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At least 919 records · Page 51Linked to original sources
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↗
PubMed2001

Astrocytes, not microglia, are the main cells responsible for viral persistence in Theiler's murine encephalomyelitis virus infection leading to demyelination.

The BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) persists in the CNS and produces a chronic inflammatory demyelinating disease that is an animal model for human multiple sclerosis (MS). The mechanisms leading to TMEV-induced demyelination are still under study but most likely involve both immune-mediated and virus induced damage to cells in the CNS, both depending on viral persistence. It is therefore important to identify the cells in which continued virus production is permitted. In this study, we looked at virus infection in primary astrocytes, microglia and oligodendrocytes, derived from brains of neonatal susceptible SJL/J mice. As evidenced by Western blots and immunocytochemistry, we were able to detect viral antigens in all these brain-derived cells. In addition, we extended the study to spinal cord tissues from mice suffering TMEV-induced disease. Immunohistochemistry staining with anti-TMEV sera and antibodies to specific cell markers detected viral antigens in all these cells. We then asked the question whether viral antigen present in these cells, particularly in microglia/macrophages, represented true viral replication or not. By using different techniques, including immunoprecipitation experiments and the very sensitive method of negative RNA detection through RNase protection assay, we show that both astrocytes and oligodendroglia permit de novo viral replication and viral protein synthesis but with only minimal cytopathic effects. Of these two cell types, astrocytes carry the brunt of viral replication. In microglia, on the other hand, viral replication is restricted since only minimal amounts of negative RNA copies can be demonstrated, while there are clear signs that some of these cells undergo apoptosis. These findings show that the main cell for viral replication is the astrocyte, rather than the microglia/macrophage. Most of the viral antigen present in macrophages, therefore, is probably the result of phagocytosis, rather than actual viral replication. In view of the demonstrated presence of viral replication in astrocytes and of great amounts of viral antigens in microglia/macrophages, it is possible that both types of cells act as antigen presenting cells during this immunopathological disease.

Animals↗
PubMed2007

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication.

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

Animals↗
PubMed1989

Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection.

In order to assign specific functions to individual gene products encoded by adenovirus type 5 early region 4 (E4), we have constructed and analyzed a set of mutant viruses that express individual E4 open reading frames or combinations of open reading frames. The results of these analyses demonstrate that the gene products of E4 open reading frames 3 and 6 have redundant effects in viral lytic infection. These E4 products independently augment viral DNA replication, viral late protein synthesis, the shutoff of host cell protein synthesis, and the production of infectious virus. The product of open reading frame 6 is more efficient in the regulation of these processes than is the product of open reading frame 3. The regulation of viral DNA replication and the control of viral and cellular protein synthesis appear to be separable functions associated with both E4 gene products. The role of early region 4 in adeno-associated virus helper function, however, is mediated only by the product of open reading frame 6. Finally, we demonstrate that E4 mutant viruses display a multiplicity-leakiness phenotype which is consistent with the regulatory role that this region plays in viral infection.

Adenoviruses, Human↗
PubMed2000

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells.

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.

Animals↗
PubMed2002

Baculovirus infection raises the level of TATA-binding protein that colocalizes with viral DNA replication sites.

During the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus, the TATA-binding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.

Animals↗
PubMed2004

Dissection of the Kaposi's sarcoma-associated herpesvirus gene expression program by using the viral DNA replication inhibitor cidofovir.

Treatment of primary effusion lymphoma cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8 [HHV-8]) with agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a lytic viral replication cycle, with an ordered gene expression program. Initial studies of the KSHV expression program following TPA induction using viral microarrays yielded useful information concerning the viral expression program, but precise kinetic assignments for some genes remained unclear. Classically, late herpesvirus genes require viral DNA replication for maximal expression. We used cidofovir (CDV), a nucleotide-analogue KSHV DNA polymerase inhibitor, to dissect KSHV expression into two components: genes expressed without viral DNA replication and those requiring it. The expression of known immediate-early or early genes (e.g., open reading frames [ORFs] 50, K8 bZIP, and 57) serving lytic regulatory roles was relatively unaffected by the presence of CDV, while known late capsid and tegument structural genes (e.g., ORFs 25, 26, 64, and 67) were CDV sensitive. Latency-associated transcript ORF 73 was unaffected by the presence of TPA or CDV, suggesting that it was constitutively expressed. Expression of several viral cellular gene homologs, including K2 (vIL-6), ORF 72 (vCyclin), ORF 74 (vGPCR), and K9 (vIRF-1), was unaffected by the presence of CDV, while that of others, such as K4.1 (vMIP-III), K11.1 (vIRF-2), and K10.5 (LANA2, vIRF-3), was inhibited. The results distinguish KSHV genes whose full expression required viral DNA replication from those that did not require it, providing additional insights into KSHV replication and pathogenesis strategies and helping to show which viral cell homologs are expressed at particular times during the lytic process.

Antiviral Agents↗
PubMed2001

Attenuation of a field Sendai virus isolate through egg-passages is associated with an impediment of viral genome replication in mouse respiratory cells.

We investigated the mechanisms responsible for attenuation of mouse pathogenicity of Sendai virus (SeV) through passages in eggs. A highly virulent clone, E0, derived from the field SeV Hamamatsu strain, was successively passaged in hen's eggs. Analysis of the mouse lethal dose 50% (MLD50) of virus clones obtained from the viruses at egg-passages 1, 15, 30 and 50 demonstrated that attenuation of E0 by egg-passage occurred due to the gradual appearance of and replacement by virus variants possessing higher MLD50. Comparison of viral replication in the mouse lung and mouse pathogenicity with the representative SeV clones, E0, E15c12, E30c12 and E50c19, obtained from the respective egg-passages revealed that the low pathogenicity of the egg-passaged clones was due to poor multi-cycle viral replication in the lung. Furthermore, MLD50s of the SeV clones were found to be negatively correlated with the replication capability in primary mouse pulmonary epithelial (MPE) cells; the egg-passaged clones with more attenuated phenotypes showed lower replication capability in MPE cells. In the MPE cells infected with the SeV clones at m.o.i. 10, however, viral protein and mRNA syntheses of the egg-passaged clones were enhanced or comparable to those of the parental E0 clone at 1 day and 2 days post infection (p.i.) but decreased more rapidly thereafter. In contrast, viral genome synthesis of the egg-passaged clones in the cells at 2 days p.i. was several times lower than that of E0. These results strongly suggest that attenuation of a virulent field SeV strain by egg-passage occurs due to the appearance and selection of virus variants possessing poor propagation capacity in mouse respiratory epithelial cells, which is caused primarily by an impediment of viral genome replication.

Animals↗
PubMed1998

Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.

Adenoviridae↗
PubMed1992

Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus.

Addition of reduced glutathione inhibited the production of Sendai virus in African green monkey kidney (AGMK) cells. This result could be accounted for by a direct action of GSH on viral replication. The inhibitory action was associated to an increase of the GSH intracellular level, while the host cell metabolism was unaffected. The antiviral effect was related to decrease and inactivation of the hemagglutinin-neuraminidase (HN) virus glycoprotein.

Animals↗
PubMed1985

An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells.

Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in G0-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in G0-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells.

Adenoviruses, Human↗
PubMed2004

Kinetics of virus-specific CD8+ T cells and the control of human immunodeficiency virus infection.

Several primate models indicate that cytotoxic T lymphocyte-inducing vaccines may be unable to prevent human immunodeficiency virus infection but may have a long-term benefit in controlling viral replication and delaying disease progression. Here we show that analysis of the kinetics of antigen-specific CD8+ T-cell expansion suggests a delay in activation following infection that allows unimpeded early viral replication. Viral kinetics do not differ between controls and vaccinees during this delay phase. An increase in virus-specific CD8+ T-cell numbers around day 10 postinfection coincides with a slowing in viral replication in vaccinees and reduces peak viral loads by around 1 log. However, this response is too little too late to prevent establishment of persistent infection.

AIDS Vaccines↗
PubMed1994

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.

Animals↗
PubMed2001

The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication.

Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.

Amino Acid Sequence↗
PubMed2002

Nuclear IE2 structures are related to viral DNA replication sites during baculovirus infection.

The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is 1 of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early-expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, I. Quadt, J. Schröer, and D. Knebel-Mörsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromodeoxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers, while IE2 alone colocalized with early replication sites, demonstrating that PML structures do not form common domains with viral DNA replication centers. Thus, we conclude that IE2 colocalizes alternately with PML and the sites of viral DNA replication. Small ubiquitin-like modifier SUMO-1 has been implicated in the nuclear distribution of PML. Similar to what was found for mammalian cells, small ubiquitin-like modifiers were recruited to PML domains in infected insect cells, which suggests that IE2 and PML colocalize in conserved cellular domains. In summary, our results support a model for IE2 as part of various functional sites in the nucleus that are connected with viral DNA replication.

Animals↗
PubMed2005

Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

Amino Acid Sequence↗