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.     

Phage lambda DNA injection into Escherichia coli pel- mutants is restored by mutations in phage genes V or H.

Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R_f and a 0.5 R_f activity. The 0.2 R_f enzyme was found only in actively growing cells, while the 0.5 R_f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R_f form of this enzyme.SV40 wild-type and SV40 tsBC and tsC mutants stimulated the levels of the 0.2 R_f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 tsA mutants (tsA7, 28, 30, 58, and 209) and two different SV40 tsD mutants (tsD202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R_f thymidine kinase activity could be detected in tsA or tsD mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R_f thymidine kinase activity at both permissive and nonpermissive temperatures.These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.     

Induction of interferon by temperature-sensitive mutants of Newcastle disease virus

Spontaneously-selected and mutagen-induced temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were used to study interferon induction in chick embryo (CE) cells at temperatures permissive (37°) and nonpermissive (42°) for virus replication. Both infectious and UV-irradiated virus were tested for interferon-inducing ability in cells pretreated or not pretreated with homologous interferon. At 37°, only UV-irradiated NDV was capable of inducing interferon in cells not treated with interferon before infection. In cells pretreated with interferon, on the other hand, both unirradiated and UV-irradiated virus stimulated the production of interferon. At 42°, the interferon-inducing phenotype for some UV-irradiated ts mutants was dependent on whether or not cells were pretreated with interferon. For example, out of 10 mutants examined, one UV-irradiated ts mutant induced interferon in both untreated and interferon pretreated cells; 7 mutants failed to induce in untreated cells but induced from 25–100% of the wild-type level of interferon in cells pretreated with interferon; and two mutants failed to induce interferon in both types of cells. In addition, one mutant (NDV₀ts-100) induced low or undetectable levels of interferon at both 37° and 42°, conditions under which wild-type virus (NDV₀) produced significant levels of interferon. Co-infection of cells with UV-irradiated ts-100 and a preparation of NDV₀ exposed to prolonged irradiation resulted in considerable production of interferon. These results suggest the possibility that more than one virus function may be involved in interferon induction by NDV in CE cells.     

Synthesis of virus-specific polypaptides by temperature-sensitive mutants of herpes simplex virus type 1.

The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4–24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA^?ts mutants which share several common polypeptide defects, (2) DNA^±ts mutants which exhibit polypeptide profiles resembling the DNA^?ts mutants, (3) DNA⁺ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39°, and (4) DNA⁺ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes.     

A replication defective mutant of Rous sarcoma virus which fails to make a functional reverse transcriptase.

ts 672 is a replication defective temperature sensitive mutant of Rous sarcoma virus which produces large yields of noninfectious viral particles (NI 672) during growth in cells at the nonpermissive temperature. This study has been directed to identifying the temperature sensitive function of ts 672 by analysis of the NI 672 particles and by comparing this mutation to other known mutations in functions affecting replication.The only structural defect observed with NI 672 was the absence of the virion-associated RNA dependent DNA polymerase activity characteristic of all infectious RNA tumor viruses. Although there are other viral mutants which exhibit an altered polymerase activity, ts 672 is unique in that the DNA polymerase is only temperature sensitive prior to or during the assembly of the virus particle. The DNA polymerase activity of the ts 672 virions produced at the permissive temperature is just as temperature stable as the wild type enzyme.Mixed infection tests performed with ts 672 and with two other DNA polymerase mutants have failed to show a complementation, thus supporting the idea that ts 672 is defective in the DNA polymerase function. Recombination studies with ts 672 and an avian leukosis virus have shown that there is a very high degree of genetic linkage between the temperature sensitive DNA polymerase function of ts 672 and the determinants for the host range of these viruses, the viral envelope proteins.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of tsBN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [³H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5°. However, when higher (41°–42°) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of tsBN75 shifted to 42° at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When tsBN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5° C), the restart of DNA synthesis preceeded the appearance of mitotic cells. These data suggest that the tsdefect of tsBN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

The use of vesicular stomatitis virus pseudotype production in the study of a temperature-sensitive murine leukemia virus.

A mutant of Moloney murine leukemia virus (MoLV), designated ts₃ was recently shown to have a temperature-sensitive defect associated with the release of mature virus particles budding from the cell membrane [Wong, P. K. Y., and McCarter, J. A., Virology58, 396–408 (1974)]. In an attempt to determine whether the defective function resides in an envelope component of the virion, the formation of pseudotypes between VSV and ts₃ was studied under nonpermissive and permissive conditions of ts₃ infection. Whereas similar levels of phenotypic mixing were observed between VSV and wild-type MoLV at both 39 and 34°, the level of pseudotypes formed between VSV and ts₃ was found to be considerably lower at 39° (nonpermissive temperature) than at 34° (permissive temperature). The results of temperature-shift experiments indicate that two separate blocks to VSV ts₃ pseudotype production may occur depending on the length of time ts₃-infected cells are incubated at the nonpermissive temperature. Preincubation of ts₃-infected cells for 24 hr at 39°, prior to superinfection with VSV at 39°, prevents pseudotype formation. In contrast, brief incubation at 39°C, coincident with VSV infection, introduces a reversible block on the release of VSV (ts₃) pseudotypes from the cell membrane. Complementation of ts₃ through ts₃ (VSV) pseudotype production was not detected at the nonpermissive temperature.     

The relationship between the formation of inclusions and viral DNA synthesis in adenovirus 12-infected cells

The relationship between the formation of inclusions and viral DNA synthesis was examined in cells infected with human adenovirus type 12 (H12) DNA-minus temperature-sensitive (ts) mutants and with H12 wild type (WT) in the presence of inhibitors of DNA synthesis. Three groups of H12 ts mutants (tsA, tsB, and tsC) are defective in initiation of viral DNA synthesis at the restrictive temperature (40°C). Inclusions were either absent or abortively formed in cells infected with the ts mutants at 40°, although inclusions comparable to those in WT-infected cells were observed in cells infected with the ts mutants at the permissive temperature (34°). The temperature shift-up experiment showed that the inclusion formed in cells infected with tsA at 34° disintegrated at 40°, while those in cells infected with tsB and tsC were retained at 40°. This observation suggests that the morphological integrity of the inclusions is dependent on gene A and the function of the inclusions on genes A, B, and C. The temperature shift-down of tsA-infected cells also showed the formation of inclusions and the recovery in viral DNA synthesis. The inclusions were not formed in WT-infected cells, when viral DNA synthesis was inhibited with either cytosine arabinoside or hydroxyurea. Viral DNA synthesis was not induced and the inclusions were not formed in infected cells after removal of cytosine arabinoside. In contrast, viral DNA synthesis was induced rapidly and the inclusions were formed concomitantly in cells after removal of hydroxyurea. It was shown that the type II inclusions were formed in H12-infected monkey cells, although neither the type IV inclusions nor virions were formed. These observations show the close relationship between the formation of inclusions and viral DNA synthesis, suggesting an important role of DNA-binding protein, the gene-A product, in the formation and the function of inclusions.     

The isolation of SV40 tsA/deletion, double mutants and the induction of host DNA synthesis

Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished.     

Biogenesis of poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis

Characterization of six is mutants of the DNA^? phenotype of vaccinia 1HD-W is described. Complementation analysis revealed that four of these map into different complementation groups while one was able to complement, albeit inefficiently, with only one among the other ts mutants tested. Judging by the rate of viral DNA synthesis it became evident that all DNA^? mutants except ts 6389 became more “leaky” for DNA synthesis at the restrictive temperature in single-cycle infection if adsorption was carried out at 4° when compared with values obtained after initiation of infection at 39.5°. This suggested that these mutants are defective in some function required very early in the virus life cycle. This notion was further supported by the observation that multiple rounds of viral DNA synthesis could occur at 39.5° provided that the temperature was raised after initiating the infection at a lower temperature. Employing density-shift experiments coupled with analysis of the size of replicative intermediates by means of alkaline sucrose gradients, it was found that progeny DNA synthesized by all but ts 6389 was indistinguishable from wild-type DNA. By contrast, DNA synthesis in ts 6389-infected cytoplasm was is throughout the period of viral DNA replication, regardless of the time when temperature was shifted up, relegating this mutant to the “fast-stop” category. This idea is supported by evidence that all of the DNA^? isolates except ts 6389 are deficient at 39.5° in some of the virus-specified DNA-binding proteins. Therefore, ts 6389 is the only isolate at hand with the characteristics expected of a mutant with a defective protein related to the growing fork of the vaccinia DNA replication apparatus.     

Analysis on the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77

Ts 334, a temperature sensitive mutant of avian sarcoma virus B77, cannot produce infectious progeny nor induce neoplastic transformation at the nonpermissive temperature. In order to clarify the relationship between these two functions of ts 334 we attempted to (1) isolate and characterize nonconditional transformation defective (td) mutants from ts 334, (2) isolate and characterize recombinants between ts 334 and RAV-1, and (3) reexamine rescue of ts 334 with RAV-1 at the nonpermissive temperature.All seven nonconditional td mutants isolated from ts 334 kept their temperature sensitive character in replication, although they had lost transforming capacity both at the permissive and at the nonpermissive temperatures. They appear to have a temperature sensitive step in virus maturation like ts 334.The helper function of these td mutants for the defective Bryan high titer strain of Rous sarcoma virus is also temperature-dependent.Two recombinants were isolated from cells coinfected with ts 334 and RAV-1. These recombinants combined the cell-transforming ability of ts 334 and of the envelope properties of RAV-1. These two recombinants were unable to induce cell-transformation but grew well at the nonpermissive temperature.In RAV-1 producing cells not only the genome of ts 334, but also the envelope property of ts 334 were rescued at the nonpermissive temperature, though cell transformation was not observed.These observations suggest that ts 334 has two mutations, one affecting reproduction and another cell-transformation capacities.     

ts Transformation mutants of avian sarcoma virus PRCII: lack of strict correlation between transforming ability and properties of the P105-associated kinase

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive (ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type (wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

Reconstitution of herpes simplex virus type 1 ribonucleotide reductase activity from the large and small subunits

An assay for the presence of functional large (RR₁) and small (RR₂) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR₁, and ts1222, which has a lesion in RR₂. In cells infected with ts1207 at 39.5°C, the defective RR₁ is unable to associate with RR₂ to form an active enzyme, and, as a result, a pool of functional RR₂ and defective RR₁ accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31°C or 39.5°C accumulate a pool of functional RR₁, but do not contain detectable RR₂. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5°C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.     

A mutant of Rous sarcoma virus with a conditional defect in the determinant(s) of viral host range.

A temperature-sensitive mutant of the Prague strain of Rous sarcoma virus of subgroup C, tsPH734PR-C, replicates much less efficiently at the nonpermissive (41°) than the permissive (35°) temperature while transforming equally well at both temperatures. In contrast, the wild-type parent, wtPR-C, is able to replicate and to transform chick embryo fibroblasts equally well at both 35° and 41°. Two lines of evidence suggest that tsPH734PR-C is defective in the synthesis or utilization of virus envelope glycoproteins GP85 and GP35. First, tsPH734PR-C appears to be defective in the incorporation of the virus envelope glycoproteins GP85 and GP35 into the noninfectious virus particles synthesized at 41°. Second, tsPH734PR-C, a host range subgroup C virus, is not complemented for replication of subgroup C virus at 41° by coinfection of cells with RAV-6 avian leukosis virus of subgroup B or by coinfection with the defective Bryan strain of Rous sarcoma virus, BH-RSV(?).     

Temperature-sensitive mutants of toxinogenic corynebacteriophage beta. II. Properties of mutant phages

Temperature shift experiments and electron microscopy were used to investigate the functions defined by toxinogenic corynebacteriophage β temperature-sensitive (ts) mutants. Temperature shift experiments between the permissive and nonpermissive temperatures showed that all ts markers involved late functions. Wild-type β was sensitive to a temperature shift-up during the course of infection. At the nonpermissive temperature phages with ts markers in genes clustered on the left side of the genetic map produced only phage tails, while phages with ts markers in genes clustered on the right side of the map produced only phage heads. All mutants tested were unimpaired in diphtheria toxin synthesis.     

Phosphorylation of pp60src and the cycloheximide insensitive activation of the pp60src-associated kinase activity of transformation-defective temperature-sensitive mutants of Rous sarcoma virus

Chick embryo fibroblasts infected with two transformation defective temperature-sensitive mutants of Rous sarcoma virus (GI 202 and GI 251), when shifted from the nonpermissive temperature (42°) to the permissive temperature (35°) show a rapid increase in the detectable pp60^src-associated kinase activity. When such cells are shifted back to the nonpermissive temperature there is an equally rapid loss of demonstrable kinase activity. The rapid increase in detectable kinase activity is not substantially hindered by cycloheximide at concentrations inhibiting all de novo protein synthesis, and is associated with a cycloheximide-insensitive phosphorylation of the mutant pp60^src.