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At least 19 recordsLinked to original sources
PubMed1981

Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.

We have determined the nucleotide sequence of portions of two circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host--vector system. The region whose sequence was determined represents the circle junction site--i.e., the site at which the ends of the unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion of the long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center of a 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA--unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat--is very similar to the structure of certain transposable elements. Several features of the sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNATrp, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.

Avian Sarcoma Viruses↗
PubMed1986

Investigation of possible vaccine-induced epizootics of infectious bovine rhinotracheitis, using restriction endonuclease analysis of viral DNA.

Viral DNA was extracted from each of 14 modified-live (ML) bovine herpesvirus 1 vaccines, representing all of the ML infectious bovine rhinotracheitis virus (IBRV) vaccines licensed by the US Department of Agriculture for use in cattle. Restriction endonucleases Pst I and Bgl II were used to establish restriction enzyme patterns for the vaccinal viruses. Viral DNA from isolates obtained from 6 field samples of IBRV (1 from Colorado, 1 from West Virginia, 3 from Wisconsin, 1 from South Dakota) were digested with restriction endonucleases, and patterns were compared to evaluate the role of vaccinal virus in these field epizootics of infectious bovine rhinotracheitis. Animals from which field samples were obtained had been vaccinated with ML IBRV vaccine before the epizootic of infectious bovine rhinotracheitis occurred in the herds. In 2 of the 6 field samples, DNA restriction endonuclease analyses patterns from the isolates were indistinguishable from the pattern for the vaccinal viruses used. In the remaining 4 field samples, DNA restriction endonuclease analyses patterns of the IBRV from isolates were different from those of the vaccinal viruses.

Animals↗
PubMed1976

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.

Adenoviruses, Human↗
PubMed2003

The (52-96) C-terminal domain of Vpr stimulates HIV-1 IN-mediated homologous strand transfer of mini-viral DNA.

Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.

Amino Acid Sequence↗
PubMed1988

Association of mitochondria DNA with viral DNA in purified preparations of poxviruses.

The presence of cellular material in purified preparations of Shope fibroma virus (SFV) and two orthopoxviruses (vaccinia and Indiana), was investigated. Mitochondria were observed in purified preparations of SFV by electron microscopy and mitochondrial (mt) DNA was identified in restricted viral DNA by Southern blot hybridization with cloned mouse mt DNA. Mitochondrial DNA was also detected in vaccinia and SFV DNAs extracted from purified virions treated with DNase I followed by core isolation. The viral and mt DNAs could be separated on the basis of their size by agarose gel electrophoresis, but not by their buoyant density by centrifugation in cesium chloride gradients. These findings led us to re-examine previously reported results showing some homology between SFV, a leporipoxvirus and Indiana, an Orthopoxvirus (Berkowitz and Pogo, Virology 142, 437-440, 1985) using cloned fragments of SFV DNA instead of the entire viral DNA. The results indicated that cross-hybridization between SFV and Indiana DNAs was due in part to mt DNA but they also revealed an unrecognized region of homology between the two poxvirus genera.

DNA, Mitochondrial↗
PubMed1977

Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells.

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.

Adenoviruses, Human↗
PubMed1984

Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication.

An adenovirus mutant, Ad2ts111, has previously been shown to be temperature sensitive for viral DNA replication in vivo and also to induce degradation of cellular DNA. Soluble nuclear extracts prepared from Ad2ts111-infected HeLa cells grown at either the permissive (32 degrees C) or the nonpermissive (39.5 degrees C) temperature are thermolabile for elongation but not for initiation of DNA replication in vitro. Adenovirus single-stranded-DNA-binding protein purified from wild-type-infected cells can complement these extracts at the restrictive temperature in vitro. The DNA-binding protein synthesized in Ad2ts111-infected cells is stable at the nonpermissive temperature and is phosphorylated, as is the wild-type protein. In contrast, the mutant DNA-binding protein synthesized in Ad5ts125-infected cells is unstable. Ad2ts111 and Ad5ts125 do not complement each other for virus growth in vivo. These results suggest that Ad2ts111 contains a mutation in the DNA-binding protein that affects viral DNA synthesis. Finally, we demonstrated that, unlike viral DNA synthesis, the induction of cellular DNA degradation in Ad2ts111-infected cells is not temperature sensitive and that this phenotype is a result of a mutation in early region 1 on the virus genome. Thus, the two phenotypes displayed in Ad2ts111-infected cells, namely, the temperature-sensitive replication of viral DNA and the degradation of cell DNA, are the result of two separate mutations.

Adenoviruses, Human↗
PubMed2003

Efficient extraction of viral DNA and viral RNA by the Chemagic viral DNA/RNA kit allows sensitive detection of cytomegalovirus, hepatitis B virus, and hepatitis G virus by PCR.

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.

Cytomegalovirus↗
PubMed1999

Detection of SV40 like viral DNA and viral antigens in malignant pleural mesothelioma.

This study investigated the presence of simian vacuolating virus 40 (SV40) deoxyribonucleic acid (DNA) in malignant pleural mesothelioma, non-neoplastic mesothelium and pleural carcinoma metastasis and correlated these data with immunohistochemistry for SV40 viral antigens. The novel Primed In Situ (PRINS) method was applied to detect the presence of SV40 DNA in situ in tissue sections of malignant mesothelioma (n = 25), non-neoplastic mesothelium (n = 30) and pleural carcinoma metastasis (n = 30). Immunohistochemistry with an SV40-specific antibody was applied for detection of the SV40 viral antigen in the same material. SV40 DNA and expression of one of the viral proteins (small t-antigen) was found in approximately 60% of the investigated mesothelioma cases in contrast to non-neoplastic mesothelium and carcinoma metastasis that were negative for both SV40 DNA and SV40 viral antigens. These results suggest that simian vacuolating virus 40 deoxyribonucleic acid may be biologically active as there was also immunoreactivity for simian vacuolating virus 40 viral antigen in those cases positive for simian vacuolating virus 40 deoxyribonucleic acid with the primed in situ reaction. Simian vacuolating virus 40 viral deoxyribonucleic acid and antigens may be potential markers for neoplastic mesothelium that may prove useful in the rather difficult histopathological differential diagnosis between malignant mesothelioma and reactive mesothelium or pleural carcinoma.

Antigens, Viral↗
PubMed1977

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.

Animals↗
PubMed1998

The UL112/113 gene products of human cytomegalovirus which colocalize with viral DNA in infected cell nuclei are related to efficient viral DNA replication.

The UL112/113 gene products of human cytomegalovirus (HCMV) were shown by transient complementation ori Lyt-dependent DNA replication assay to be early viral proteins required for efficient viral DNA synthesis. By immunofluorescence analysis followed by fluorescence in situ hybridization, we showed that UL112/113 gene products of HCMV are colocalized with viral DNA prior to and during viral DNA replication in infected cell nuclei. We have used an anti-sense RNA approach for functional analysis of the UL112/113 gene in HCMV. The astrocytoma cell line U373-MG was used for permanent expression of the anti-sense UL112/113 gene. Expression of the anti-sense RNA in this cell line significantly blocked expression of UL112/113 gene products and viral DNA replication, indicating that the UL112/113 gene products are related to efficient viral DNA replication.

Cell Line↗
PubMed1982

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.

Animals↗
PubMed1984

Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA.

Recombinant viral genomes carrying a selectable drug resistance marker have been constructed by insertion of a hybrid gene for neomycin resistance into the helper-independent adenovirus vector, delta E1/X. The hybrid gene consists of sequences coding for the aminoglycoside 3'-phosphotransferase II from Tn5, under the control of the simian virus 40 early promoter, and renders mammalian cells resistant to the neomycin analog, G-418. Most of adenovirus early region 1 is deleted from delta E1/X (nucleotides 455 to 3330), and recombinant viral genomes carry the hybrid gene in its place. The large and small XbaI fragments of delta E1/X were ligated to the hybrid gene, and the mixture was transfected into 293 cells. Single plaques were isolated and subsequently passaged in 293 cells to produce virus stocks. The recombinant viruses efficiently rendered cultured rat (Rat2) and simian (CV1) cells resistant to G-418. Cloned cell lines selected for resistance to G-418 contained viral DNA integrated into the host cell genome, demonstrating that early region 1 is not essential for integration of the viral genome. Southern transfer experiments revealed that (i) the sites of integration in the host genome were not unique; (ii) in general, transformed CV1 cell lines contained single-copy, full-length viral genomes, colinear with the infecting virus; (iii) transformed Rat2 cell lines generally contained one to several copies of full-length viral genomes integrated colinearly with the infecting viral DNA; and (iv) three of these five lines of transformed Rat2 cell lines contained tandemly repeated viral DNA sequences in which the right and left ends of the viral genome were joined to each other.

Adenoviruses, Human↗
PubMed1991

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.

Alkaline Phosphatase↗
PubMed1976

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.

Animals↗