Viable deletion mutants in the simian virus 40 early region.

For the purpose of isolating hr-t-like mutants of simian virus 40, we have constructed variants that have lost the unique site for the restriction enzyme Taq I at 0.565. Five mutants have been isolated and characterized by restriction enzyme analysis. All of them produce a normal size T antigen. Four produce a t antigen reduced in size as well as in amount; the fifth one does not seem to make any t antigen at all. The ability of these mutants to transform mouse cells in vitro, as tested by anchorage dependence, is clearly altered; however, the defect is only partial. In the same test, the mutants can complement a tsA mutant for transformation and therefore define a second complementation group in the simian virus 40 early region.     

The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.     

Mapping of the late promoter of simian virus 40.

Mapping of the simian virus 40 (SV40) late promoter was carried out in the absence of the viral early protein, large tumor (T) antigen, and replication of the viral DNA template. SV40 late control region DNA fragments, containing specific deletions, were cloned directly upstream from the coding region of the herpes simplex virus-1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The promoter activities of the fragments were determined by measuring the tk transformation frequencies of the chimeric tk constructs in mouse L TK- APRT- (adenine phosphoribosyltransferase-negative) and human 143B TK- cell lines. The following results were obtained. (i) The SV40 control region functions with equal efficiencies in the early and late promoter orientations. (ii) A major late control element has been localized within the G+C-rich 21-base-pair (bp) repeat. Thus, in conjunction with our earlier results, the 21-bp repeat is a bidirectional promoter element functioning as a major component of both the early and late promoters and is an element that enhances the replication efficiency of SV40 DNA. (iii) Minor late promoters have been localized within the minimal replication origin and the 72-bp repeat. (iv) The minimal replication origin is not per se a constituent of the major late promoter; however, both the minimal replication origin and the 21-bp repeat are required for obtaining high levels of late gene expression observed at late times after infection by SV40. (v) The 72-bp repeat exerts a 4- to 5-fold enhancement of late promoter expression.     

Mouse primase initiation sites in the origin region of simian virus 40.

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.     

Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells

Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple genetically marked viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene.In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.     

Organization and expression of early genes of simian virus 40

The early region of simian virus 40 codes for at least two immunologically related polypeptides: large-T and small-t, with apparent molecular weights of 90,000-100,000 and 15,000-20,000, respectively. Because small-t shares methionine-containing tryptic peptides with large-T, the two polypeptides are probably coded, in part, by a common nucleotide sequence. To locate the coding sequences for large-T and small-t in the DNA, the production of these proteins was examined after infection of CV-1 cells with wild-type and deletion mutants of simian virus 40. We found that a deletion at the distal portion of the early region alters the structure of large-T but not of small-t; but deletions within the region between map coordinates 0.59 and 0.55 result in an alteration or absence of small-t and a normal large-T. These findings have been rationalized by a model that proposes the existence of two early mRNAs, one coding for large-T and the other for small-t. Both mRNAs span virtually the entire early region; but the mRNA coding for large-T lacks the nucleotide sequence between map coordinates 0.59 and 0.54. We suggest that small-t is translated from the larger of the two mRNAs, beginning at or near its 5' end and terminating at a termination codon at about map coordinate 0.54. Larger-T, on the other hand, is translated from the shorter mRNA, beginning at the same initiator codon, and, because of the deletion of the terminator codon at 0.54, translation proceeds to the terminator codon at or near map position 0.18.     

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.     

Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.

Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei ofSV80 cells, a continuous line of simian virus 40-transformed human fibroblasts. A five-step purification method was used. Its apparent molecular weight (in sodium dodecyl sulfate/polyacrylamide gels) is approximately 90,000-94,000. It contains a detectable amino-terminal residue.     

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.     

Expression of early genes of origin-defective mutants of simian virus 40.

The nucleotide sequences of eight origin-defective mutants of simian virus 40 have been determined. All of the mutants have suffered deletions, which range in size from 4 to 241 nucleotides. Some of the mutants induce the synthesis of tumor (T) antigen, others do not. Viral mRNA extracted from rat cells transformed by two of the T-antigen-positive mutants has been analyzed by the S1 nuclease technique of Berk and Sharp. Irrespective of the size or the location of the deletions, the 5' ends of viral mRNAs are located approximately the same distance from the A+T-rich region (A-T-T-T-A-T) rather than at a specific site in the viral genome.     

New region of the simian virus 40 genome required for efficient viral transformation.

Viable mutants of simian virus 40 with deletions in three regions of the virus genome (map coordinates 0.21-0.17, 0.59-0.54, and 0.67-0.74) have been tested for their ability to transform rat fibroblasts to anchorage independence. Only those mutants whose deletions occur between 0.59 and 0.55 in the proximal part of the early region are defective in transforming ability. The most severely defective of these transform with less than one-hundredth the efficiency of wild type. They retain their defect when tested in Chinese hamster lung cells and when infection is initiated with viral DNA instead of intact virions. Complementation for transformation can be observed between these transformation-defective deletions and a simian virus 40 temperature-sensitive A mutant.     

Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen.

Simian virus 40 large tumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.     

Role of the amino-terminal domain of simian virus 40 early region in inducing tumors in secretin-expressing cells in transgenic mice

BACKGROUND & AIMS: The early region of simian virus 40 (SV40) encodes 2 transforming proteins, large T (Tag) and small t antigen, that produce neuroendocrine tumors in the intestine and the pancreas when expressed in secretin cells of transgenic mice. METHODS: Two SV40 early-region transgenes containing a deletion that eliminated expression of the small t antigen were expressed in transgenic mice under control of the secretin gene. The 2 lines of mice, one expressing the native large T antigen and the other T antigen with a mutation in its N-terminal J domain, were examined to determine which biological activities of the SV40 early region were required for tumorigenesis. RESULTS: Most animals expressing wild-type large T antigen developed pancreatic insulinomas and lymphomas and died between 3 and 6 months of age. However, small intestinal neoplasms were extremely rare in the absence of small t antigen expression. Transgenic lines expressing the J domain mutant failed to develop tumors. CONCLUSIONS: Transformation of secretin-producing enteroendocrine cells by SV40 requires functional cooperation between intact large T and small t oncoproteins. In contrast, large T antigen alone is sufficient to induce tumors in the endocrine pancreas and thymus.     

Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression.

The effect of DNA methylation on polyoma virus and simian virus 40 gene expression was investigated. For this purpose, the cytosines of all C-G dinucleotides of the viral DNAs were methylated by the use of rat liver methylase and the completeness of methylation was verified by dinucleotide analysis and restriction endonuclease treatment. The biological activity of unmethylated and fully methylated DNAs was tested by microinjecting them into tissue culture cells. The functions analyzed included early and late viral gene expression, viral DNA replication, oncogenic transformation efficiency, and virus maturation. No difference in any of these biological functions was observed between methylated and unmethylated DNA. Early gene expression of methylated DNA is not the result of demethylation because viral DNA reextracted from the injected cells, under nonpermissive conditions, retained the methylation pattern of the input DNA. In contrast, viral DNA extracted from transformed cells or from intact virus particles was partially or completely demethylated.