Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

Complementation Analysis of Simian Virus 40 Mutants

Seventy six new temperature-sensitive mutants of simian virus 40 have been isolated. A simple modification of the standard plaquing technique permits complementation analyses to be performed readily. By using this technique the new mutants have been divided into four complementation groups.     

Physical and Genetic Characterization of Deletion Mutants of Simian Virus 40 Constructed In Vitro

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.     

Proposal for a Uniform Nomenclature for Simian Virus 40 Mutants

A uniform nomenclature for simian virus 40 mutants has been developed. This nomenclature should evolve into a comprehensive nomenclature as new mutants and information are obtained. Hopefully, his nomenclature will also stimulate the development of a comprehensive nomenclature for all animal virus mutants.     

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.     

Plaque Formation with Simian Virus 40: Enhancement by Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) added to agar overlays during plaque assays of simian virus 40 (SV40) in CV1 monkey cells increases the plaque size and number and enables plaques to be read several days earlier than usual. DMSO appears to act during development of plaques, perhaps by causing cell lysis at smaller burst sizes in the presence of near-lethal DMSO concentrations. It does not act synergistically in determining virus inactivation with UV light and is equally effective on wild type and a late mutant of SV40.     

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.     

Isolation and Characterization of Simian Virus 40 Ribonucleic Acid

Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19S viral RNA species was observed. Late in the lytic cycle, 16S and 19S viral species were found. The 16S and 19S species of viral RNA were localized in the cytoplasm. High-molecular-weight heterogeneous RNA, containing viral sequences, was isolated from the nuclear fraction of infected cells late in the lytic cycle. This RNA may contain non-viral sequences linked to viral sequences. The formamide hybridization technique can be used to isolate intact late lytic viral RNA which is at least 99% pure.     

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.     

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.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

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.     

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.     

Characterization of an Endonuclease Associated with Simian Virus 40 Virions

An endonucleolytic activity associated with purified simian virus 40 (SV40) virions has been found. The enzyme is present in virions prepared from a number of different host lines. The enzyme is present in all early and late temperature-sensitive mutants examined. Some aspects of the endonucleolytic activity have been examined with SV40 deoxyribonucleic acid as substrate.     

Characterization of Small Plaque Mutants of Mouse Hepatitis Virus,JHM Strain

Two small plaque mutants designated as 1a and 2c were isolated from DBT cells persistently infected with the JHM strain of mouse hepatitis virus. Unlike the wild type JHM, these two mutant viruses grew more slowly with no prominent cell fusion. The buoyant densities of the mutants were slightly lower and 2c was revealed to have fewer peplomers than JHM by electron microscopy. The purified JHM contained five polypeptides with molecular weights (M.W.) of 260,000, 105,000 (GP105), 65,000, 60,000 (P60), and 23,000 (GP23). In addition to two polypeptides, P60 and GP23, which were common to JHM and the mutants, 1a was found to contain three other specific polypeptides with M.W. of 180,000 (GP160), 110,000, and 95,000 (GP95), while 2c had GP180, GP105, GP95, and one with a M.W. of 175,000. All of these polypeptides were shown to be glycosylated except for P60. After bromelain treatment, all these viruses lost the peplomers and contained P60 and another new 18,000 dalton polypeptide.     

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.     

Structural Polypeptides of Simian Virus 40

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.     

Airborne Stability of Simian Virus 40

The influence of relative humidity on the airborne survival of simian virus 40 (SV40) was studied by allowing virus aerosols to age in rotating drums at 21 or 32 C and at a relative humidity (RH) value ranging from 22 to 88%. Airborne SV40 virus was stable at every RH tested at 21 C, but aerosols maintained at 32 C were inactivated within 60 min at mid-range RH values. The unusual stability at 21 C over a broad RH range indicates that potentially biohazardous situations may occur under laboratory conditions if this virus becomes accidentally airborne.