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PubMed2002

Analysis of an Autographa californica multicapsid nucleopolyhedrovirus lef-6-null virus: LEF-6 is not essential for viral replication but appears to accelerate late gene transcription.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAc(lef6KO)) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two "repair" AcMNPV BACmids (vAc(lef6KO-REP-P) and vAc(lef6KO-REP-ie1P)) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc(lef6KO) BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAc(lef6KO)) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

Animals↗

PubMed1995

Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

Amino Acid Sequence↗

PubMed2002

Non-lethal viral challenge of influenza haemagglutinin and nucleoprotein DNA vaccinated mice results in reduced viral replication.

Influenza DNA vaccines have been widely studied in experimental animal models and protection documented after lethal viral challenge. In this study, we have investigated the humoral response after a non-lethal viral challenge of mice vaccinated with plasmids encoding the influenza haemagglutinin (HA) or nucleoprotein (NP) genes. BALB/c mice were immunized intramuscularly with three doses (100 microg) of HA, NP or backbone plasmid at 3-week intervals, or alternatively infected intranasally, before being challenged with homologous virus 13 weeks later. Mice were then sacrificed at weekly intervals and the antibody-secreting cell response was examined systemically (spleen and bone marrow) and in the respiratory tract (nasal associated lymphoid tissue (NALT) and lungs). Sera were collected after each dose of vaccine and at sacrifice and analyzed by ELISA, haemagglutination inhibition and virus neutralization assays. We found that previous viral infection apparently elicits sterilizing immunity. Vaccination with HA or NP DNA significantly reduced viral replication in the nasal cavity after viral challenge, however, increases in serum antibody titres were observed after challenge. Prior to challenge, specific antibody-secreting cells were observed in the systemic compartment after HA or NP DNA vaccination but were also found in the NALT after viral challenge. In conclusion, intramuscular DNA vaccination resulted in immunological memory in the systemic compartment, which was rapidly reactivated upon viral challenge.

Animals↗

PubMed2002

Impact of suppression of viral replication by highly active antiretroviral therapy on immune function and phenotype in chronic HIV-1 infection.

We compared immune phenotypes, lymphocyte proliferation (LP), and delayed type hypersensitivity (DTH) responses in 28 male antiretroviral treatment-naive and experienced HIV-1-infected patients, matched pair-wise according to age and CD4+ T-lymphocyte count. Median CD4+ T-lymphocyte counts were 441 cells/microL and 483 cells/microL and median CD4+ T-lymphocyte nadirs were 435 cells/microL and 150 cells/microL in both groups, respectively. Absolute numbers of circulating T-lymphocyte subpopulations and proportions of naive and memory T-lymphocytes were comparable in the two groups. Untreated patients had greater proportions of activated CD4+ (p <.05) and CD8+ (p <.01) T-cells expressing human leukocyte antigen (HLA)DR and CD38 and fewer CD8+ cells expressing CD28 (p <.05). DTH and LP responses were comparable in both groups except for HIVp24, LP responses, and mumps DTH responses, which were of greater magnitude in the group treated with highly active antiretroviral therapy (HAART) (p <.05). Thus, HIV-1-infected patients who experienced substantial increases in CD4+ T-lymphocyte counts after suppression of viral replication on HAART had fewer activated lymphocytes and similar immune function when compared with findings in untreated patients with similar CD4+ T-cell counts. HIV replication has minimal real-time effect on CD4+ T-cell function in response to non-HIV antigens but helper T-cell responses to HIV-gag antigen are impaired during ongoing viral replication and may be restored by antiretroviral therapy.

ADP-ribosyl Cyclase↗

PubMed1988

Evidence of restricted viral replication in adult mink infected with Aleutian disease of mink parvovirus.

Strand-specific hybridization probes were used in in situ molecular hybridization specifically to localize cells containing replicative intermediates of Aleutian disease of mink parvovirus (ADV). When adult mink of Aleutian genotype were infected with ADV Utah I, the largest number of cells positive for viral replication (i.e., containing replicative-form DNA and RNA) were found in the mesenteric lymph nodes and spleens at 10 days after infection. The localization of positive cells in the middle of germinal centers suggested that they were B lymphoblasts. Circulating leukocytes and bone marrow cells also contained viral RNA, but the levels of replicative-form DNA were below detectability. The levels of viral DNA and RNA in adult mink cells replicating ADV were decreased compared with those in permissively infected cell cultures or neonatal mink, suggesting that the replication of ADV in adult mink might be semipermissive or restricted at some early stage of viral gene expression. The low level of viral replication and transcription in lymphoid cells might provide a mechanism for the development of immune disorders and for the maintenance of persistent infection. Single-stranded virion DNA was found in other organs, but the strand-specific probes made it possible to show that this DNA represented virus sequestration. In addition, glomerular immune complexes containing virion DNA were detected, suggesting that ADV virions, or perhaps free DNA, may have a role in the development of ADV-induced glomerulonephritis.

Aleutian Mink Disease Virus↗

PubMed1999

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.

Cloning, Molecular↗

PubMed2003

Selective replicating viral vectors : potential for use in cancer gene therapy.

Treatment of cancer is limited by toxicity to normal tissue with standard approaches (chemotherapy, surgery and radiotherapy). The use of selective replicating viral vectors may enable the targeting of gene-modified viruses to malignant tissue without toxic effect. Studies of these vectors have demonstrated tumour-selective replication and minimal evidence of replication in normal tissue. The most advanced clinical results reported involve gene-modified adenoviral vectors. Several completed, histologically confirmed responses to local/regional injection have been induced, particularly in recurrent squamous cell carcinoma involving the head and neck region. Dose limiting toxicity above 10(13) viral particles per injection has been observed. Anti-tumour effect is demonstrable in animal models without evidence of significant toxicity when these vectors are used alone or in combination with chemotherapy, radiation therapy or as gene delivery vehicles. Preliminary clinical trials, particularly with E1B-deleted adenoviruses, report evidence of clinical activity in comparison with expected historical responses. Enhancement in replication selectivity to malignant tissue is also demonstrated preclinically and clinically with an E1B-deleted adenovirus utilising a prostate-specific antigen promoter. Other selective replicating viral vectors such as herpes simplex virus and vaccinia virus have also been explored clinically and suggest evidence of activity in patients with cancer. Modifications may one day enable more aggressive use of these new and exciting therapeutics as systemic gene delivery vehicles.

Animals↗

PubMed1994

High resolution localization of replicating viral genome in adenovirus-infected HeLa cells.

Previous autoradiographical and in situ hybridization experiments have revealed that the replication of viral genomes in adenovirus type 5 infected HeLa cells induces changes in nuclear structure of which one of the more striking is the formation of distinctive replicative foci. The latter consist of a viral ssDNA accumulation site and a surrounding fibrillogranular network. We have reexamined these structures and processes by a more direct and higher resolution approach, that is, incorporation of bromodeoxyuridine (BrdU) by the infected cells and subsequent immunogold detection of the BrdU incorporated into DNA. Short pulses with BrdU in pulse-chase experiments confirmed that viral DNA replication at the early stage of nuclear transformation was confined within small virus-induced structures, the so-called early replicative sites, and revealed the persistence of the newly synthesized viral DNA at those sites for at least 2 h. At intermediate and late stages of nuclear transformation, the intensity of viral DNA replication, which varies from one type of replicative focus to another, was most intense in that layer of the fibrillogranular network which was in closest proximity to the viral single-stranded DNA (ssDNA) accumulation sites, whereas replication was weakest over the latter. Subsequently, BrdU-containing viral DNA molecules became more widely distributed in the viral ssDNA accumulation sites and the fibrillogranular network, the two constituents of the viral replicative machinery. Two hours later, some labeled molecules attained the viral genome storage site and/or became encapsulated. The most striking observation is the presence of a limited region in the replicative focus which is the preferential site for viral genome replication. The data also indicated that viral DNA molecules which were labeled during the short pulses remained in the replicative foci themselves, to be replicated and transcribed prior to attaining the pool of inactive genomes and/or becoming encapsulated.

Adenoviruses, Human↗

PubMed2003

Overexpression of interferon-gamma-inducible GTPase inhibits coxsackievirus B3-induced apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway and inhibition of viral replication.

Our previous studies using differential mRNA display have shown that interferon-gamma-inducible GTPase (IGTP), was up-regulated in coxsackievirus B3 (CVB3)-infected mouse hearts. In order to explore the effect of IGTP expression on CVB3-induced pathogenesis, we have established a doxycycline-inducible Tet-On HeLa cell line overexpressing IGTP and have analyzed activation of several signaling molecules that are involved in cell survival and death pathways. We found that following IGTP overexpression, protein kinase B/Akt was strongly activated through phosphorylation, which leads to phosphorylation of glycogen synthase kinase-3 (GSK-3). Furthermore, in the presence of CVB3 infection, the intensity of the phosphorylation of Akt was further enhanced and associated with a delayed activation of caspase-9 and caspase-3. These data indicate that IGTP expression appears to confer cell survival in CVB3-infected cells, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell viability assay. However, the ability of IGTP to induce phosphorylation of Akt and to promote cell survival was attenuated by the phosphotidylinositol-3 kinase (PI3-K) inhibitor LY294002. Transient transfection of the cells with a dominant negative Akt construct followed by doxycycline induction and CVB3 infection reversed Akt phosphorylation to basal levels and returned caspase-3 activity to levels similar to those when the PI3-K inhibitor LY294002 was added. Moreover, IGTP expression inhibited viral replication and delayed CVB3-induced cleavage of eukaryotic translation initiation factor 4G, indicating that IGTP-mediated cell survival relies on not only the activation of PI3-K/Akt, inactivation of GSK-3 and suppression of caspase-9 and caspase-3 but also the inhibition of viral replication.

Animals↗

PubMed1969

The pathogenesis of Aleutian disease of mink. I. In vivo viral replication and the host antibody response to viral antigen.

Mink inoculated with 1 x 10(5)ID(50) of Aleutian disease virus revealed very high virus titers in the tissues 8-18 days later. The highest virus titers observed were 5 x 10(8)ID(50) per g of spleen and 1 x 10(9)ID(50) per g of liver 10 days after inoculation. Concomitant with the increase in infectious virus titers, viral antigen(s) was found in the cytoplasm of macrophages in the spleen and lymph nodes and in Kupffer cells in the liver. Antiviral antibody was assayed by indirect immunofluorescence, using sections of infected liver as the source of antigen. A few mink infected for 9 days and all those infected 10 days or more developed antibody to Aleutian disease virus antigen(s). By 60 days after infection, when hypergammaglobulinemia was marked, the mink had an exceptionally high mean antibody titer of 100,000. The pathogenesis of the glomerulonephritis of Aleutian disease is apparently related to formation of viral antigen-antibody-complement complexes which lodge in glomerular capillaries. No evidence was found that viral infection of the kidney took place, and no autoimmune responses were found. In this "slow-virus" disease the virus replicates rapidly and the morphologic and biochemical manifestations of disease are apparently due to the continuing interplay between a replicating antigen and the host immune response.

Animals↗

PubMed2002

Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication.

Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HIV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.

Animals↗

PubMed1998

Effect of HIV constructs containing protease-reverse transcriptase fusion proteins on viral replication.

OBJECTIVE: To determine whether disruption of the cleavage site between protease and reverse transcriptase (RT) or the HIV-1 frameshift site could yield trans-dominant negative HIV-1 variants that interfere with wild-type viral replication. DESIGN: Residues at the cleavage site between the HIV-1 protease and RT coding regions were mutagenized to produce a protease-RT (PR-RT) fusion protein that was expressed in the context of a full-length provirus. The PR-RT cleavage site mutation was also combined with a read-through mutation at the frameshift site in order to overexpress the mutant Gag-Pol polyproteins. METHODS: COS-7 cells were transiently transfected with the mutant constructs to produce viruses harbouring the PR-RT fusion protein. In addition, we performed cotransfection studies in various cell types to analyze the inhibition of wild-type replication by the mutant constructs. RESULTS: Immunoblot analysis revealed that this novel mutation prevented cleavage between the two proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. While both the protease and RT components of this fusion protein remained functionally active, viruses containing the cleavage site mutation were less infectious in tissue culture than wild-type viruses produced by COS-7 cells. This defect was further pronounced when the cleavage site mutation between protease and RT was overexpressed as a consequence of an additional mutation that interfered with frameshifting. Cotransfection of COS-7 cells with the mutant constructs and wild-type HIV-1 interfered with the replication of the latter and reduced the infectiousness of the virus particles produced. CONCLUSIONS: HIV-1 constructs harbouring a cleavage site mutation between protease and RT can probably act as trans-dominant negative mutants to interfere with wild-type viral replication.

Animals↗

PubMed2003

High rates of human immunodeficiency virus type 1 recombination: near-random segregation of markers one kilobase apart in one round of viral replication.

One of the genetic consequences of packaging two copies of full-length viral RNA into a single retroviral virion is frequent recombination during reverse transcription. Many of the currently circulating strains of human immunodeficiency virus type 1 (HIV-1) are recombinants. Recombination can also accelerate the generation of multidrug-resistant HIV-1 and therefore presents challenges to effective antiviral therapy. In this study, we determined that HIV-1 recombination rates with markers 1.0, 1.3, and 1.9 kb apart were 42.4, 50.4, and 47.4% in one round of viral replication. Because the predicted recombination rate of two unlinked markers is 50%, we conclude that markers 1 kb apart segregated in a manner similar to that for two unlinked markers in one round of retroviral replication. These recombination rates are exceedingly high even among retroviruses. Recombination rates of markers separated by 1 kb are 4 and 4.7% in one round of spleen necrosis virus and murine leukemia virus replication, respectively. Therefore, HIV-1 recombination can be 10-fold higher than that of other retroviruses. Recombination can be observed only in the proviruses derived from heterozygous virions that contain two genotypically different RNAs. The high rates of HIV-1 recombination observed in our studies also indicate that heterozygous virions are formed efficiently during HIV-1 replication and most HIV-1 virions are capable of undergoing recombination. Our results demonstrate that recombination is an effective mechanism to break the genetic linkage between neighboring sequences, thereby reassorting the HIV-1 genome and increasing the diversity in the viral population.

Cells, Cultured↗

PubMed1996

A cellular homolog of hepatitis delta antigen: implications for viral replication and evolution.

Hepatitis delta virus (HDV) is a pathogenic human virus whose RNA genome and replication cycle resemble those of plant viroids. However, viroid genomes contain no open reading frames, whereas HDV RNA encodes a single protein, hepatitis delta antigen (HDAg), which is required for viral replication. A cellular gene whose product interacts with HDAg has now been identified, and this interaction was found to affect viral genomic replication in intact cells. DNA sequence analysis revealed that this protein, termed delta-interacting protein A (DIPA), is a cellular homolog of HDAg. These observations demonstrate that a host gene product can modulate HDV replication and suggest that HDV may have evolved from a primitive viroidlike RNA through capture of a cellular transcript.

Adaptor Proteins, Signal Transducing↗

PubMed1987

In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.

Aleutian Mink Disease Virus↗

PubMed1997

Enhanced (cytomegalovirus) viral replication after transplantation for fulminant hepatic failure.

Fulminant hepatic failure (FHF) is an established indication for liver transplantation. A pretransplant diagnosis of FHF may be a risk factor for subsequent development of cytomegalovirus (CMV) infection, although the mechanism of this association is not understood. FHF is associated with very high levels of tumor necrosis factor alpha (TNF-alpha), and TNF-alpha may directly promote viral replication. We have used the polymerase chain reaction (PCR) to examine sequentially collected buffy coats from 106 consecutive adult liver transplant recipients. PCR evidence of CMV replication was found for 13 of 18 patients who underwent transplantation for FHF (c.f. 23/88 non-FHF patients; P < .01). Ten of 12 patients who received transplants transplanted for fulminant seronegative hepatitis were buffy-coat PCR-positive, sometimes during the first posttransplant week. TNF-alpha was measured by enzyme-linked immunosorbent assay in selected sera, and results were examined in the context of a quantitative PCR assay. Serum TNF-alpha levels increased and decreased in concert with viral titers. High levels of TNF-alpha were not found in the early posttransplant period. We conclude that FHF (in particular, seronegative hepatitis) is associated with enhanced CMV replication in the posttransplant period. Results also suggest that viral replication may be enhanced before transplantation. These patients may be at special risk for development of symptomatic CMV infection.

Cytomegalovirus↗

PubMed2006

Mechanistic link between the anti-HCV effect of interferon gamma and control of viral replication by a Ras-MAPK signaling cascade.

Interferon-gamma (IFN-gamma) exerts potent antiviral activity in the hepatitis C virus (HCV) replicon systems. However, the mechanisms underlying the direct antiviral effect have not been determined. We found that the type II transcriptional response to IFN-gamma could be suppressed by inhibition of MEK1/2 kinase activity by MEK1/2 inhibitor U0126 in the hepatoma cell line Huh-7. Using a bicistronic HCV replicon system expressing a luciferase reporter gene in Huh-7 cells (RLuc-replicon), we showed that inhibition of MEK1/2 kinase activity is sufficient to counteract the antiviral activity of IFN-gamma. Expression of a constitutive active form of Ras inhibited the luciferase activity of RLuc-replicon, whereas a dominant-negative mutant of Ras enhanced the reporter activity, indicating that the Ras-MAPK pathway has a role in limiting replication of the viral RNA. Consistent with the involvement of the Ras-MAPK pathway, treatment with epidermal growth factor suppressed HCV protein expression in the RLuc-replicon cells, an effect that could be abolished by U0126. Inhibition of MEK1/2 kinase activity correlated with reduced phosphorylation of the HCV NS5A protein and enhanced RLuc-replicon luciferase reporter activity, in line with recent reports that phosphorylation of NS5A negatively modulates HCV RNA replication. Finally, genetic deletion analysis in yeast supported the role of a MEK-like kinase(s) in the regulation of NS5A phosphorylation. In conclusion, the direct anti-HCV effect of IFN-gamma in cell culture is, at least in part, mediated through the Ras-MAPK signaling pathway, which possibly involves a direct or indirect modulation of NS5A protein phosphorylation.

Antiviral Agents↗

PubMed2005

Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 confers efficient viral replication.

We showed previously that infection of herpes simplex virus type 1 (HSV-1) rapidly induced the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, in the amnion cell line FL. Thus, HSV-1 suppresses the interferon (IFN) signaling pathway at the step of IFN-induced phosphorylation of janus kinases during an early infection stage. In the present study, we examined SOCS3 induction by HSV-1 infection in several types of human cell lines. FL cells and the T-cell line CCRF-CEM strongly induced SOCS3 during HSV-1 infection. The virus rapidly propagated in both cell lines and produced a lytic infection. On the other hand, the monocytic cell lines U937 and THP-1, and the B-cell line AKATA showed neither SOCS3 induction nor suppression of IFN-induced STAT1 phosphorylation during HSV-1 infection. These cell lines resulted in a persistent or prolonged infection, which continuously produced a low titer of infectious virus. The induction of SOCS3 by HSV-1 should occur via STAT3 activation immediately after HSV-1 infection. SOCS3 induction was inhibited by the addition of a Jak3 inhibitor WHI-P131. Treatment with WHI-P131 or transfection of antisense oligonucleotides specific for SOCS3 dramatically suppressed replication of HSV-1 in FL cells. The suppression of viral replication by WHI-P131 was released in the presence of neutralizing anti-IFN-alpha and anti-IFN-beta antibodies. In conclusion, suppression of IFN signaling by HSV-1-induced SOCS3 is required for efficient replication and lytic infection of HSV-1. The SOCS3 induction varied among cell lines, indicating that it is an important factor determining the cell type specificity of efficient HSV-1 replication.

Amnion↗