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The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.

Physical maps of the genomes of the two newly discovered primate papovaviruses, SA12 and stump-tailed macaque virus (STMV), were generated by restriction endonuclease analysis. The base sequence homologies among the genomes of SA12, stump-tailed macaque virus, and simian virus 40 (SV40) were studied by heteroduplex analysis. Heteroduplexes between SA12 and SV40 DNAs and stump-tailed macaque virus and SV40 DNAs were constructed and mounted for electron microscopy in various amounts of formamide to achieve a range of effective temperatures. At each effective temperature, the regions of duplex DNA in the heteroduplexes were measured and localized on the SV40 physical and functional maps. By analyzing the data from this study and rom our previous study (N. Newell, C. J. Lai, G. Khoury, and T. J. Kelly Jr., J. Virol. 25:193-201, 1978) on the base sequence homology between the genomes of BK virus and SV40, some general conclusions have been drawn concerning the evolutionary relationships among the genomes of the primate papovaviruses. The extent of homology among the viral genomes does not reflect the phylogenetic relationships of their hosts. At comparable effective temperatures Tm - 33 degrees C), the heteroduplexes between the DNAs of BK virus and SV40 contained the largest amount of duplex (about 90%). The heteroduplexes made between SA12 and SV40 DNAs were slightly less homologous, containing about 80% duplex. The heteroduplexes made between SV40 and stump-tailed macaque virus DNAs were only 20% duplex under the same conditions. When the various heteroduplexes were mounted for microscopy at effective temperatures greater than Tm - 33 degrees C, the fraction of the duplex DNA decreased in each case, indicating the existence of considerable base mismatching in the homologous regions. When specific coding or noncoding regions of the viral genomes were compared, the data indicated that the extent of sequence divergence differed markedly from one region to another. In all the heteroduplexes studied, there were two regions, located near the junctions between early and late regions on the SV40 map, which were essentially nonhomologous. All of the heteroduplexes studied showed significantly greater homology in the late region than in early region. Within the late region, the sequences coding for the major capsid polypeptide, VP1, were the most highly conserved.

The 5' terminus of each nascent daughter strand of adenovirus DNA replicated in vitro is covalently linked to a protein with an apparent Mr of 80,000. This protein may represent a precursor to the 55,000-dalton protein known to be linked to the 5' ends of mature adenovirus DNA strands. Partial proteolysis experiments indicate that the 80,000-dalton and 55,000-dalton proteins are structurally related. Furthermore, both proteins are attached to DNA by the same linkage: a phosphodiester bond between the beta-OH of a serine residue in the protein and the 5'-OH of the terminal deoxycytidine residue of the DNA. The role of the 80,000-dalton protein in adenovirus DNA replication is not yet clear, although one reasonable possibility is that it serves as the primer for daughter strand synthesis.

The base sequence homology between the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV) was studied by the heteroduplex method of Ferguson and Davis (J. Mol. Biol. 94:135-149, 1975). When mounted for microscopy in 30% formamide (Tm-35 degrees C), BKV/SV40 heteroduplexes were an average of 92% double-stranded and contained only two small nonhomologous regions that mapped near the junctions between the early and late regions of the SV40 Genome. At higher formamide concentrations, the fraction of duplex DNA in the BKV/SV40 heteroduplexes decreased, indicating significant base mismatching in the homologous regions. The strongest regions of homology were located in the late region.

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.

We have determined the structure of host DNA and viral DNA at the site of integration of Simian virus 40 (SV40) in a line of transformed Balb/c-3T3 cells (SVB400) isolated by single cell cloning after virus infection. Recombinant phage containing integrated viral DNA and flanking host DNA were purified from a genomic library and, in conjunction with restriction endonuclease cleavage analysis of the transformed cell DNA, were used to determine the organization of the integrated viral sequences. There is heterogeneity in the arrangement of the viral sequences resulting from tandem duplications of all or part of the SV40 genome with preservation of the viral-host junctions. The predominant arrangement is the result of tandem duplication of 41% of the SV40 genome from 0.64 to 0.23. Analysis of the structure of integrated viral DNA in SVB400 at different passage numbers and in single cell clones derived from the 20th passage indicated that rearrangements of viral DNA occur after the integration event and continue with passage of the cells. The organization of host sequences before and after the integration of SV40 was determined by restriction endonuclease cleavage analysis of parental 3T3 DNA and SVB400 DNA, and by analysis of recombinant phage isolated from genomic libraries. A deletion of at least 15 X 10(3) bases of host DNA occurred at the site of integration, which indicates that viral integration was not a result of a simple insertion of SV40. Nucleotide sequence analysis of the virus-host junctions showed that retained SV40 sequences were colinear with the viral genome, and that the junctions with SV40 DNA occurred at nucleotide numbers 1377 and 3610. There was no evidence of duplications of viral or host sequences at the junctions, and a comparison of the flanking mouse sequences with the deleted SV40 sequences revealed no significant homology at the point of joining of the two genomes.

The simian virus 40 (SV40) DNA segments present in a series of adenovirus-SV40 hybrids have been mapped with respect to the sites of cleavage of SV40 DNA by restriction endonucleases. Two approaches have been used. First, nucleic acid hybridizations were performed between equimolar quantities of the denatured DNAs of SV40 and each hybrid virus and the radiolabeled transcripts of 11 DNA fragments obtained by cleavage of SV40 DNA by restriction endonuclease from Hemophilus influenzae. Secondly, selected fragments of SV40 DNA produced by the H. influenzae or H. parainfluenzae restriction endonucleases were used to form heteroduplex DNA molecules with adenovirus and adenovirus-SV40 hybrid DNA, which were then analyzed by electron microscopy. The two sets of data were consistent and have permitted alignment of the map of the SV40 segments of the hybrid viruses with the H. influenzae and H. parainfluenzae cleavage maps of SV40. Since cells infected with some of the hybrid viruses contain one or more SV40-specific antigens, the genetic determinants of these antigens could be localized on the cleavage map.

The E46(+) strain of Adenovirus 7 is a mixed-virus population containing defective Adenoviurs 7-SV40 hybrid particles and helper, nonhybrid Adenovirus 7 particles. We have applied electron microscopic mapping techniques to obtain a physical map of the genome of the hybrid particles present in E46(+)PL1, a substrain of E46(+) derived from a single two-hit plaque. DNA molecules extracted from purified E46(+)Pl1 virions were found to be linear duplexes, with a mean lenght of 10.9 mum. When these molecules were denatured and renatured, a unique heteroduplex was formed that presumably derived one of its strands from an Adenovirus 7-SV40 hybrid molecule and the other from a nonhybrid Adenovirus 7 molecule. This heteroduplex was double-stranded, except for a short region near one end where the two strands were not paired. On the basis of measurements of the lengths of the single-and double-stranded regions in the heteroduplex, the structure of the Adenovirus 7-SV40 hybrid genome can be reconstructed as follows: The hybrid genome contains 16% less Adenovirus 7 DNA than the nonhybrid Adenovirus 7 genome. This deletion consists of the segment of DNA that maps between 0.05 and 0.21 molecular lenghts in the nonhybrid Adenovirus 7 DNA molecule. The deleted DNA has been partially replaced by an amount of heterologous DNA equivalent to 75% of the complete SV40 genome. A model for the generation of the hybrid genome is presented.

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.