Mapping temperature-sensitive mutants of simian virus 40: Rescue of mutants by fragments of viral DNA

Five temperature-sensitive (ts) mutants of simian virus 40 (SV40), isolated and characterized by Tegtmeyer and Ozer (1971), have been mapped by marker rescue using endo R fragments of wild-type SV40 DNA. For each mutant a specific fragment corrected the ts defect, from which we infer that the mutation is within the genome segment corresponding to the active fragment. Since the position of each fragment in the SV40 cleavage map is known, the mutational sites could be localized. Of the five ts mutants examined, two were “early” mutants and were in complementation group A of Tegtmeyer; these mapped in contiguous Hin fragments H (tsA30) and I (tsA28), which had been shown previously to be part of the “early” region of the SV40 genome. Three ts mutants were “late” mutants and were in complementation group B of Tegtmeyer; these mapped in contiguous Hin fragments F (tsB8), J (tsB4), and G (tsB11), which had been shown previously to be part of the “late” region of the SV40 genome.     

Interference in SV40 DNA infections: a possible basis for cellular competence.

At the restrictive temperature, DNA from temperature-sensitive mutants of simian virus 40 (SV40) (in the A or B/C genes) interferes with plaque formation by wild-type SV40 DNA. This interference occurs early in the first cycle of infection. Nonhomologous DNA also can interfere with SV40 DNA infection. The mode of interference by ts SV40 DNAs and nonhomologous DNAs appears to be the same. In conjunction with other observations the interference by nonhomologous DNA and the similarity of interference by tsA DNA and tsB DNA suggest that interference does not require expression of any viral gene product. Since interference is observed even when the infections by interfering DNA and wild-type DNA are separated in time, interference must occur after the DNAs interact with cells. Furthermore, the different efficiencies with which structurally similar DNAs (SV40, PM2 and φX174 replicative form) interfere indicate that interference depends at least in part on nucleotide sequence. Those two observations suggest that interference is an intracellular, perhaps nuclear, phenomenon. The possible relevance of this interference phenomenon to observations on cellular competence for SV40 DNA infections is discussed.     

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

An early SV40 temperature-sensitive (ts) mutant, tsA58, and a late mutant, tsB11, each expressed homotypie 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, tsD101 did not display interference activity. Nevertheless, each of the is mutants promoted the survival of CV-1 cells coinfected with WT at 37° and, consequently, facilitated the establishment of persistent 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 for ts⁺ 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 were 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.     

Recombination between endogenous and exogenous simian virus 40 genes. III. Rescue of SV40 tsA and tsBC mutants by passage in permissive transformed monkey lines

Growth of the SV40 temperature-sensitive mutants tsA209 and tsBC210 at the permissive temperature on permissive SV40-transformed monkey CV-1 cells generated temperature-resistant virus. The plaque-forming ability of such rescued mutants was indistinguishable from that of wild-type virus upon replating on normal CV-1 cells at both the restrictive and permissive temperatures. The DNAs of the rescued (temperature resistant) mutants were analyzed by restriction endonuclease cleavage to determine whether the phenotypic change arose from recombination with the endogenous SV40 genome in the transformed cell. Two out of six independently rescued tsA209 mutants were shown to contain a segment derived from the endogenous SV40 genome, as judged by the R. HinIII and R. Hinf cleavage pattern. The exchange was confined to the region where the tsA209 mutation is located; other segments of the rescued mutant's genome were characteristic of the parental ts virus. Although substantial amounts of tsBC210 virus were rescued by growth on transformed cells (10²- to 10³-fold greater than the spontaneous reversion rate in normal CV-1 cells), the HinII + III, Hinf and HaeIII cleavage patterns of five independently rescued mutant DNAs were indistinguishable from that of the parental ts DNA. It is suggested that these rescued tsBC210 genomes represent a class of recombinants in which the segment derived from the endogenous SV40 genome of the transformed cell (and which is responsible for the reversal of the ts phenotype) contains no sequence modifications that affect the mobilities of the restriction fragments examined. The relationship between the genotype of the endogenous SV40 genome in the transformed cell and the extent to which different classes of SV40 ts mutants can be rescued in these cells are discussed.     

A detailed genetic analysis of the late complementation groups of simian virus 40

A simple mixed-plaque assay procedure for determining complementation between pairs of SV40 mutants is described. Since the data obtained can be quantified with respect to both the relative numbers and sizes of plaques, this method is less likely than other complementation assay procedures to yield false-positive results and gives some indication as to the efficiency of the complementation. This quantitative assay procedure, which works with both viral DNA and virions, was used to examine the complementation properties of a variety of late region temperature-sensitive and deletion mutants of SV40. Conclusions reached from these studies included (i) the complementation observed between tsB and tsC mutants is definitely intragenic; (ii) D mutants define a true complementation group; (iii) sequences within the last 68 carboxyl-terminal amino acids of VP-3 are necessary for expression of the D function; and (iv) when linked to the last 19 carboxyl-terminal amino acids of VP-1, the first 136 amino-terminal amino acids of VP-2 are sufficient for expression of the E function. Lastly, the collection of deletion mutants described here may be useful in identifying both structural and regulatory functions of the virion proteins and in analyzing some aspects of viral mRNA biogenesis.     

Host cell DNA synthesis as a possible factor in the enhancement of replication of human adenoviruses in simian cells by SV40

Temperature-sensitive mutants of SV40, ts-701 and ts-702, have been isolated after treatment of wild-type virus with nitrous acid and nitrosoguanidine, respectively. Although neither mutant produces infectious virus at the nonpermissive temperature, both can induce SV40 T and V antigens, virus DNA synthesis, and the stimulation of host cell DNA synthesis. Both are late mutants and appear to be in the same SV40 complementation group. Both mutants can also complement the replication of human adenovirus type 7 at the elevated temperature, as can another late mutant, pm-301, and an early mutant, tsA7. Therefore, the adenovirus complementation step appears to be prior to SV40 virus DNA synthesis. Since a BSC-1 clone in which SV40 infection stimulates host cell DNA synthesis allows the complementation of adenovirus type 7 by SV40 while parental BSC-1 cells neither support complementation of adenovirus type 7 by SV40 nor are stimulated to replicate their DNA after SV40 infection, the stimulation of host cell DNA synthesis appears to be critical to adenovirus enhancement. This stimulation appears to be specific since stimulation of DNA synthesis after nutrient deprivation does not induce the replication of human adenoviruses in simian cells.     

A temperature-sensitive mutant of the baculovirus Autographa californica nuclear polyhedrosis virus defective in an early function required for further gene expression

Fifteen temperature-sensitive (ts) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) have been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins synthesized in infected cells. One of the mutants, tsB821, was found to be defective in a very early function. Seven virus-induced proteins were synthesized by 2 hr postinfection. In marked contrast to wild-type virus and the other 14 ts mutants, the synthesis of further virus-induced proteins did not occur in tsB821-infected cells at the restrictive temperature (33 degrees ). Host protein synthesis continued as normal after transient expression of the seven early proteins. Viral-specific DNA synthesis was blocked or significantly delayed in tsB821-infected cells at 33 degrees . The relative synthesis of certain viral-induced proteins, particularly P31, P32, P42, P66, and P69, varied considerably in the remaining 14 mutants at 33 degrees. Three mutants exhibited alterations in specific polypeptides; P75 was approximately 1 kDa smaller in tsB1075, P40 was approximately 1 kDa smaller in tsB951, and P25 was greatly reduced in quantity or altered in tsB305.     

Temperature-sensitive B mutants of SV40 disassemble intracellular encapsidation particles at elevated temperature

We have investigated the effect of elevated temperature on intracellular SV40 nucleoprotein complexes. Shifting cell cultures infected with strains 777, Rh911, or established tsB mutants to 41° for 3 hr before extracting viral nucleoprotein from cell nuclei, led to the selective, irreversible loss of viral encapsidation-oriented structures, including mature virions, from nuclear extracts. Returning cells to 37° after a shift to elevated temperature allowed for encapsidation to resume with newly synthesized nucleoprotein being normally processed into virions. Encapsidation complexes lost during the shift to elevated temperature were not recoverable in nuclear extracts. Using a simplified complementation assay, we found mutation in the B region to be responsible for this response. Electron microscopic investigation showed that shift of intact cells infected with tsB mutants to elevated temperature, led to massive disassembly of capsids. No effect on capsid integrity was noted on extracted nucleoprotein shifted to elevated temperature in vitro. Attachment of virus both before and after temperature shift to a filamentous nuclear structure supports a role for it in the disassembly response.     

Biological properties of simian virus 40 host range mutants lacking the COOH-terminus of large T antigen

Three mutants of simian virus 40 (SV40), with deletions near the 3' end of the A gene, displayed a host range phenotype for growth and virus production in various African green monkey kidney cell lines. The mutants formed plaques in CV-1P cells at 40.5 degrees, in BSC-1 cells at 37 and 40.5 degrees, and in Vero cells at 32, 37, and 40.5 degrees. Virus yields in these three lines were cold sensitive: the burst size was greatest at 40.5 degrees and least at 32 degrees, but some progeny was produced under all conditions examined. Mutant yields never exceeded 10% of wild-type yields under the most permissive conditions (Vero cells at 37 or 40 degrees) and were less than 1% of wild type under the most restrictive conditions (CV-1P cells at 32 degrees). These mutants can be complemented by any SV40 mutant which produces a large T antigen containing a normal COOH-terminus. Mutants whose T antigens could not be transported to the nucleus were most efficient at complementation. Mutant virus production in a line of rhesus monkey kidney cells and in primary cultures of African green and rhesus monkey kidney cells was also substantially below wild type. These mutants were also completely defective for adenovirus helper function. Our data suggest that the host range property and adenovirus helper function represent the same activities of large T antigen.     

Enhancement of the replication of human adenovirus in simian cells by a series of temperature-sensitive mutants of simian virus 40.

Simian virus 40 (SV40) temperature-sensitive mutants of complementation groups A, B, C, and BC and enhance the replication of human adenovirus 7 in simian cells at nonpermissive temperatures whereas members of group D cannot.     

Different histone families in intracellular SV40 nucleoprotein complexes with respect to the acetylation turnover

The turnover of normal and butyrate-induced acetylation in the histones complexed with intracellular SV40 DNA was analyzed in infected CV-1 cells. The analysis concerned the two principal structures containing the intracellular SV40 chromatin: the 75 S complexes and the provirions. It was found that, while the normal acetylation in the 75 S chromatin turns over at a lower rate than that of the host-cell chromatin, the hyperacetylation induced by butyrate turns over at a rate very close to that of cell chromatin. This distinguishes two families of 75 S histones, presumably corresponding to nucleosomes with different accessibility to histone-modifying enzymes. A third type of turnover is shown by the provirion histones, in which the normal acetylation occurs at a low rate but is highly conserved. The response to butyrate in these structures is either absent or similar to that of the 75 S histones. In the chromatin of the late SV40 mutant tsB11, histone hyperacetylation does not occur when infection proceeds at the restrictive temperature for the coat-protein assembly. The hyperacetylation of histones which characterizes the morphogenesis of SV40 appears to be an assembly-dependent process, starting in the 75 S chromatin and building up mainly in the provirions.     

Augmentation in the infectivity of SV40 virus DNA by amphotericin B methyl ester

Summary The methyl ester of amphotericin B (100 µg/ml) enhanced the infectivity of SV40 virus DNA for CV-1 cells up to 300-fold. Amphotericin B methyl ester may prove useful for augmenting cellular penetration of macromolecules and for probing biological mechanisms.With 2 Figures     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) III. Involvement of SV40 gene A in the expression of TrAg in permissive cells.

Temperature-sensitive (ts) mutants of simian virus (SV40) which map in the early region of the SV40 genome were used to determine the role of the viral genome in the expression of SV40-specific transplantation rejection antigen (TrAg). The results indicated that tsA mutants (1612, 1637, 7, and 28) did not induce the expression of SV40-TrAg at the surface of infected permissive African green monkey kidney cells (TC-7) at 41° but did induce the expression of TrAg at the permissive temperature (33°) in TC-7 cells. Wild-type SV40 and late SV40 temperature-sensitive mutants (tsBC1602, tsBC1606, tsB8, and tsC219) induced SV40-TrAg in TC-7 cells at nonpermissive and permissive temperatures with equal efficiency. One of the mutants belonging to complementation group D (tsD1601) was defective in inducing SV40-TrAg at 41°. Kinetic studies indicated that SV40-TrAg appears by 18 hr after infection at 41° and 38 hr post-infection at 33°, paralleling closely the synthesis of T antigen. The synthesis of immunoreactive T antigen in TC-7 cells infected with tsA mutants at nonpermissive temperature did not correlate with the inability of tsA mutants to express TrAg at nonpermissive temperature. We conclude that the expression of TrAg in SV40-infected cells depends upon normal functioning of the A gene.     

The expression and properties of polyoma virus middle-T antigen in simian cells

SV40 late replacement vectors containing the polyoma middle-T coding sequences have been constructed. Mixed hybrid virus stocks have been obtained through complementation with a defective SV40 helper genome (dl 1055) following DNA transfection into CV-1 cells. Middle-T antigen is expressed in the infected simian cells at about 5-10 fold higher levels than in polyoma virus-infected mouse cells and has the pp60c-src-associated tyrosine-specific protein kinase activity in vitro. However, the 'specific activity' of the kinase in extracts of the infected CV-1 cells is lower than that observed in polyoma infected 3T6 cell extracts. The half-life of middle-T antigen in the CV-1 cells is about 4 h but the in vitro kinase activity associated with middle-T has a half-life of at least 8 h and hence appears to be stabilized. The in vivo phosphorylated species of middle-T has been shown by sucrose gradient analyses to be largely distinct from the middle-T with associated protein kinase activity in vitro.     

Mutagenic activity of BKV and JCV in human and other mammalian cells

Summary We present data suggesting that human polyomaviruses BKV and JCV, widely distributed throughout human populations, are able to induce gene mutations in cultured cells. In this study, using different infecting agents, cell lines to be infected, mutation expression periods, and selection systems, we observed mutagenic effects of varying extent with values of spontaneous mutant frequencies being increased after BKV infection up to 100-fold in BHK cells (6-thioguanine resistance) and nearly 35-fold in virus-transformed human Lesch-Nyhan cells (ouabain resistance). In experiments with BKV the viral mutagenic potential was found to be raised both in moderately uv-irradiated cells, or when wild-type virus was replaced by the variant BKV-IR isolated from a human tumor [27]. Since BKV-IR is defective in the expression of small-t antigen, the viral mutagenicity does not require this protein to be active. BKV was shown to mutate, besides different established cell lines, human peripheral blood lymphocytes. Moreover, as demonstrated by comparing mutagenicities of DNAs from BKV, JCV, and the related polyomavirus SV40, the mutagenic effects of the three viruses do not appear to be essentially different. Implications of these findings are discussed.     

Integration of SV40 DNA and induction of cellular DNA synthesis after a ts SV40 infection

The early temperature-sensitive mutant of simian virus 40 (SV40), ts1101, was characterized in nonpermissive Chinese hamster (ChH) and permissive monkey CV-1 cells for integration of viral DNA and induction of cellular DNA synthesis. At 42° in CV-1 cells, this mutant did not induce the synthesis of T antigen or cellular and viral DNA. The viral DNA did not integrate into the CV-1 DNA, although nuclear penetration occurred. In ChH cells the mutant induced T antigen at 40° and 42°, and the viral DNA integrated into ChH DNA, but ChH DNA synthesis was not induced. These findings suggest that the integration of SV40 DNA does not depend on the replication of cellular DNA and that, at least functionally, integration precedes the induction of cellular DNA synthesis.     

Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype.

Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.     

Functional interaction between the early viral proteins of simian virus 40 and adenovirus

Complementation studies using early temperature sensitive mutants of SV40 and adenovirus suggest that heterologous DNA-protein interactions can occur between these viruses in coinfected cells. This conclusion is based on the following experimental results: (a) The replication defective adenovirus 5 mutant ts125 can grow in African green monkey kidney cells at the nonpermissive temperature in the presence of preinfection with wild type SV40. (b) The overproduction of early SV40 RNA by the early SV40 temperature sensitive mutant tsA58 can be suppressed by superinfection of the monkey cells with wild type adenovirus. (c) The SV40 helper function for the growth of adenovirus in monkey kidney cells can be provided by the early SV40 mutant tsA58, even at the nonpermissive temperature. We suggest that viral DNA-protein cross-reactions may be responsible for these biological phenomena and show by DNA-DNA hybridization under nonstringent conditions (where hybrid DNAs with up to 35% base mismatch will be thermally stable) that certain regions of the Ad2 and SV40 genomes share significant sequence homology.