Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Altered Protein Metabolism in Infection by the Late tsB11 Mutant of Simian Virus 40

The DNA of the temperature-sensitive mutant tsB11 is replicated at the same rate as the DNA of wild-type virus in infection at the restrictive temperature. The progeny mutant DNA cannot be distinguished from wild-type DNA by gel electrophoresis and is assembled into a nucleoprotein complex with the same velocity sedimentation characteristics as the wild-type complex. Analysis of in vivo protein synthesis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoprecipitation techniques demonstrated that the capsid components VP1, VP2, and VP3 of the mutant and wild-type virus are synthesized at a similar rate, but VP1 fails to accumulate within cells infected by tsB11. Furthermore, VP1 is located predominantly in the cytoplasmic rather than in the nuclear fraction of extracts from cells infected by the mutant. Immunofluorescent studies localized virion antigen within the nucleolus as well as the cytoplasm. The altered intracellular distribution and stability of VP1 suggest that it may be the mutant protein of tsB11. The synthesis of a 72,000 dalton protein is consistently induced in significant quantity in cells infected by tsB11 at the restrictive temperature. A protein of the same apparent molecular weight is present in smaller quantities in uninfected cells and is only slightly increased in quantity in cells infected by wild-type virus.     

Temperature-Sensitive Mutants of Simian Virus 40: Infection of Permissive Cells

Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants.     

Antigenic Phenotypes and Complementation Groups of Temperature-Sensitive Mutants of Simian Virus 40

The antigenic phenotypes of several temperature-sensitive mutants of simian virus 40 were determined by an immunofluorescence microtechnique that allowed a very high degree of internal control for the conditions of virus infection and antigenic staining. The tumor (T), U, capsid protein (C), and virion (V) antigens were investigated. Productive infection in monkey cells and abortive infection in mouse cells were simultaneously monitored for antigen production at both permissive and restrictive temperatures. Complementation analyses of the mutants demonstrated two complementing groups (A and B) and one noncomplementing group ((*)). One of the complementing groups could be subdivided into two subgroups having very different antigenic phenotypes. The following phenotypes were observed at the restrictive temperature in monkey cells. (i) The noncomplementing group produced none of the antigens. (ii) Group A induced T antigen in moderately but consistently reduced numbers of cells. Other antigens were markedly reduced or absent. (iii) Some of the group B mutants produced T antigen but little or no U and V antigens. The C antigen appeared in the nucleolus and cytoplasm of this subgroup. (iv) In the other group B mutants, antigen synthesis was not altered. Similar phenotypes were observed in mouse cells, except that U, C, and V antigens could not be detected during either the mutant or wild-type virus infections at any temperature.     

Viral DNA Synthesis in Cells Infected by Temperature-Sensitive Mutants of Simian Virus 40

Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.     

Complementation Between BK Human Papovavirus and a Simian Virus 40 tsA Mutant

Complementation tests between BK human papovavirus and SV40 temperature-sensitive mutants tsA58 and tsB11 were performed. Under the reported experimental conditions, BKV complemented the "early" mutant tsA58 but failed to complement the "late" mutant tsB11.     

Genetic Analysis of Simian Virus 40 IV. Inhibited Transformation of Balb/3T3 Cells by a Temperature-Sensitive Early Mutant

The temperature-sensitive early mutant, ts(*)101, was characterized during productive infection in monkey cells, and the results are presented in an accompanying paper. This paper demonstrates that although 101 mutant virions adsorb normally to confluent Balb/3T3 mouse cells at both permissive (33 C) and restrictive (38.5 C) temperatures, T antigen synthesis and transformation, abortive and stable, are inhibited at both temperatures (host-range inhibition). T antigen synthesis is temperature sensitive, whereas abortive and stable transformation are not. Clones of 101-transformed Balb/3T3 cells were isolated, and virus was rescued from all clones at both permissive and restrictive temperatures. The rescued virus was as temperature sensitive as the original transforming 101 virions.     

Genetic Analysis of Simian Virus 40 III. Characterization of a Temperature-Sensitive Mutant Blocked at an Early Stage of Productive Infection in Monkey Cells

A temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, has been characterized during productive infection in monkey kidney cells. The mutant virion can adsorb to and penetrate the cell normally at the restrictive temperature, but cannot induce the synthesis of cellular deoxyribonucleic acid (DNA) nor initiate the synthesis of SV40-specific tumor, virion, or U antigens or viral DNA. First-cycle infection with purified ts(*)101 DNA is normal at the restrictive temperature, but the resulting progeny virions are still temperature-sensitive. The mutant neither complements nor inhibits other temperature-sensitive SV40 mutants or wild-type virions. The affected protein in the ts(*)101 mutant may be a regulatory structural protein, possibly a core protein, that is interacting with the viral DNA.     

Simian Virus 40-Host Cell Interactions. I. Temperature-Sensitive Regulation of SV40 T Antigen in 3T3 Mouse Cells Transformed by the ts*101 Temperature-Sensitive Early Mutant of SV40

BALB/3T3 and Swiss/3T3 mouse cells transformed at permissive temperature (33 C) by the early temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, exhibited a temperature-dependent modulation of SV40 tumor (T) antigen as assayed by immunofluorescence. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells was reduced at restrictive temperature (39 C) when compared to 33 C and to wild-type SV40 transformed cells at either 33 C or 39 C. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells returned to the 33 C control level when the cells were shifted from 39 to 33 C. The ts(*)101 transformed cells could be superinfected with wild-type, but not ts(*)101, virions at 39 C as assayed by an increase in T antigen-positive nuclei.     

Isolation of Defective Lysogens from Simian Virus 40-transformed Mouse Kidney Cultures

Rescue of simian virus 40 (SV40) from hamster and murine cell lines transformed by nonirradiated or by ultraviolet (UV)-irradiated SV40 (10(-3) to 10(-5) survival) was studied. A combination of tests was employed to detect induction of SV40 synthesis: (i) co-cultivation with susceptible monkey kidney (CV-1) cells; (ii) treating mixtures of transformed and CV-1 cells with UV-irradiated Sendai virus (UV-Sendai) prior to co-cultivation; and (iii) plating untreated or UV-Sendai-treated mixtures of transformed and CV-1 cells with freshly trypsinized CV-1 cells. The first and second tests provided a measure of the total infectious SV40 yield per culture, and the third test provided a measure of the frequency of induction (fraction of transformed cells giving rise to infectious centers). With the combination of tests, SV40 was rescued in all trials from TSV-5 hamster cells, mKS-BU100 mouse cells, and from several lines of mouse kidney cells transformed by UV-irradiated SV40 (mKS-U lines). The frequency of induction was about 7 x 10(-2) for TSV-5 cells, about 3 x 10(-3) for mKS-BU100 cells, greater than 10(-4) for the mKS-U lines which were "good" yielders, and about 10(-5) to 10(-4) for the mKS-U lines which were "average" yielders. SV40 of a plaque type different from parental virus was rescued from four of the mKS-U cell lines. Virus was also easily rescued from: (i) tumor cells produced from the mKS-A line of transformed mouse kidney cells; (ii) mouse kidney cells transformed by SV40 which had been rescued from mKS-BU100 cells; and (iii) tumor cells (HATS) which had been produced by inoculating newborn hamsters with SV40 rescued from mKS-BU100 cells. The frequency of induction of HATS cells was of the same order of magnitude as the frequency of induction of TSV-5 cells. In a study of the kinetics of virus induction, it was shown that SV40 could be detected 28, 40, and 48.5 hr after UV-Sendai treatment of mixtures of CV-1 and TSV-5, HATS, or mKS-BU100 cells, respectively. Although all of the mKS-U lines contained the SV40-specific tumor antigen, some were poor virus yielders (SV40 was recovered in less than 50% of the trials) and five lines were rare virus yielders (SV40 recovered only once in four or more trials). Forty-eight mKS-U lines were nonyielders; SV40 was never recovered by any test used thus far. UV-Sendai-treated mixtures of pairs of nonyielder mKS-U lines with CV-1 cells also did not yield infectious virus. Various factors affecting rescue have been discussed. The mKS-U lines which were poor virus yielders, rare yielders, or which never yielded virus have been classified tentatively as "defective lysogens" which contain mutational lesions at loci essential for detachment of SV40 from integration sites or for SV40 replication, or for both.     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.