Molecular cloning of covalently closed circular DNA of bovine leukemia virus

The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs.     

Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

Structure and Functions of the Kirsten Murine Sarcoma Virus Genome: Molecular Cloning of Biologically Active Kirsten Murine Sarcoma Virus DNA

The unintegrated closed circular form of viral DNA prepared from NIH3T3 cells infected with Kirsten murine sarcoma virus was cloned into bacterial plasmid pBR322. The closed circular DNA, which consisted of two different-sized populations, was enriched from the virus-infected cells, linearized with BamHI, and inserted into pBR322 DNA. Four different recombinant DNAs (clones 2, 4, 6, and 7) were obtained, and a physical map of each was constructed by using various restriction enzymes. Clone 4 DNA had the largest insertion, corresponding to a complete copy of the linear DNA. This suggested that this insertion contained two copies of the 0.55-kilobase pair long terminal redundant sequence. Clone 2 and clone 6 insertion DNAs had deletions of 0.2 and 0.5 kilobase pair, respectively, which mapped near the right end (3' side of viral RNA) of the linear DNA. Clone 7 DNA appeared to have a deletion of a single copy of the large terminal redundant sequence. Transfection of BALB3T3 cells with the clone 4 DNA insertion showed that this DNA had transforming activity. The efficiency of transfection with clone 4 Kirsten murine sarcoma virus DNA was enhanced eightfold by inserting EcoRI-cleaved viral DNA into the EcoRI site of pBR322. The EcoRI-inserted DNA produced foci with single-hit kinetics, suggesting that a single molecule of Kirsten murine sarcoma virus DNA can induce transformation. Results of transfections with EcoRI-inserted Kirsten murine sarcoma virus DNA cleaved with various restriction enzymes suggested that the first 3.3-kilobase pair region at the left end of the linear DNA is important for the initiation of transformation or maintenance of transformation or both.     

Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus.

Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.     

Deletion analysis of the DNA sequence required for the in vitro initiation of replication of bacteriophage lambda

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Integration of viral DNA into the genome of the adenovirus type 2-transformed hamster cell line HE5 without loss or alteration of cellular nucleotides

Hamster cell line HE5 has been established from primary LSH hamster embryo cells by transformation with adenovirus type 2 (Ad2) (1). Each cell contains two to three copies of integrated Ad2 DNA (2, 3). We cloned and sequenced the sites of junction between viral and cellular DNAs. The terminal 10 and 8 nucleotides of Ad2 DNA were deleted at the left and right sites of junction, respectively. The integrated viral DNA had an internal deletion between map units 35 and 82 on the Ad2 genome. At the internal site of deletion, the remaining viral sequences were linked via a GT dinucleotide of unknown origin. From HE5 DNA, the unoccupied sequence corresponding to the site of insertion was also cloned and sequenced. Part of this sequence was shown to be expressed as cytoplasmic RNA in HE5 and primary LSH hamster embryo cells. The viral DNA had been inserted into cellular DNA without deletions, rearrangements or duplications of cellular nucleotides at the site of insertion. Thus, insertion of Ad2 DNA appeared to have been effected by a mechanism different from that of bacteriophage lambda in Escherichia coli and from that of retroviral genomes in vertebrates. It was conceivable that the terminal viral protein (4) was somehow involved in integration either on a linear or a circularized viral DNA molecule.     

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.     

Isolation and characterization of recombinant DNA clones of avian retroviruses: size heterogeneity and instability of the direct repeat

Unintegrated proviral DNA of Schmidt-Ruppin B Rous sarcoma virus was cloned in the bacteriophage lambda vector Charon 21A. A total of 12 independent recombinant lambda SRBtd clones which were derived from the transformation-defective component in the viral preparation were analyzed with restriction endonucleases and molecular hybridization techniques. Three classes of clones were observed. Type I clones contained a 5.0-megadalton insert of viral DNA, type II clones contained phage with two size classes of inserts (5.0 and 5.2 megadaltons), and one type III clone contained only a 5.2-megadalton insert. The smaller insert present in type II clones appeared to be derived by deletion of one copy of a directly repeated sequence which was present in the larger insert. Mapping data indicated that the deletion includes all or part of the terminal repeat found in linear double-stranded proviral DNA. Similar results were obtained from lambda RAV2 recombinant clones derived from Rous-associated virus type 2. Analysis of DNA from type II and type III clones of lambda SRBtd and lambda RAV2 revealed limited heterogeneity in the size of the direct repeat.     

Sequence organization of feline leukemia virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.     

Organization of type C viral DNA sequences endogenous to baboons: analysis with cloned viral DNA.

Unintegrated linear and circular forms of baboon endogenous type C virus M7 DNA were prepared from M7-infected cells by chromatography on hydroxyapatite columns, and the circular DNAs were purified in cesium chloride-ethidium bromide equilibrium density gradients. The circular DNAs were linearized by digestion with EcoRI, which had a unique site on the viral DNA. The linearized DNA was then inserted into lambda gtWES. lambda B at the EcoRI site and cloned in an approved EK2 host. Molecularly cloned full-length M7 DNA was restricted with BamHI, and the resulting five subgenomic fragments were then subcloned individually in plasmid pBR322. The organization and sites of integration of the approximately 100 copies of M7 DNA sequences endogenous to baboons were investigated by digesting the DNA with restriction enzymes and identifying the virus-specific fragments by hybridization to labeled probes made by using the molecularly cloned full-length and subgenomic fragments of the viral DNA. We found that most of the endogenous sequences had sizes and organizations similar to those of the unintegrated viral DNA and therefore approximately similar to the RNA of the infectious virus. A few of the multiple sequences had deletions in the 3' end (envelope region), and some of the sequences either lacked or contained modified BamHI restriction sites on the 5' end of the viral DNA. The endogenous viral DNA sequences were nontandem, uninterrupted, and colinear with the DNA of the infectious virus, and they were integrated at different sites in the baboon DNA, like the M7 proviral DNA sequences acquired upon infection.     

Common sites for recombination and cleavage mediated by bacteriophage T4 DNA topoisomerase in vitro

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.     

In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of Moloney murine leukemia virus.

Detergent-disrupted virions of Moloney murine leukemia virus synthesize a 9 kbp double-stranded infectious DNA. It contains mainly full-length, single-stranded DNA, and its infectivity and size are insensitive to digestion by the single-strand-specific S1 nuclease. Analysis of fragmentation of the DNA using restriction endonucleases has shown that it is indistinguishable from the linear double-stranded DNA synthesized in infected cells. On the basis of the positions of the cleavage sites for a number of enzymes, the 9 kbp DNA has a 575 base direct terminal repetition. It is longer than the viral RNA at both ends, evidently due to repetitive copying of segments of the RNA. Virions also synthesize an 8.4 kbp double-stranded circular DNA that lacks one copy of the terminal repetition, as well as viral DNA longer than 9 kbp. The enzymatic machinery in the virions of retroviruses therefore appears to be responsible for all the steps involved in making fully double-stranded linear and one form of circular DNA.     

Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization with a cloned viral DNA fragment. The size of the viral DNA was estimated at ca. 158 kilobase pairs. Restriction endonuclease patterns suggested structural similarities to cottontail herpesvirus DNA.     

Rearrangements in unintegrated retroviral DNA are complex and are the result of multiple genetic determinants.

We used a replication-competent retrovirus shuttle vector based on a DNA clone of the Schmidt-Ruppin A strain of Rous sarcoma virus to characterize rearrangements in circular viral DNA. In this system, circular molecules of viral DNA present after acute infection of cultured cells were cloned as plasmids directly into bacteria. The use of a replication-competent shuttle vector permitted convenient isolation of a large number of viral DNA clones; in this study, over 1,000 clones were analyzed. The circular DNA molecules could be placed into a limited number of categories. Approximately one-third of the rescued molecules had deletions in which one boundary was very near the edge of a long terminal repeat (LTR) unit. Subtle differences in the patterns of deletions in circular DNAs with one versus two copies of the LTR sequence were observed, and differences between deletions emanating from the right and left boundaries of the LTR were seen. A virus with a missense mutation in the region of the pol gene responsible for integration and exhibiting a temperature sensitivity phenotype for replication had a marked decrease in the number of rescued molecules with LTR-associated deletions when infection was performed at the nonpermissive temperature. This result suggests that determinants in the pol gene, possibly in the integration protein, play a role in the generation of LTR-associated deletions. Sequences in a second region of the genome, probably within the viral gag gene, were also found to affect the types of circular viral DNA molecules present after infection. Sequences in this region from different strains of avian sarcoma-leukosis viruses influenced the fraction of circular molecules with LTR-associated deletions, as well as the relative proportion of circular molecules with either one or two copies of the LTR. Thus, the profile of rearrangements in unintegrated viral DNA is complex and dependent upon the nature of sequences in the gag and pol regions.