Nonviable mutants of simian virus 40 with deletions near the 3'' end of gene A define a function for large T antigen required after onset of viral DNA replication

Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40.     

A biochemical method for increasing the size of deletion mutations in simian virus 40 DNA.

A biochemical procedure has been developed for increasing the size of deletion mutations in closed-circular, double-stranded DNA. Specifically, the deletion in a simian virus 40 (SV40) mutant (dl892), a viable deletion mutant lacking about 35 base-pairs at 0.675 to 0.68 SV40 map units, has been enlarged to produce a series of new mutants lacking from 45 to 90 base-pairs. To enlarge the deletion, the following steps were involved: mutant and wild-type SV40 DNAs were cleaved with the EcoRI restriction endonuclease to form full-length linear molecules, and then they were mixed, denatured and annealed to reform duplex structures. The linear heteroduplex DNAs were re-circularized by treatment with DNA ligase. These closed-circular molecules, half of which contain a small deletion loop at 0.675 to 0.68 map units, were treated with S1 endonuclease, which cleaves at the site of the deletion loops to produce linear molecules with ends at 0.675 to 0.68 map units. Mutants containing enlarged deletions were obtained by infecting permissive monkey kidney cells with the linear DNA. The location of the enlarged deletion in each mutant was compared to that of the parental mutant, dl892. One end of the parental deletion (at about 0.675 map units) remained essentially unmoved; the deletions were enlarged almost entirely in the opposite direction. Since these mutants were all selected for viability, 0.675 map units very likely marks the boundary between a region of the genome previously shown to contain non-essential sequences (from 0.675 to about 0.74 map units) and a portion of the genome required for lytic growth.     

The physical and genetic organization of a viral genome.

Mutants of SV40 with deletions of a few to several thousand base pairs have been constructed in vitro and cloned in cultured monkey cells. The location and size of these deletions has been determined by restriction endonuclease mapping and electron microscopic and enzymatic analysis of DNA heteroduplex molecules. Analysis of the phenotype of these deletion mutants permits us to specify the locations of the known SV40 genes, in particular, the novel organization of SV40s two early genes that are required for oncogenesis.     

Construction and Characterization of Viable Deletion Mutants of Simian Virus 40 Lacking Sequences near the 3′ End of the Early Region

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3′ end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases HinfI or MboII, followed by removal of approximately 25 base pairs of DNA from the 5′ termini using λ-5′-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (dl2401 through dl2405) was prepared, using as a parent SV40 mutant dl862, which has a deletion at the single HpaII site (0.725 map unit). A second set (dl2491 through dl2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental dl862, although mutant progeny yields were usually lower than yields of dl862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants dl2402, dl2492, and dl2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.     

Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene.

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.     

Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells.

Mutants of SV40 with deletions of various sizes mapping between 0.54 and 0.59 on the genome grow at a rate equal to or slightly slower than that of wild-type virus, in a range of host cells. Their ability, however, to induce transformation in several mouse, rat and rabbit cell lines is impaired. The extent of transformation observed is dependent upon the assay used to measure it, but in general, the ability of the mutants to transform falls as the size of the deletion increases. In addition, rat embryo fibroblasts transformed by deletion mutants have fewer of the characteristics of a fully transformed phenotype (for example, growth in low serum, increased saturation density, growth in semi-solid medium) than those transformed by wild-type virus. During lytic infection, immunoprecipitable T antigen produced by the deletion mutants is of the same size as that seen during infection with wild-type virus, and is present at a similar level. Mutant virus-coded small t protein, however, is reduced in size compared with that from wild-type virus. For each mutant, the reduction in protein size is dependent upon the amount of DNA deleted, but not on the relative position of the deletion in the genome. These results demonstrate that the DNA sequences mapping between 0.54 and 0.59 on the viral genome code for the small t protein, and that SV40-induced transformation is at least partially dependent upon the expression of this protein.     

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.     

Mapping of Simian Virus 40 Early Functions on the Viral Chromosome

The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2(+)ND(4), is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R(1) restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2(+)ND(4), called Ad2(+)ND(4del), was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R(1) endonuclease cleavage point.     

Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.     

Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome.

Two mutants of simian virus 40, dl-1261 and dl-1262, have deletions that map between coordinated 0.90 and 0.95 (Cole et al., J. Virol 24:277--294, 1977). Both affect the structure of the two minor proteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence analysis. Mutant dl-1261 is deleted of 54 base pairs, is temperature sensitive for the protein defined by the D complementation group, and promotes the synthesis of shorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of temperature and has a deletion of 36 base pairs, 23 of which overlap the deletion in dl-1261. Since these mutants produce normal VP1, the deleted regions probably have no function in the splicing of precursor RNA to the VP1 mRNA.     

Polyoma viruses with mutations at endonuclease HindII site 1: alterations at the COOH terminus of VP1.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.     

Mutagenesis of cauliflower mosaic virus

A series of insertion mutants of cauliflower mosaic virus (CaMV) DNA has been constructed in vitro. These insertions consist of a short DNA sequence (10 or 22 bp) containing a restriction endonuclease site (SmaI) not represented on the viral DNA. Viral infectivity was analyzed by inoculating plants with the mutated cloned viral DNA and observing symptoms. Insertions within ORFVII, and in one site within the large intergenic region, did not interfere with viral infectivity, whilst insertions within ORFII and at the end of ORFIV retarded the development of viral symptoms. All other insertion mutants analyzed were lethal. CaMV with a deletion of 105 bp within ORFVII was viable. Such viable mutants can be used to construct additional deletions or to insert foreign DNA into the viral genome.     

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.     

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).     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

SV40 deletion mutants lacking the 21-bp repeated sequences are viable, but have noncomplementable deficiencies.

We have constructed deletion mutants of simian virus 40 (SV40) lacking the two tandemly repeated copies or all three copies of the 21-bp repeated sequence located in the origin region. The mutants were viable, but had lower infectivities compared to the wild type. The mutant lacking two copies of the 21-bp repeat grew fairly well indicating that the one copy of the 21-bp repeat it contains is adequate. The other mutant lacking all the three copies of the 21-bp repeat was also viable but grew poorly. The viability of this mutant suggests that the upstream 72-bp repeated sequence compensates, though only partially, for the absence of the 21-bp repeat. The growth deficiencies of the deletion mutants could not be overcome by complementation with temperature-sensitive helper mutants providing either the early or the late functions of the virus, suggesting that the deficiencies lie in both early and late gene expression and/or in replication.     

A single cleavage of Simian virus 40 (SV40) DNA by a site specific endonuclease from Thermus aquaticus, Taq I.

A site specific endonuclease from Thermus aquaticus, Taq I, cleaves Simian virus 40 (SV40) DNA at a single site. The cleavage site was localized on the physical map by double digestions, using the previously characterized fragments produced by digestion with Hae II, Hae III, AluI, HhaI, HinfI, or BstI. The Taq I site is located at the position that is 56.5% of the unit length from the Eco RI site.     

Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function.

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.