Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VII. Induction of SV40 TrAg in nonpermissive mouse cells by early viable SV40 deletion (o.54/0.59) mutants

Early viable deletion (0.54/0.59) mutants were tested for their ability to induce SV40 transplantation antigen (TrAg) in infected or transformed nonpermissive mouse cells in an attempt to determine the requirement for small t antigen in the expression of TrAg at the cell surface. The results indicate that dl (0.54/0.59) mutants are as efficient as wild-type SV40 in generating specific cytotoxic lymphocytes in C57BU6 mice and in immunizing BALB/c mice against an SV40 tumor cell challenge. Mouse (C57BU6) cells transformed by these mutants were also susceptible to lysis by the specifically sensitized lymphocytes. It can, therefore, be concluded that the synthesis of small t antigen is not an absolute requirement for expression of SV40 TrAg in SV40-infected or -transformed nonpermissive cells.     

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.     

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.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IV. Inhibition by human interferon of expression of SV40 TrAg in SV40-infected monkey cells.

Human interferon inhibits the synthesis of SV40 transplantation rejection antigen (TrAg) in SV40-infected but not in SV40-transformed monkey cells. The synthesis of SV40 tumor antigen as detected by the indirect immunofluorescence test and the large and small T antigens as detected by the immunoprecipitation with sera from tumor-bearing hamsters and electrophoresis in SDS-gels was similarly affected in SV40-infected monkey cells. These results suggest that the induction of SV40-specific TrAg in the cytolytic cycle depends upon a viral, rather than a host, message.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Modification of simian virus 40 large tumor antigen by glycosylation

The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin-layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein.     

Biological activities of deletion mutants of simian virus 40.

Mutants of Simian virus 40 (SV40) with large deletions in the early or late regions of the genome were tested for biological activity. Deletion mutants lacking portions of both late genes (B/C and D), but with an intact early genomic segment, were able to induce T antigen in infected cells, replicate their DNA in the absence of helper virus, stimulate thymidine incorporation into cellular DNA, and transform mouse and hamster cells. Cells transformed by late deletion mutants were shown to contain the mutant genome by a fusion-complementation rescue procedure. Deletion mutants lacking substantial portions of the early genomic region, including those segments where tsA mutants map, lacked all of the above activities. However, both early and late deletion mutants interfered with SV40 DNA replication.     

Nuclear preparations of SV40-transformed cells contain tumor-specific transplantation antigen activity.

Nuclear preparations from human SV40-transformed cells containing high levels of the tumor antigen (TA) were found to be enriched for tumor-specific transplantation antigen (TSTA).     

Expression of type C virus p30 in mouse cells infected with herpes simplex virus.

Expression of type C virus p30 in BALB/c and NIH Swiss mouse cells infected with live (productive infection) or uv-irradiated herpes simplex virus (uv-HSV) types 1 and 2 was studied by use of either immunofluorescent (FA) or radioimmunoassay (RIA) procedures. No evidence for enhanced expression of p30 was obtained with either of these procedures although activation of type C virus in uv-HSV-infected BALB/c cells was readily demonstrated at low frequencies (～10^?4) by infectious center assay. While FA staining with rabbit anti-murine leukemia virus (MuLV) p30 serum was observed in mouse cells productively infected with HSV or infected with uv-HSV, this was shown to be nonspecific. It was concluded that activation of type C virus in uv-HSV-infected BALB/c cells does not occur in significantly more cells than detected by infectious center assay.     

Interferon production in L cells persistently infected with hemagglutinating virus of Japan (HVJ).

The present study shows that when L cells persistently infected with hemagglutinating virus of Japan (HVJ) (L-HVJ cells) were incubated at 32°, the interferon-producing capacity of the cells was suppressed and was restored by the temperature shift-up to 38°. Synthesis of envelope protein antigen was not detected in the L-HVJ cells incubated at 38° which could produce interferon. Moreover, glutamine-starved L-HVJ cells did produce interferon even at 32°. The relationship between the suppressed state of interferon production and synthesis of viral envelope protein is discussed.     

Surface structure of virions budding from L1210(V) gln- mouse leukemia cells.

The surface structure of virions budding from L1210(V) gln^? murine leukemia cells was studied by freeze-drying intact cells, and two types of virus particles were detected. One type possessed random 10-nm surface projections, similar to budding viruses on cells producing only murine leukemia virus (MuLV). The other type possessed regularly arranged 5-nm projections, similar to budding viruses on cells producing only murine mammary tumor virus (MuMTV). Results of double antibody labeling are in agreement with the indication that individual viral envelopes on L1210(V) gln^? cell viruses are homogeneous in their surface structure.     

Lactic dehydrogenase virus replicates in somatic cell hybrids of mouse peritoneal macrophages and SV40-transformed human fibroblasts

Hybrid clones obtained from mouse peritoneal macrophages and SV40-transformed human Lesch-Nyhan fibroblasts are permissive for the replication of lactic dehydrogenase virus. Immunofluorescent assays show that all of the cells are infected. These hybrid cells produce interferon in response to LDV infection as had previously been shown for LDV-infected macrophages.     

Studies on the in vitro formation of infectious DNA-proteins aggregates from SV40 components.

Complete SV40 virions, disrupted in vitro at pH 10.6, were reassociated by HCl neutralization into a heterogeneous group of DNA-protein aggregates. These contained lower, varying ratios of DNA:protein than control virions. After “reassociation” a portion of the DNA was sensitive to DNase treatment. This treatment led to a loss of the infectivity which had been found in the reassociated aggregates. Nucleic acid is not essential for physical reassociation since aggregates reconstituted from disrupted complete virions had varying amounts of DNA and further aggregates were readily assembled from disrupted empty shells. Disruption by the relatively mild conditions used indicates that the capsid is held in its conformation in part by noncovalent linkage. The rapid reassociation as well as the absence of low molecular weight protein after disruption suggests that there is not a total breakdown by the disruption procedure used. The nucleic acid is probably bound to the capsomeres by noncovalent linkages since disruption does lead to loss of a large amount of the DNA. The rapid in vitro reconstitution of disrupted empty particles to protein aggregates similar to native empty shells strongly suggests that orientation of capsomeres into a tertiary conformation is fairly specific when unencumbered by the presence of DNA. Successful attempts were made to synthesize infectious aggregates in vitro using disrupted [¹⁴C]amino acid-labeled empty shells and [³H]thymidine-labeled SV40 nucleoprotein complex obtained from infected cells 25 hr postinfection.     

Domains of simian virus 40 large T-antigen exposed on the cell surface

The orientation of SV40 large T on the surface of SV40-transformed mouse cells and of human cells infected with nondefective adenovirus 2 SV40 hybrid viruses has been studied. Using antibodies against a synthetic peptide corresponding to a region of 11 amino acids at the carboxyterminus of large T, the surface of formaldehyde-fixed SV40-transformed cells could be specifically stained by indirect immune fluorescence. Staining was inhibited by an excess of the peptide. These data suggest that the carboxyterminus of large T is exposed on the surface of formaldehyde-fixed cells. Antibodies against the carboxyterminus of large T also stained the surface of cells infected with the hybrid viruses Ad2⁺ND1, Ad2⁺ND2, and Ad2⁺ND4. Thus, the carboxytermini of the SV40-specific proteins synthesized in hybrid virus-infected cells are also exposed on the cell surface. When analyzed with an antiserum against purified denatured large T, which among many other determinants also recognizes the large T carboxyterminus, surface fluorescence was observed in cells infected by all three hybridviruses. The surface fluorescence of Ad2⁺ND1-infected cells, expressing an SV40-specific protein of 28 K, and Ad2⁺ND2-infected cells expressing SV40-specific proteins of 42 K and 56 K molecular weight, was completely inhibited by carboxyterminal peptide. However, the surface fluorescence of Ad2⁺ND4-infected cells, expressing SV40-specific proteins up to nearly full size large T, was unaffected by carboxyterminal peptide. Our data suggest that a major portion of large T, located between a region near the carboxyterminus and a region corresponding to the aminoterminus of the 56 K protein, is not exposed on the surface of hybridvirus-infected cells. However, some parts of the aminoterminal one-third of large T appear to be exposed again. We conclude that SV40 large T on the surface of SV40-transformed cells is oriented in a specific manner, suggesting that it is specifically associated with the plasma membrane.     

Simian virus 40 specific cytotoxic lymphocyte clones localize two distinct TSTA sites on cells synthesizing a 48 kD SV40 T antigen

Simian virus 40 specific antigenic sites (TSTA) which react with SV40 specific cytotoxic lymphocytes (CTL) were localized on the surface of mouse embryo fibroblasts (H-2b) transformed with a recombinant plasmid which contain SV40 large T antigen coding DNA sequences (0.65-0.37 map units). These cells (B6/pSV-20-GV) synthesize a large T antigen polypeptide of 48 kD and could be lysed with two independently isolated CTL clones which recognize distinct antigenic sites on SV40 transformed cell surface. These results suggest that at least two distinct TSTA sites are present in cells synthesizing only the amino terminal half of SV40 T antigen.     

Characterization of a temperature-sensitive, DNA-positive, nontransforming mutant of simian virus 40

We have characterized an early mutant of SV40, tsA1642 (M. J. Tevethia and L. W. Ripper, 1977, Virology, 81, 192–211), which differs from other tsA mutants both in its location on the genome and in its phenotype. Marker rescue experiments position tsA1642 between 0.304 and 0.325 map units in the unique coding region for large T antigen. DNA sequencing within this region reveals a single nucleotide substitution at position 1782. Unlike other tsA mutants, the tsA1642 mutation does not result in the metabolic instability of large T antigen at nonpermissive temperature, nor does it lead to a defect in autoregulation of early gene expression. At nonpermissive temperature in the lytic cycle, tsA1642 accumulates viral DNA, T antigens, and late proteins at near wild-type levels, but produces only low levels of infectious virus. In addition to its defect in lytic growth, tsA1642 is markedly defective in transformation. It is unable to transform Brown Norwegian rat kidney (B/NRK) cells to anchorage-independent growth at 40.5°. However, using the same assay, tsA1642 is nonconditionally defective in the transformation of a continuous line of C57B1/6 mouse embryo fibroblasts. TsA1642 generates transformed B/NRK cell lines some of which are temperature sensitive and some temperature resistant for the maintenance of the transformed phenotype. This mutant, therefore, appears to genetically separate, at least partially, some of the functions of large T antigen required for lytic growth from those required for transformation.