were in evidence by the end of Week 2, representing about 0.02% cell survival. There was little, if any, CPE in this culture at all subsequent times. The tsBl1 -infected culture showed only slight CPE during Weeks 1 and 2 and little, if any, CPE at all subsequent times. Extensive CPE deveiu$ed in the tsDlOlinfected culture during Week 2. However, a total of 17 clones emerged, corresponding to about 0.01% survival of the original cells. This culture was subsequently free of CPE until Week 5 when many detached cells were observed over an apparently healthy monolayer. This phenomenon was characteristic of the infection until Week 12, after which time it displayed little, if any, CPE. The above results show that each ts mutant is more able than WT to establish persistent infections of CV-1 cells under condi-

tions of single infection at 3’7”. The persistent infections established by single infection of CV-1 cells with tsA58, tsBl1, or tsDlO1 (ts PIs) will hereafter be referred to, respectively, astsA58 PI, tsBl1 PI, and tsDl01 PI. Pattern

of Infectivity

and Virus Production

on a per Cell Basis in Persistently

Infected Cultures

Samples of the persistent infections were stained at various times for the SV40 T or V antigens by the indirect fluorescent antibody method (Fig. 4). During times when little, if any, CPE was evident in the cultures only a few percent of the cells produced T or V antigen. During periods of extensive CPE, as many as 60% of the attached cells produced T antigen with somewhat fewer cells producing V antigen. Nevertheless, at most times (as late as 19 weeks in the ts + WT PIs and 27 weeks in the ts PIs) only a few percent of the cells produced T antigen or structures that could react with antiserum prepared against virions. A Week 19 sample of tsA58 + WT PI, in which approximately 10% of the cells produced T or V antigen, and a Week 27 sample of tsA58 PI, in which about 1% of the cells

PERSISTENT

INFECTION

WITH SV40 ts MUTANTS

381

FIG. 4. Fluorescent antibody staining of persistently infected CV-1 cells. (A) a clone of tsA5R PI stained for T antigen at 9 weeks. (B) tsDlO1 PI stained for T antigen at 24 weeks. (C) tsDZO2 PI stained for V antigen at 24 weeks. (D) WT PI stained for V antigen at 18 weeks.

produced T or V antigen, were each superinfected with WT SV40 at an input of 100 PFU/cell. Superinfection did not lead to a change in the fraction of T or V antigen-

producing cells at 48 hr nor did it affect the 72-hr viral yields, which were about 5 x 106 PFU/ml in each instance. Thus, all susceptible cells were already producing virus,

382

LEONARD

apparently to their capacity, before superinfection. A number of clones of tsA58 PI, tsBl1 PI, and WT PI were readily isolated in the absence of antiserum. The ease with which the clones were obtained is probably explained by the pattern of resistance described above. These clones were also examined for T and V antigen by indirect FA staining. Each of the clones contained cells which displayed viral antigens and cells which did not (e.g., Fig. 4a). No clones were observed in which all of the cells or none of the cells exhibited viral antigens. All of the clones also contained cells showing CPE as well as healthy looking cells. These Carrier Interferon

Cultures Do Not Produce

This was indicated by their susceptibility to direct challenge with VSV. Following infection with VSV (m.o.i.= 1 PFUicell), the 24-hr VSV yields in the persistently infected CV-1 cultures and in normal CV-1 cultures were each of the order of lo7 PFUI ml, as determined by plaque assay on LLCMK, cell monolayers.

C. NORKIN

more efficient at producing plaques at 33 than at 37” (Fig. 3). Thus, it is important to note that the tsDlO1 stock used as the inoculum of this system produced lo”-fold more virus when grown at 33 than at 41”, under conditions in which WT yields were equivalent at each temperature. However, this stock produced about loo-fold more virus at 37 than at 41”. The leakiness of tsDl01 at 37 and the apparently strong selection for ts+ virus in these infections probably account for the high initial yields in this persistent infection. The virus stocks produced by tsD101 PI at later times tended to be even less temperature-sensitive than those produced earlier, as indicated by their relative efficiencies of plaque production at 37 versus 33” (Fig. 3), and by their relative yields at 41 versus 33” when used as inocula (Table 3). There was an even more rapid selection of ts+ virus in the ts + WT PIs, as ts+ virus became predominant in these systems during the first week. Moreover the ts + WT PIs did not produce ts stocks at any subsequent time up to 13 weeks. This was indicated by the relative efficiencies of plaque production by the ts + WT PI stocks at 33 versus 37” (data not shown). TABLE

Temperature Sensitivity of the Virus Produced by the Persistently Infected Cultures

Early stocks produced by tsA.58 PI and tsBl1 PI contained mostly ts virus, as indicated by their efficiencies of plaque production at 33 versus 37” (Fig. 3). However, there was a tendency for the relative amounts of ts+ virus to increase afterwards in both of these persistent infections. The weekly infectious virus yields, which were initially low in tsA58 PI and tsBl1 PI, also tended to subsequently increase, in parallel with the emergence ofts+ virus. It should be noted that while the virus produced by tsB11 PI became ts’ for plaque production at 37”, it remained ts for growth at 41” (Table 3), indicating that it was not true WT. High viral yields were produced by tsD101 PI from the outset. Furthermore, the Week 1 tsDlO1 PI stock was only 30-fold

3

TEMPERATURE SENSITIVITY OF PI VIRUS USED AS INOCULA”

Virus tsA58 PI (22 weeks) tsBl1 PI (22 weeks) tsDlO1 PI (22 weeks)

Yield efficiency in cv-1 cells (41”/33” ratio) 0.47

2.1 x 10-4 0.19

tsA.58 + WT PI (13 weeks) tsBl1 + WT PI (9 weeks) tsDlO1 + WT PI (13 weeks)

0.03

WT

1.0

0.31 0.10

” Duplicate cultures of CV-1 cells ~vere infected with each inocula at inputs of about 0.1 PFLJicell. In each instance adsorption was at 33” for 2 hr. One set of cultures was subsequently incubated at 41” and harvested 3 days postinfection. The other set was incubated at 33” and harvested 5 days postinfection. Yields were titered on CV-1 monolayers at 33”.

PERSISTENT

INFECTION

Although ts+ virus became predominant in the ts + WT PIs during the first week, the Week 1 viral yields were lower in these systems than in the corresponding WT infection (Fig. 2). This shows that the ts virus in the mixed inocula did serve to modulate the infections. However, the Week 1 yields of tsA.58 + WT PI and tsBl1 + WT PI were considerably higher than those of tsA58 PI and tsBl1 PI (Figs. 2 and 3), consistent with the early predominance of ts+ virus in the ts + WT PIs. As in the case of tsBl1 PI, the ts+ virus produced by tsB11 + WT PI was mostly not true WT, since tsBl1 + WT PI stocks remained temperaturesensitive for growth at 41” (Table 3).

(Relative

PI concentration

WITH

SV40

Interference Stocks

ts

MUTANTS

Activity

383

in Persistent

Infection

Virus samples from the persistent infections were tested for their capacities to interfere with the replication of WT SV40. It can be determined from the yield reduction curves in Fig. 5 that the Week 22 samples from tsB11 PI and tsDlOl PI each contained interfering particle titers greater than 2 x 10’ DIP/ml. The infectious particle titers of these samples were about 4 x 10’ PFUi ml, indicating that these stocks contained an excess of nonproductive interfering particles. Although the Week 22 sample from tsA.58 PI did not reduce the yield of WT to a

VIRUS DOSE of undiluted

stock

= 100)

FIG. 5. Effect of dilution ofts PI virus stocks on virus yields in cultures coinfected with 1 cells were coinfected at 37” with WT virus at a m.o.i. of 0.1 PFIJicell and with twofold of Week 22 samples of tsA.58 PI, tsBl1 PI, and tsDlOl PI. Lysates were harvested at yields were titered by plaque assay on CV-1 monolayers at 37”. The single infection WT 2.4 x 10’ PFUlml.

WT virus. CVserial dilutions 3 days and viral virus yield was

LEONARD

384

level below that of the single infection WT yield, this sample clearly displayed autointerference activity (Fig. 5). In contrast to the findings with the persistent infections established by single infection with the ts mutants, virus stocks from tsBl1 + WT PI and tsDl01 + WT PI showed no evidence of interference activity, while a stock from tsA58 + WT PI displayed only slight autointerference activity (data not shown). of Viral Persistent Infections

Characterization

DNA

in

(‘. NORKIX 1

2

3 4

5

6 7

8

9 1011

12 13 14 15 16

the

The presence of DI virus in some of the ts PI stocks was confirmed by polyacrylamide gel electrophoresis of the Hind111 digests of viral DNA from these stocks (Fig. 6). The digests of samples from tsA58 PI, tsBl1 PI, and tsDlO1 PI each contained large amounts of aberrant fragments in addition to the

E’lG. 6. Polyacrylamide gel electrophoresis of wildtype SV40 DNA and viral DNA from persistent infection stocks, each digested with HindIII. The samples in each slot are as follows: (1) WT; (2) tsAS + WT PI, 13 weeks; (3) tsBll + WT PI, 8 weeks; (4) fsDlO1 + WT PI, 13 weeks; (5) WT PI, 13 weeks; (6) WT; (7) tsA.58 PI, 22 weeks; (8) tsBl1 PI, 22 weeks; (9) t.sDlOl PI, 22 weeks: (10) WT.

FIG;. 7. Xgarose gel rlectrophoresis of wild-type SV40 DNA and viral DS.4 from persistent infection stocks either undigested or digested with @II. EcoRI. or Hir/dIII. The samples in each slot are as follows: (1) WT, undigested: (2) W’T, BglI; (3) bVT. EcoRI; (1) WT, Hi~dI11: (5) tsrl:i# PI, unidigrsted; (6) tsA.iX PI, Hg/I; (7) tsA.i~ PI, EcoRI; (x) tsA.iR -C WT PI:, undigested; (9) tsA.i?l + WT PI. BgII; (10) tsA.58 + WT PI, EcoRI: (11) tsRf1 PI, undigested; (1”) tsBlz PI, RgII; (13) tsL311 PI, EcoRI; (14) tsDlO1 PI, undigested; (1.5) tsDlO1 PI. &$I; (16) fsDlO1 PI. EcoRI. The tsA.iX PI, tsRlf PI. and tsDIU2 PI samples are from Week 22. The inA% 4 WT PI sample is from Week 13.

standard SV40 Hind111 fragments. In contrast, the samples from TsBll + WT PI and tsDl01 + WT PI, which did not display interference activity, yielded standard HindII1 fragments only, while the sample from tsA.58 + WT PI yielded relatively low levels of aberrant Hind111 fragments. Viral DNA samples from tsA.58 PI, tsAS8 + WT PI, tsBl1 PI, and tsDlO1 PI were further characterized by electrophoretic analysis in agarose gels (Fig. 7). Unrestricted WT DNA (lane 1) displays two major bands in agarose gels. The more rapidly migrating of these corresponds to form I (supercoiled) DNA and the other to form II (relaxed circles) DNA (Tegtmeyer and Macasaet, 1972). The intermediate minor band corresponds to full-length linear form III molecules. While the unrestricted WT sample displayed homogeneous bands, each of the unrestricted PI samples (lanes 5, 8, 11, and 14) displayed notable heterogeneity.

PERSISTENT

INFECTION

BglI makes a single cut on the WT SV40 genome at 0.67 map units (Zain and Roberts, unpublished results) near to the origin for DNA replication (Danna and Nathans, 1972; Fareed et al., 1972). Restriction with BgZI thus converts nearly all of the WT circular DNA to genome-length linear molecules (lane 2). In contrast, BgZI restriction of the PI samples (lanes 6,9, 12, and 15) produced short fragments ranging in size between 33 and 13% as long as the WT SV40 genome. These fragments were probably produced by the restriction of grossly aberrant viral genomes which contained multiple Bg21 sites and, consequently, multiple replication origins. Larger aberrant BglI fragments in these samples, which ranged in size from 87 to 62% as long as the WT genome, represent either short genomes containing a single Bgl I site or portions of larger genomes which also yield a shorter Bgl I fragment. EcoRI makes a single cut on the WT genome at 0.0 map units in that portion of the late region which codes for the major viral protein VP1 (Morrow and Berg, 1972; Mulder and Delius, 1972; Lai and Nathans, 1975). While EcoRI restriction converted all of the form I and form II WT DNA to fulllength linear molecules, much of the viral DNA in the samples from tsBl1 PI (lane 13) and tsDlO2 PI (lane 16) was resistant to EcoRI. Lesser amounts of EcoRI-resistant molecules were present in the tsAg58 PI (lane 7), and tsA58 + WT PI (1an.e 10) samples. Viral genomes containing reiterations of the BglI site might be expected to be missing the EcoRI site since these are separated by 33 map units on the WT genome. Thus, one might have expected to find relatively more EcoRI-resistant molecules in the tsA58 PI and tsA58 + WT PI samples. In this regard, the tsA58 + WT PI and tsBl1 PI samples each yielded Bgl I and EcoRI fragments having similar electrophoretic mobilities. This might be explained if both a BglI and an EcoRI cleavage site were present on the same reiterated sequences, as is suggested by the similar intensities of the comigrating BglI and EcoRI fragments. Such a sequence could result from either a complex rearrangement of viral sequences or from

WITH

SV40 ts MUTANTS

385

the incorporation of cellular sequences. Consequently, the samples in Fig. 7 were tested by blot hybridization for the presence of sequences which are homologous to the highly reiterated (Y component of monkey DNA (Rosenberg et al., 1978) as well as for a sequence that is infrequently repeated in the monkey genome but which is present in several independently isolated defective variants of SV40 (Oren et al., 1976). No evidence could be obtained for the presence of these host sequences in the viral DNA samples described here (not shown). DISCUSSION

The objectives of this investigation were to determine whether representatives of well-characterized groups of SV40 ts mutants might be able to interfere with the replication of WT and, if so, to determine whether this capacity might correlate with the ability to aid in the establishment of persistent infections of GMK cells. One early mutant (tsA58) and two phenotypically distinct late mutants (tsBl1 and tsD101) were each examined. The lesion in tsA58 maps in the genome region which codes for the A gene product (T antigen) (Lai and Nathans, 1975; Fiers et al., 1978; Reddy et al., 1978). T antigen functions in the initiation of viral DNA replication (Tegtmeyer, 1972) and in cellular transformation (Tegtmeyer, 1975). The lesion in tsBl1 maps in the region coding for the major capsid protein, VP1 (Lai and Nathans, 1975; Cole et al., 1977). Although tsB11 makes normal amounts of a protein which reacts with VPl-specific antibodies, the mutant fails to assemble capsids (Tegtmeyer et al., 1974). The lesion in tsD101 maps in the region of the genome coding for the minor capsid proteins, VP2 and VP3 (Lai and Nathans, 1975; Cole et al., 1977). TsDlOl is unable to synthesize viral mRNA at 41” because of a presumed block at some stage of uncoating (Robb and Martin, 1972). As reported above, tsA58 and tsBl1 are each able to interfere with the replication of WT SV40. In contrast, tsDlO1 does not interfere measurably with the replication of WT, presumably because interference requires mutant genome activity (above and

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Norkin, 1979a). Nevertheless, each of the mutants is able to facilitate the establishment of persistent infections of CV-1 cells which have been coinfected with WT at 37 (ts + WT PIs). Furthermore, each mutant is more able than WT to establish persistent infections under conditions of single infection at 37” (ts PIs). The seemingly contradictory results with tsDlO1 are probably explained, at least in part, by the leakiness of this mutant at 37”. Consistent with this, the initial viral yields and levels of CPE were greater in tsD101 + WT PI than in tsA58 + WT PI or tsBll + WT PI. Homotypic interference by the mutageninduced mutants, tsA58 and tsBl1, is similar to that expressed by DI particles and small plaque variants from an SV40/rhesus monkey kidney cell carrier system (Norkin, 1979a, b) in that it results from a simple competitive interaction between variant and WT. Thus, our findings are consistent with the suggestion of Youngner and Quagliana (1976) that ts mutants are defectives of a sort even though they are not deletion mutants and that they might play a role in persistent infections similar to that of DI particles. It is interesting in this regard that both early A gene mutants and late B gene mutants express homotypic interference. no particular SV40 gene Furthermore, product appears to be required at WT functional levels for the establishment of persistent infection, although we have not yet tested that class of early mutants which fails to produce the small-t antigen (Crawford et al., 1978). Regardless, the capacity to express homotypic interference or to establish persistent infection is not restricted to any particular mutant group. It is worth noting that homotypic interference in the SV40 system is a phenomenon fundamentally different from homotypic interference as expressed by the much studied DI particles of VSV. Interference in the VSV system is expressed in an all-or-none manner on a per cell basis (Marcus and Sekellick, 1974). The underlying basis for the enhanced capacity of the SV40 ts mutants to establish persistent infections under conditions of single infection is not yet clear although it

C. NORKIX

probably involves the relatively slow growth of the mutants at 37” and their consequent diminished cytopathogenicity (Norkin, 1977a). This might delay cell destruction for a time sufficient to allow for the expression of other factors such as the emergence of DI particles and/or cells which express partial or transient resistance (below). The emergence of ts’ revertants and the eventual predominance of DI particles in the established ts PIs suggests that DI particles play a more important role than ts virus at later times in those carrier systems. TsBll + WT PI and tsDlO1 + WT PI stabilized despite the fact that the virus populations in those systems rapidly became ts+ without the concomitant emergence of detectable levels of DI particles. Furthermore, none of the SV40 carrier systems produced interferon. Although tsB11 + WT PI and tsDl01 + WT PI were tested at only one time (13 weeks) for aberrant viral DNA and DI particle activity, at that time these systems were free of CPE and yet were producing weekly viral yields of about lox PFUi ml. These results might best be explained by the induction and/or selection of cells which express a partial or transient resistant state. The presence of a large fraction of such cells is implied by the per cell pattern of virus production in these systems, as discussed below. Only a few percent of the cells in these persistent infections produced SV40 T or V antigen. Furthermore, the fraction of T antigen-producing cells and the virus yields were unchanged after high input superinfection with WT. Thus, while homotypic interference by ts mutants and DI particles is not expressed as an all-or-none phenomenon during the initial infection of normal CV-1 cells, the block to virus production on a per cell basis in the established persistent infections is expressed in an all-or-none way. The presence of a small fraction of T and V antigen-producing cells within clones indicates that resistance is transient or incomplete and that new virus-producing cells are recruited from the population of T antigennegative nonproducing cells. Fluctuation analysis suggests that rather

PERSISTENT

INFECTION

than being induced by SV40 (Hahn and Sauer, 1971; Hahn, 1972), resistant cells arise spontaneously during the growth of normally susceptible CV-1 cells (Wilson et al., 1976). If so, then A gene mutants might be expected to be less able than B mutants or WT to select for resistant cells because the block to infection in resistant cells is expressed at an early stage. This could explain our reproducible finding that tsA58 PI did not stabilize until the emergence of ts+ virus, while tsA58 + WT PI was established with relatively little CPE. In contrast, tsBl1 PI was established with much less CPE than tsBl1 + WT PI. Regardless, these results suggest that T antigen might play a role in the establishment of SV40 persistent, infections in GMK cells. Many viral genomes in the PI stocks which displayed DI particle activity contained multiple BglI cleavage sites and/or were shorter than WT genomes. Since the single Bgl I cleavage site maps very near to the unique origin for DNA replication, variant genomes with multiple BgZI sites almost certainlv contain duplications of the origin. The variants which emerge in SV40 carrier systems prevail over, and thereby int,erfere with WT by effectively competing with and replicating at the expense of WT (Norkin, 1979a). Those variant genomes which contain duplications of the origin probably benefit from a selective advantage since they are able to initiate replication more frequently than genomes with a single origin (Lee and Nathans, 1979). Short genomes should also enjoy a selective advant.age in replication. Consequently, while many different types of defectives probably arise in these persistent infections, those which are likely to benefit from a selective advantage in replication appear to become predominant. Defective genomes with repeat units containing the origin for DNA replication also become predominant in SV40 stocks generated by serial undiluted passages (Brockman et al., 1973; Lee et al., 1975). ACKNOWLEDGMENTS This investigation was supported by Public Health Service Research Grant ROl AI14049 from the National Institute of Allergy and Infectious Diseases.

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We are grateful to Peter Tegtmeyer and Harvey Ozer for kindly providing the SV40 ts mutants. We also thank Maxine Singer and Ronald Thayer for the blot hybridization test for the detection of cellular DNA sequences and for their critical reading of this manuscript. The excellent technical assistance of Cheryl Goguen and R. Bruce Register is very gratefully acknowledged.

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