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At least 577 records · Page 32Linked to original sources
PubMed1989

An insertion mutation in the pol gene of Moloney murine leukemia virus results in temperature-sensitive pol maturation and viral replication.

An insertion mutation in the pol gene of Moloney murine leukemia virus (M-MuLV) was found to render the virus temperature-sensitive for replication. A provirus containing a 12-bp insertion at the boundary between the reverse transcriptase (RT) and integrase (IN) domains induced the formation of mutant virions containing a partially processed RT-IN fusion protein. Some proteolytic processing to form mature RT and IN was observed at 32 degrees, but only aberrantly processed proteins were detected at 39 degrees. The uncleaved precursor was found to exhibit DNA polymerase activity, even though it could not support replication of the virus in vivo at 39 degrees.

Animals↗
PubMed2004

Pseudorabies virus UL3 gene codes for a nuclear protein which is dispensable for viral replication.

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily Alphaherpesvirinae: To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in Escherichia coli as a glutathione S-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-DeltaUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.

Animals↗
PubMedDate not supplied

Inhibition of HIV by an anti-HIV protease synthetic peptide blocks an early step of viral replication.

The processing of the human immunodeficiency virus (HIV) gag and gag-pol precursor proteins by the virus-encoded protease is an essential step in maturation of infectious virus particles. Like most retroviral proteases, the HIV protease belongs to the aspartyl-protease family and can be inhibited by specific inhibitors. Twenty-four synthetic peptides known to be inhibitors of human renin were tested for inhibition of HIV replication in tissue cultures. One of them, a synthetic peptide analogue, SR41476, which has been shown to be a specific inhibitor of purified recombinant HIV1 protease in vitro, totally blocked infection with different isolates including the HIV1 LAV prototype, the highly cytopathic Zairian isolate HIV1 NDK, and HIV2 ROD, both in primary blood lymphocytes (PBL) and in the lymphoid cell lines MT4 and CEM, for at least 3 weeks. It also significantly reduced virus replication in chronically infected CEM cells, without any effect on cell proliferation. Radioimmunoprecipitation assay revealed that the inhibitor blocked processing of polyprotein precursors p55 gag and p40 gag into a mature form of gag proteins, p25 and p18. Synthetic peptide analogue SR 41476, when added before infection, efficiently inhibited formation of HIV DNA provirus and successfully suppressed synthesis of HIV-specific proteins. These results imply that the HIV protease inhibitor not only inhibited virus maturation in the late phase of the HIV replication cycle, but also interfered in the early phase, before the provirus was formed. This mechanism of antiviral activity provides new possibilities and strategies for AIDS chemotherapy.

Amino Acid Sequence↗
PubMed1997

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.

Animals↗
PubMed2005

ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture.

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Amino Acid Sequence↗
PubMed2001

Low-dose IFN-alpha monotherapy in treatment-naive individuals with HIV-1 infection: evidence of potent suppression of viral replication.

To evaluate the safety and antiviral action of interferon-alpha (IFN-alpha) in HIV-1 infection, we undertook a proof of concept study in 27 treatment-naive patients. Eligible patients comprised two groups: the IFN-alphaT group (n = 17), which received 5 MIU IFN-alpha s.c. daily for 32 consecutive days, and the IFN-alphaNT group (n = 10), which did not receive IFN-alpha prior to highly active antiretroviral therapy (HAART), which was commenced on day 28 in both groups. IFN-alphaTreatment was well tolerated in 14 of the 17 patients of the IFN-alphaT group who completed the study. The mean HIV RNA reduction in the IFN-alphaT group on day 14 was 1.1 log(10). Viral load suppression was inversely associated with baseline viral load (p = 0.031). Four weeks after initiation of HAART, IFN-alphaT and IFN-alphaNT group patients had 2.40 and 1.82 log(10) HIV RNA reduction from baseline, respectively (p < 0.001). There was no evidence of cross-resistance with existing antiretrovirals in patients with HIV-RNA rebound after initial plasma viral load decline > or = 1 log(10) during IFN-alpha monotherapy. Thus, low daily IFN-alpha exhibits potent anti-HIV-1 activity in vivo without serious adverse effects. These properties render IFN-alpha an attractive candidate for further assessment as a constituent of HAART.

Anti-HIV Agents↗
PubMed1997

VP5 of infectious bursal disease virus is not essential for viral replication in cell culture.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, encodes in its bisegmented double-stranded RNA genome four structural virion proteins, VP1, VP2, VP3, and VP4, as well as a nonstructural protein, VP5. Recently, the establishment of an infectious cRNA system for IBDV has been described (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). Here, we report the isolation of a VP5- IBDV mutant constructed by site-directed mutagenesis of the methionine start codon of VP5, followed by cRNA transfection. The resulting virus mutant was replication competent in cell culture, which indicates that VP5 is not required for productive replication of IBDV. Absence of VP5 expression was verified by lack of reactivity with newly established anti-VP5 monoclonal antibodies and polyclonal sera. VP5- IBDV exhibited a delay in replication in chicken embryo cells compared to the VP5+ parental virus. However, final yields were similar. Our results thus show that VP5 is nonessential for IBDV replication, which makes it a prime candidate for the construction of deleted, marked vaccines.

Amino Acid Sequence↗
PubMed2003

Evidence for immune-mediated reduction of viral replication in Macaca nemestrina mucosally immunized with inactivated SHIV(89.6).

Although most HIV-1 infections worldwide result from heterosexual transmission, most vaccine candidates have focused on induction of systemic immunity and protection. We hypothesized that combining systemic priming with mucosal boosting would induce mucosal immunity that would protect from intravaginal challenge. Macaques were primed systemically with recombinant vaccinia viruses and boosted mucosally using inactivated SHIV(89.6) plus adjuvant. Other animals received protein boosts with adjuvant alone. Priming and boosting induced antiviral IgG and IgA antibodies. Such antibodies were induced to a lesser degree in animals receiving boosts alone. Anti-SHIV T cell responses were induced only in the prime-boost animals. Immunized animals and controls were challenged intravaginally with SHIV(89.6) and significant reductions in proviral and viral RNA loads were observed in the prime-boost animals. The boost-only animals did not have significant viral load reductions. These data suggest that cellular immunity was required for protection from intravaginal challenge. This immunization regimen provides a promising lead for vaccine development.

AIDS Vaccines↗
PubMed1995

Turnover of circulating virion RNA and of cell-associated viral DNA reflects active viral replication in human immunodeficiency virus type 1-infected individuals.

A quantitative assessment of human immunodeficiency virus type 1 turnover in patient cell-free virion and infected-cell compartments under the dynamic conditions imposed by an effective antiviral therapy was performed. The turnover was rapid, and following a temporal lag, the extent of viral population replacement was eventually similar in both compartments. Each compartment therefore reflects considerable active virus replication.

Antiviral Agents↗
PubMed2002

Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype.

Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.

Cells, Cultured↗
PubMed1993

HIV-specific CD8+ T-cell immune responses and viral replication.

AIM: To review current knowledge of CD8+ T cells in relation to their effect on the replication of HIV and on disease progression. PRESENT KNOWLEDGE: Both CD8+ cytotoxic T lymphocytes capable of killing cells expressing HIV antigens and CD8+ lymphocytes that suppress HIV replication in vitro are detectable in response to HIV infection. CONCLUSION: These CD8+ T cells may help to maintain a low viral load in vivo, thus allowing a long asymptomatic period of infection.

CD4-Positive T-Lymphocytes↗
PubMed2004

The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication.

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.

Animals↗
PubMed1987

PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS.

HIV↗
PubMed1977

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class.

Adenoviridae↗
PubMed2005

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication.

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

3' Untranslated Regions↗