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PubMed2001

In vivo accumulation of cyclin A and cellular replication factors in autonomous parvovirus minute virus of mice-associated replication bodies.

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases alpha and delta in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.

Animals↗

PubMed1984

The interaction of Escherichia coli replication factor Y with complementary strand origins of DNA replication. Contact points revealed by DNase footprinting and protection from methylation.

A defined region of the viral (+) strand of phi X174 and of each strand of pBR322 DNA serves as an effector for the ATPase activity of replication factor Y from Escherichia coli. These loci can also function as complementary strand origins of DNA replication in a single-stranded circular leads to replicative form pathway whose protein requirements are characteristic of phi X174 DNA. Despite this functional similarity, these three sites possess no extensive sequence homology. To uncover a possible common structural determinant, factor Y recognition sequences were treated with pancreatic DNase or dimethyl sulfate in the presence and absence of this replication protein. When factor Y was present, the action of the nuclease was altered in a similar manner on each of the three templates, indicating that factor Y was bound to the entire length of its effector site. Factor Y-mediated modification of the dimethyl sulfate methylation patterns gave evidence of specific, tight protein-DNA contacts. Protection maps, devised by plotting the results of the methylation and footprinting experiments on duplex structures, suggest that tertiary interactions are either involved in the formation of a factor Y effector site or are induced by the binding of the protein.

Adenosine Triphosphatases↗

PubMed1998

A novel filamentous phage, fs-2, of Vibrio cholerae O139.

A novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14. The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide. The purified phage formed a turbid plaque when spotted on a lawn of the host organisms. The plaque-formation activity was stable following heating to 70 degrees C but was inhibited by treatment with chloroform. fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell. Almost all V. cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant. The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages. Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2. The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V. cholerae CTX phi sequence which is speculated to be required for integration of the phage into the V. cholerae chromosome at a specific site.

Animals↗

PubMed1994

Interstitial nephritis in Aleutian mink disease. Possible role of cell-mediated immunity against virus-infected tubular epithelial cells.

Aleutian mink disease (AD) has been characterized by immune complex glomerulonephritis associated with persistent infection of Aleutian mink disease parvovirus (ADV). Histopathological examination of kidneys from ADV-infected mink in this study revealed that interstitial nephritis characterized by prominent damage of renal tubuli and lymphocyte infiltration was also common in AD along with glomerulonephritis. By using strand-specific in situ molecular hybridization technique, replication of ADV was observed in tubular epithelial cells, in addition to epithelial cells of Bowman's capsules and some glomerular cells of the infected mink. Analysis of tubular lesions by a combination of immunohistochemistry and in situ hybridization revealed that the renal tubuli positive for virion DNA or replicative form DNA/mRNA of ADV were also positive for an activation marker of immunocompetent cells, which is shared by B lymphocytes and thymic epithelial cells. Infiltration of a subpopulation of T lymphocytes around infected renal tubuli were observed but deposition of immune complexes in these tubular lesions was not demonstrable. ADV replication in epithelial cells of renal tubuli and cell-mediated immune responses to the infected epithelial cells may play a role in the pathogenesis of interstitial nephritis in Aleutian mink disease.

Aleutian Mink Disease↗

PubMed1997

Replication of rep-cap genes is essential for the high-efficiency production of recombinant AAV.

Adenoassociated virus (AAV) has been developed as a vector for gene transfer because of its advantageous features: it is nonpathogenic, naturally replication-defective; it infects growth-arrested cells, and can transfer the therapeutic gene without co-delivery of any viral genes. However, a major obstacle in conducting systematic studies of AAV-mediated gene transfer in animal models is the difficulty of obtaining large quantities of recombinant virus. Recent development of AAV packaging cell lines has simplified the procedure of producing recombinant AAV (rAAV). However, the efficacy of producing large quantities of rAAV with these cell lines is yet to be demonstrated. In this study we have analyzed the difference between the replication of wild-type AAV and the production of rAAV. Using a combined single-plasmid system that carries both an AAV vector and the rep-cap genes, we have demonstrated that the AAV vector replicates to high number of copies whereas the rep-cap sequences remain unamplified in the virus-producing cells, When the copy number of rep-cap genes was increased by varying the vector/rep-cap ratio in the transfection mixture, the titer of rAAV increased proportionally. Thus, the titer of rAAV is limited by the low copy number of the rep-cap genes that results in an insufficient expression of the Rep and Cap proteins. We have also shown that generation of double-stranded replicating form of the vector DNA is accompanied by an amplified transgene expression. We propose that the increased gene expression from the accumulating double-stranded viral DNA is likely to be the mechanism by which wild-type AAV produces a large number of particles necessary to package the self-replicating AAV genomes. We conclude that mimicking this amplified expression of rep-cap genes may provide the key to produce high titers of rAAV.

Adenoviridae↗

PubMed1977

Effects of pyrimidine derivatives on RNA-dependent RNA polymerase of mengovirus-infected Fogh and Lund (FL) cells.

Two newly synthesized pyrimidine derivatives were found to possess antiviral activity against Mengovirus in Fogh and Lund (FL) cells and in a cell-free system. The inhibitory effect on RNA-dependent RNA polymerase of Mengovirus-infected FL cells was assayed using 14C-UTP as precursor. Addition of 50 or 100 muM of the inhibitors in a cell-free system of crude enzyme and nucleoside triphosphate medium for 60 min incubation at 37 degrees C resulted in about 40 to 60% lower counting rates for drug-treated reaction mixtures. The analysis of the polymerase synthesis product (virus RNA extracted from the cell-free reaction mixture and deproteinized by the phenol-SDS method) was carried out by means of agarose-acrylamide gel electrophoresis. The main finding was a reduction of single-stranded Mengovirus RNA (RNase-sensitive and LiCl-precipitable). The rates of synthesis of the replicative intermediate (LiCl-precipitable) and the replicative form of RNA (LiCl-soluble) were not significantly influenced.

Antiviral Agents↗

PubMed1984

Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Animals↗

PubMed1999

Neoplastic transformation-associated stimulation of the in vitro resolution of concatemer junction fragments from minute virus of mice DNA.

Minute virus of mice (MVM) shows an oncotropic behavior reflected by its ability to amplify its genome more efficiently in a number of transformed versus normal cells. In vivo and in vitro studies revealed that the major effect of cell transformation on MVM DNA replication occurs at the level of double-stranded replicative-form amplification. In particular, resolution of MVM DNA concatemers into monomers was found to be highly sensitive to neoplastic transformation.

Cell Transformation, Neoplastic↗

PubMed1978

Time-course of cowpea chlorotic mottle virus RNA replication.

In a synchronous infection of cowpea chlorotic mottle virus in cowpea leaves, the three largest viral RNA components were synthesized throughout the infection at relatively constant ratios of two molecules of component 3 to one each of components 1 and 2. Component 4 (the coat protein message) was produced minimally during the initial stages of the infection, and as the infection progressed the proportion of component 4 continuously increased. The replicative forms of components 1, 2, and 3 were produced with kinetics similar to those of the corresponding single-stranded molecules.

Kinetics↗

PubMed1989

SOS processing of unique oxidative DNA damages in Escherichia coli.

phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

Binding Sites↗

PubMed1980

Expression of a DNA strand initiation sequence of ColE1 plasmid in a single-stranded DNA phage.

In order to investigate initiation of H-strand (lagging strand) replication of the plasmid ColE1, the origin region fragment (Hae II-E) of ColE1 was inserted into the intergenic region of filamentous DNA phage M13 and cloned. A site capable of promoting DNA strand initiation on a single-stranded DNA template has been detected on the L-strand (leading strand) of the cloned fragment. The site, named rri-1 rifampicin-resistant initiation), directs conversion of chimeric phage single-stranded DNA to parental replicative form in the presence of rifampicin, which blocks the function of the complementary strand origin of M13. The function of rri-1 is dependent on both the dnaG and dnaB gene products. It is postulated that rri-1 might be an initiation site for synthesis of the lagging DNA strand during unidirectional replication of ColE1 DNA.

Bacterial Proteins↗

PubMed2005

Emerging evidence of hepatitis C virus neuroinvasion.

It has been reported that hepatitis C virus (HCV) infection is associated with cognitive dysfunction, fatigue and depression, which do not correlate with the severity of liver disease and cannot be accounted for by hepatic encephalopathy or drug abuse. There is also emerging evidence that HCV infection can have negative neurocognitive effects in HIV-infected cohorts. Magnetic resonance spectroscopy has suggested the likely existence of a biological basis for these effects. HCV replicative forms have recently been detected in autopsy brain tissue and the infected cells have been identified as CD68-positive (macrophages/microglia). These findings raise the possibility that HCV infection of the brain could be directly related to the reported neuropsychological and cognitive changes. HCV is not strictly hepatotropic, as it can also replicate in leukocytes, including monocytes/macrophages. The latter cells could provide access of HCV into the central nervous system ('Trojan horse' mechanism) in a process similar to that postulated for HIV-1. In support of this hypothetical mechanism come reports showing a close relationship between HCV sequences present in the brain and cerebrospinal fluid and sequences found in lymph nodes and peripheral blood mononuclear cells. However, despite some similarities there is a fundamental difference between HIV-1 and HCV infection as the latter does not progress into AIDS-type dementia.

Brain↗

PubMed1974

Isolation and function of the gene A initiator of bacteriophage phi-chi 174, a highly specific DNA endonuclease.

The gene A product of Escherichia coli bacteriophage varphiX 174, necessary for initiation of varphiX DNA synthesis, has been identified and purified to near homogeneity. The gene A protein is shown to be a highly specific DNA endonuclease which breaks one phosphodiester bond in the viral strand of either double-stranded, circular varphiX replicative form I (RFI), or in single-stranded, circular varphiX viral DNA; it is inactive against all other species of DNA tested.

Carbon Radioisotopes↗

PubMed1999

The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I.

The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After dephosphorylation in early G(1), the level of Ser66 phosphorylation is minimal in G(1), increases progressively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G(1) and S phase but not in G(2)/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.

3T3 Cells↗

PubMed1982

Adenoviral protein-primed initiation of DNA chains in vitro.

The initiation of DNA chains by the 80-kilodalton form of the adenovirus terminal protein has been studied. This protein, which can be covalently linked to dCMP, is isolated complexed to a 140-kilodalton protein possessing DNA polymerase activity. In the presence of adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex requires the addition of ATP and nuclear extract from uninfected cells in addition to Mg2+ and dCTP. When single-stranded DNA is used in place of the adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex occurs in the absence of ATP and nuclear extract. In the presence of the four dNTPs, the complex yields DNA chains of various sizes between 100 and 300 nucleotides. The products formed with bacteriophage phi X174 single-stranded circular DNA as the template are site specific, predominantly derived from the sequences between nucleotides 2363 and 2977 and between nucleotides 3760 and 4206. These small dNA chains are blocked at their 5' ends with the 80-kilodalton protein but possess free 3'-OH ends that are susceptible to degradation by exonuclease III and can be elongated to replicative form II products with DNA polymerase I of Escherichia coli or eukaryotic DNA polymerase beta preparations. A protein priming model explaining the different requirements for initiation with adenovirus DNA-protein and with phi X174 DNA is presented.

Adenoviruses, Human↗

PubMed1988

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin.

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.

Bacteriophage phi X 174↗

PubMed2004

Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study.

IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells.

Animals↗

PubMed1999

Lysogenic conversion of environmental Vibrio mimicus strains by CTXPhi.

The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.

Animals↗