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.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.     

Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells.

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.     

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.     

Interlocking of plasmid DNAs due to Lac repressor-operator interaction.

The presence of a single lac repressor binding sequence on plasmid DNAs is shown to mediate the formation of interlocked dimers in E. coli. The presence of both homo- and hetero-interlocked dimers suggests that the lac repressor complex can bring together randomly two plasmid DNA molecules to facilitate gyrase-mediated interlocking. The exclusive formation of multiply intertwined dimers also suggest that the lac repressor complex may bind simultaneously to a pair of replicated daughter plasmid molecules prior to their segregation. The formation of interlocked plasmid DNAs can be indicative of interaction between two DNA bound proteins in vivo.     

Alternate polypurine tracts affect rous sarcoma virus integration in vivo

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.     

Physical map of infectious baboon type C viral DNA and sites of integration in infected cells.

Three species of unintegrated viral DNAs were found in permissive cells infected with baboon type C virus. The major species was a 9.0-kilobase (kb) linear DNA that was infectious. A restriction endonuclease map of this DNA was constructed and oriented with respect to the viral RNA. The linear DNA had a 0.6-kb sequence repeated at each terminus. These terminal repeat sequences were required for infectivity of the viral DNA. The minor species of the unintegrated viral DNAs were covalently closed circles of 9.0 and 8.4 kb. The smaller circle was in two- to threefold excess over the larger circle. The difference appeared to be that the smaller circle lacked one of the two 0.6-kb repeat sequences found in the larger circle. Restriction endonuclease maps of the integrated viral DNAs were constructed, and the sequences on both viral DNA and cellular DNA that are involved in integration were determined. The integrated viral DNA map was identical to that of the unintegrated infectious 9.0-kb linear DNA. Therefore, a specific site in the terminal repeat sequence of the viral DNA was used to integrate with the host cell DNA. The sizes of the cellular DNA fragments were different from clone to clone but stable with cell passage. Therefore, many sites in the cell DNA can recombine with the viral DNA.     

Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.     

Correlation between cell killing and massive second-round superinfection by members of some subgroups of avian leukosis virus.

Avian leukosis viruses of subgroups B, D, and F are cytopathic for chicken cells, whereas viruses of subgroups A, C, and E are not. The amounts of unintegrated linear viral DNA in cells at different times after infection with cytopathic or noncytopathic viruses were determined by hybridization and transfection assays. Shortly after infection, there is a transient accumulation of unintegrated linear viral DNA in cells infected with cytopathic avian leukosis viruses. By 10 days after infection, the majority of this unintegrated viral DNA is not present in the infected cells. The transient cytopathic effect seen in these infected cells also disappears by this time. Low amounts of unintegrated linear viral DNA persist in these cells. Cells infected with noncytopathic viruses do not show this transient accumulation of unintegrated viral DNA. Cells infected with cytopathic viruses and subsequently grown in the presence of neutralizing antibody do not show the transient accumulation of unintegrated viral DNA or cytopathic effects. These results demonstrate a correlation between envelope subgroup, transient accumulation of unintegrated linear viral DNA, and transient cell killing by avian leukosis viruses. The cell killing appears to be the result of massive second-round superinfection by the cytopathic avian leukosis viruses.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.     

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.