Electron microscope study of the base sequence homology between simian virus 40 and human papovavirus BK.

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.     

Evolutionary relationships of the primate papovaviruses: base sequence homology among the genomes of simian virus 40, stump-tailed macaque virus, and SA12 virus.

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.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

Addition of extra DNA sequences to simian virus 40 DNA in vivo.

The possible addition of extra sequences to simian virus 40 (SV40) DNA was analyzed by electron microscopy in two different cell systems, productively infected monkey cells and activated heterokaryons on monkey and transformed mouse 3T3 cells. We found that the closed circular DNA fraction, extracted from monkey cells at 70 h after infection with nondefective SV40 at a multiplicity of infection of 6 PFU/cell, contained oversized molesules (1.1 to 2.0 fractional lengths of SV40 DNA) constituting about 8% of the molecules having lengths equal to or shorter than SV40 dinner DNA. The oversized molecules had the entired SV40 sequences. The added DNA was heterogeneous in length. The sites of addition were not specific with reference to the EcoRi site. These results suggest that recombination between monkey and SV40 DNAs or partial duplication of SV40 DNA occurs at many sites on the SV40 chromosome. The integrated SV40 DNA is excised and replicates in activated heterokaryons. In this system, besides SV40 DNA we found heterogeneous undersized and oversized molecules containing SV40 sequences in the closed circular DNA population. Additions differeing in size appeared to be overlapping and to have occurred at a preferential site on the SV40 chromosome. These results support the hypothesis that host DNA can be added to SV40 DNA at the site of integration at the time of excision.     

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.     

Serial enrichment of heteroduplex DNA using a MutS-magnetic bead system

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: III. Heteroduplex analysis of the C. luciliae minicircles

We have investigated the nature of the sequence heterogeneity of the minicircles of Crithidia luciliae kinetoplast DNA by EM heteroduplex analysis of minicircles cleaved with endonuclease HindIII. Approximately 40% of the minicircles showed—after denaturation and reannealing—structures indicative of sequence rearrangements: the majority contained heteroduplex “eyes” interpreted as due to inversions; about 10% of the heteroduplexes yielded structures interpreted as due to translocations and a similar fraction showed insertions and deletions. The category of “eyed” molecules was analyzed in detail: four minicircle segments were found that displayed a high incidence of such eyes indicating that the rearrangements were not scattered at random over the minicircles. Moreover, since also “eyes” were found overlapping two or three of the four regions, we postulate that these segments are capable of recombining with each other. We conclude that specific segmental rearrangements form the main basis of the minicircle sequence heterogeneity in Crithidia.     

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.     

Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VII. Characterization of the Simian Virus 40 RNA Species Induced by Five Nondefective Hybrid Viruses

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.     

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.     

Structure and formation of circular dimers of simian virus 40 DNA.

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.     

Structure of Replicating Simian Virus 40 Deoxyribonucleic Acid Molecules

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.