The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells.

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.     

Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.     

Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent about one-half of the coding region for large T antigen. We present evidence here that F8dl is able to transform mouse cells in a focus assay and that cell lines derived from these foci exhibit fully transformed phenotypes, have integrated mutant genomes, and express mutant-encoded proteins. This result implies that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for the maintenance of transformation. In addition, we have found that cells fully transformed by F8dl produce a 53,000-dalton nonviral tumor antigen (p53) that is as unstable as the p53 of untransformed cells. From this result we infer that transformation by simian virus 40 does not require the stabilization of p53.     

A fragment of the simian virus 40 early genome can induce tumors in nude mice.

Cell lines transformed by simian virus 40 mutant F8dl (deleted from 0.168 to 0.424 map units, corresponding to the carboxy-terminal 62% of the wild-type simian virus 40 large tumor antigen) are tumorigenic in nude mice. Four of five C3H10T1/2 cell lines transformed by F8dl were tumorigenic in nude mice, whereas two of two wild-type transformants were tumorigenic.     

Isolation and characterization of simian virus 40 early region deletion mutants.

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

A cis-acting sequence promotes removal of simian virus 40 DNA from the replication pool.

Pulse-labeled simian virus 40 (SV40) DNA is removed from the pool of molecules available for replication (i.e., it ceases to reenter replication) a few hours after synthesis. We studied this cessation of reentry with mutants containing different deletions in the structural genes of SV40. The DNAs of two independent deletion mutants, dl-1007 (24% deletion) and dl-1003 (8% deletion), were used as templates for further DNA synthesis (i.e., they reentered replication) to a greater extent than was wild-type DNA. The alteration in reentry kinetics was not because the DNAs were smaller; other deletion mutations that were from 76 to 85% of the length of wild-type DNA (dl-BE and dl-1133 with a deletion in the late region and F8dl with a deletion in the early region) did not reenter replication to a greater extent than the wild type did. Cotransfection experiments showed that the mutant phenotypes of dl-1007 and dl-1003 were poorly complemented, if at all, by the wild type. Thus, we propose that there is a cis-acting sequence located in the HindIII E fragment of SV40, not present in either of these mutants, that promotes the efficient removal of DNA from the replication pathway.     

Simian virus 40 T-antigen: identification of tryptic peptides in the C-terminal region and definition of the reading frame

T-antigen (the simian virus 40 A cistron protein) was purified by immunoprecipitation and electrophoresis on polyacrylamide gels from monkey kidney CV-1 cells infected with simian virus S (SV-S), dl1263, or dl1265 and digested with trypsin. The tryptic peptides, labeled with [35S]methionine, [35S]cysteine, or [3H]proline, were fractionated either by chromatography on Chromobead-P resin or by two-dimensional electrophoresis and chromatography on cellulose thin layers. The T-antigen of SV-S was shown to give rise to a proline-rich (approximately 6 mol of proline) tryptic peptide which was absent in dl1265 T-antigen and hence, on the basis of DNA sequence data, must originate from the C-terminus of the SV-S protein. T-antigen from dl1265, but not SV-S, yielded a cysteine-rich terminal tryptic peptide. The presence of these cysteines caused the protein to be retarded during electrophoresis under the usual conditions in polyacrylamide gels. The T-antigen of dl1263 possessed the proline-rich tryptic peptide; the data are consistent with there being only one peptide altered by the deletion. Both deletion mutants produced a T-antigen that had a higher electrophoretic mobility than SV-S T-antigen but still a larger apparent molecular weight than was predicted by the DNA sequence. The major form of T-antigen found in several lines of 3T3 cells transformed by these mutants was indistinguishable from the T-antigen found in infected cells, and in addition seemed to associate normally with the host-coded 53,000-dalton protein. Except for a minor form of T-antigen with a slightly lower mobility in gels but the same C-terminus, no other polypeptides were detected among the extracted and immunoprecipitated proteins whose electrophoretic mobility was affected by either deletion.     

Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

The sequences between 0.59 and 0.54 map units on SV40 DNA code for the unique region of small t antigen.

To demonstrate directly that the carboxy terminal portion of simian virus 40 (SV40) small t is encoded by a sequence of nucleotides from the region between 0.59-0.54 map units on SV40 DNA, we characterized the putative shortened forms or fragments of small t produced by mutants of SV40 (dl 884, dl 885, dl 890) with deletions in this region of the genome. Attempts to isolate the putative fragments of small t from mutant-infected cells, or from cell-free systems primed with mRNA from mutant-infected cells, resulted in only low yields of the fragments. Experiments using purified SV40 mRNA in low background cell-free systems, in which large T and small t could be detected without immunoprecipitation, suggested that these low yields were accounted for by reduced amounts of mRNA coding for the shortened forms of small t present in the mutant-infected cells. Larger amounts of putative fragments of small t were produced by translation of deletion mutant cRNA (complementary RNA synthesized in vitro using purified deletion mutant DNA and E. coli RNA polymerase). Fingerprint analysis of the proteins produced showed that they contain most, if not all, of the methionine peptides common to small t and large T. Furthermore, the fragments of small t produced in response to dl 884 and dl 890 lack two methionine peptides that are present in small t but not in large T. These data provide direct evidence that the region between 0.59-0.54 map units on SV40 DNA codes for polypeptide sequences that are unique to small t, and establishes that the nucleotide sequences from the region between 0.59-0.54 map units are both a coding sequence (for small t) and an intervening sequence (for large T).     

Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis.

The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.     

Genomic arrangement of an adenovirus-simian virus 40 hybrid virus, Ad2+ND4del.

Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.