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PubMed2026

MARK1 suppresses infectious bursal disease virus replication via phosphorylating VP3.

Infectious bursal disease virus (IBDV) of the Birnaviridae family is a non-envelope, double-stranded RNA virus that encodes a VP3 protein with multiple functions, which controls viral genome replication, IFN-β production, and virus traffic in infected cells. Posttranslational modifications (PTMs), such as ubiquitination, of VP3 have been demonstrated for affecting its function and stability. To clarify the mechanism by which VP3 is regulated in IBDV infected cells, we focused on the phosphorylation of VP3. Mass spectrometry analysis identified that microtubule-affinity regulating kinases 1 (MARK1) was a kinase interacting protein of VP3. Inhibitory function of MARK1 in affecting viral replication was validated. We describe the phosphorylation event at the serine 130 (S130) and serine 163 (S163) residues of VP3 mediated by MARK1 via mass spectrometry analysis. Alanine replacement of the phosphorylation sites in VP3 significantly enhanced its RNA-binding activity. Additionally, the mutation of two serine residues led to remarkably improved in its polymerase-enhancing function. We then incorporated the two mutations to rescue recombinant IBDV. Viral growth curve analysis revealed that replication of mutant IBDV was significantly enhanced relative to wild type (WT) virus. In conclusion, we found that VP3 functions are specifically regulated by MARK1 mediated phosphorylation at S130 and S163 and that this regulation suppresses IBDV replication ultimately.

Infectious bursal disease virus↗
PubMed2003

Glucocorticoid regulation of lactate dehydrogenase-elevating virus replication in macrophages.

Lactate dehydrogenase-elevating virus (LDV) is a macrophage-tropic arterivirus which generally causes a persistent viremic infection in mice. LDV replication in vivo seems to be primarily regulated by the extent and dynamics of a virus-permissive macrophage population. Previous studies have shown that glucocorticoid treatment of chronically LDV-infected mice transiently increases viremia 10-100-fold, apparently by increasing the productive infection of macrophages. We have further investigated this phenomenon by comparing the effect of dexamethasone on the in vivo and in vitro replication of two LDV quasispecies that differ in sensitivity to immune control by the host. The single neutralizing epitope of LDV-P is flanked by two N-glycans that impair its immunogenicity and render LDV-P resistant to antibody neutralization. In contrast, replication of the neuropathogenic mutant LDV-C is suppressed by antibody neutralization because its epitope lacks the two protective N-glycans. Dexamethasone treatment of mice 16 h prior to LDV-P infection, or of chronically LDV-P infected mice, stimulated viremia 10-100-fold, which correlated with an increase of LDV permissive macrophages in the peritoneum and increased LDV infected cells in the spleen, respectively. The increase in viremia occurred in the absence of changes in total anti-LDV and neutralizing antibodies. The results indicate that increased viremia was due to increased availability of LDV permissive macrophages, and that during a chronic LDV-P infection virus replication is strictly limited by the rate of regeneration of permissive macrophages. In contrast, dexamethasone treatment had no significant effect on the level of viremia in chronically LDV-C infected mice, consistent with the view that LDV-C replication is primarily restricted by antibody neutralization and not by a lack of permissive macrophages. beta-Glucan, the receptor of which is induced on macrophages by dexamethasone treatment, had no effect on the LDV permissiveness of macrophages.

Animals↗
PubMed1990

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.

Animals↗
PubMed1992

Effects of bone marrow stimulatory cytokines on human immunodeficiency virus replication and the antiviral activity of dideoxynucleosides in cultures of monocyte/macrophages.

Cells of the monocyte lineage are important targets for the replication of human immunodeficiency virus (HIV). Our group and others have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates HIV replication in monocyte/macrophages, but that it also enhances the anti-HIV activity of 2',3'-dideoxy-3'-azidothymidine (AZT). In the present study, we have explored the effects of other bone marrow stimulatory cytokines on the replication of HIV and on the anti-HIV activity of certain dideoxynucleosides in human peripheral blood monocyte/macrophages (M/M). Like GM-CSF, macrophage CSF (M-CSF) enhanced HIV replication in M/M. In contrast, granulocyte CSF (G-CSF) and erythropoietin (Epo) had no such effects. The anti-HIV activity of zidovudine (AZT) was increased in M/M exposed to GM-CSF. In contrast, the anti-HIV activity of AZT was unchanged in M/M exposed to M-CSF, and the activities of 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddl) were unchanged or slightly diminished in M/M stimulated with GM-CSF or M-CSF. These differential activities of AZT and ddC were paralleled by differential effects of the cytokines on the anabolism of these drugs to their active 5'-triphosphate moieties. GM-CSF increased the levels of AZT-5'-triphosphate (at least in part through an increase in thymidine kinase activity) and overall induced an increase in the ratio of AZT-5'-triphosphate/thymidine-5'-triphosphate. In contrast, M-CSF-induced increases in AZT-5'-triphosphate were roughly matched by increases in thymidine-5'-triphosphate. Also, GM-CSF- or M-CSF-induced increases in the levels of ddC-5'-triphosphate were associated with parallel increases in the levels of deoxycytidine-5'-triphosphate (the physiologic nucleoside that competes at the level of reverse transcriptase), so that there was relatively little net change in the ddC-5'-triphosphate/deoxycytidine-5'-triphosphate ratio. Thus, bone marrow stimulatory cytokines may have a variety of effects on HIV replication and on the activity and metabolism of dideoxynucleosides in M/M.

Antiviral Agents↗
PubMed1993

Inhibition of herpes simplex virus replication by genistein, an inhibitor of protein-tyrosine kinase.

Genistein, an inhibitor of protein-tyrosine kinase, inhibited the replication of herpes simplex virus type 1 (HSV-1) at genistein concentrations of more than 25 microM, whereas the related compounds, which do not inhibit protein-tyrosine kinases, did not affect the replication of HSV-1. In the presence of genistein, the phosphorylation of tyrosine residues in specific viral polypeptides was markedly reduced. These results indicate that the phosphorylation of tyrosine residues in viral polypeptides may be essential for the replication of HSV-1.

Animals↗
PubMed1988

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures.

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.

Base Sequence↗
PubMed1983

Contribution of macrophage arginase in the intrinsic restriction of herpes simplex virus replication in permissive macrophage cultures induced by gamma-interferon containing products of activated spleen cells.

Cultured peritoneal macrophages (PM) from adult mice of strain DBA/2 (but not C57BL/6) supported productive replication of HSV, as monitored by infectious virus yield and electron microscopy. In contrast, PM of DBA/2 origin, when pretreated with supernatants of Con A-stimulated spleen cells containing immune interferon (IFN-gamma) activity, manifested remarkable intrinsic restriction to HSV replication. The acquisition of this intrinsic restriction to virus replication correlated with the generation of appreciably elevated levels of arginase in supernate of treated PM. Addition of incremental doses of exogenous arginine in the culture medium led to the abrogation of induced restriction to HSV replication in DBA/2 PM, indicating the critical role of arginase in the phenomenon. Furthermore, anti-HSV activity of arginase levels released into the culture medium of IFN containing supernate-treated PM became apparent when assayed on PM, which were precultured in arginine-deficient medium for 24 hours before virus infection and exposure to arginase containing preparation. Taken together, these observations indicate that macrophage arginase can mediate intrinsic restriction to HSV replication in PM.

Animals↗
PubMed1993

Analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins play essential roles in viral DNA replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. By using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the Rep78 protein that mediate binding to the AAV origin of replication in vitro. Deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 abolished binding. Alterations in several other regions of the protein affected the binding affinity of the mutant proteins. All of the mutant proteins that support AAV DNA replication or p40 trans activation bound to the terminal repeat sequence, thus verifying the importance of binding for these functions. Several mutant rep genes that failed to suppress oncogene-mediated cellular transformation produced proteins that were capable of binding to the AAV terminal repeat sequences.

DNA Mutational Analysis↗
PubMed2004

Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication.

This study evaluated whether intensification of standard antiretroviral therapy with abacavir, with or without efavirenz, leads to better viral suppression and acceleration of the rate of HIV-1 decay. Ten HIV-1-infected individuals were enrolled in a prospective, open-label study and received standard, combination antiretroviral therapy with either 3 or 4 agents. The rate of decay of the HIV-1 latent reservoir and the frequency of intermittent viremia were compared between 5 patients who underwent treatment intensification and 5 control subjects with comparable baseline characteristics. When compared with control patients, the median half-life (t1/2) of the latent reservoir decreased from 31 to 10 months (P = 0.016) in subjects who had treatment intensification. The frequency of intermittent viremia/year also decreased in 4 of 5 individuals following intensification (2.4/y vs. 0.8/y). These data suggest that ongoing virus replication during standard antiretroviral therapy is due, in part, to the inadequate antiviral potency of current regimens. Despite better viral suppression, treatment intensification did not completely block viral replication, as evidenced by continuing intermittent viremia in some individuals. Additional studies are needed to understand the host- and pathogen-related determinants of incomplete pharmacologic control of HIV-1 replication.

Adult↗
PubMed2005

Inhibition of simian/human immunodeficiency virus replication in CD4+ T cells derived from lentiviral-transduced CD34+ hematopoietic cells.

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

Adenosine Deaminase↗
PubMed2006

Resveratrol inhibition of varicella-zoster virus replication in vitro.

Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30 h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.

Antiviral Agents↗
PubMed1986

The effect of loss of regulation of minus-strand RNA synthesis on Sindbis virus replication.

During the replication cycle of Sindbis virus minus-strand synthesis stops normally at the time that plus-strand synthesis reaches a maximum rate. We have isolated and characterized revertants of ts24, a member of the A complementation group of Sindbis HR mutants, that we had demonstrated previously to have a temperature-sensitive defect in the regulation of minus-strand synthesis. These revertants of ts24 replicated efficiently at 40 degrees but nevertheless retained the temperature sensitive defect in the regulation of minus-strand synthesis: they continued to synthesize minus strands late in the replication cycle at 40 degrees but not at 30 degrees and in the presence or absence of protein synthesis. Although failure to regulate the synthesis of minus strands resulted in continuous minus-strand synthesis and in the accumulation of minus strands, the rate of plus-strand synthesis was not increased concertedly. Minus strands synthesized after the rate of plus-strand synthesis had become constant were demonstrated to be utilized as templates for 26 S mRNA synthesis. Thus, the change from an increasing to a constant rate of plus-strand synthesis during the alphavirus replication cycle cannot be governed solely by the number of minus strands that accumulate in infected cells. We present a model for the preferential utilization of minus strands as a mechanism for the cessation of minus-strand synthesis that occurs normally during alphavirus replication.

Animals↗
PubMed2003

Regulation of hepatitis B virus replication by the ras-mitogen-activated protein kinase signaling pathway.

The replication of hepatitis B virus (HBV) can be regulated by a variety of factors, including hormones, growth factors, and cytokines. However, the molecular mechanisms of these regulations are largely unknown. Ras is a small GTPase that responds to many of these external stimuli. In this study, we investigated the possible effect of Ras on the replication of HBV. Our results indicated that activated Ras could suppress the replication of HBV in both Huh7 and HepG2 cells. This suppression was independent of the X protein and most likely occurred at the transcriptional level. Deletion-mapping analysis of the HBV core promoter and its upstream ENI and ENII enhancers revealed multiple elements responsive to activated Ras. This suppression of HBV replication by activated Ras was apparently mediated by the mitogen-activated protein (MAP) kinase pathway, as it was accompanied by activation of ERK1/2 and abolished by the MEK1/2 inhibitor U0126. Our results thus indicate that external stimuli may suppress HBV replication through the Ras-MAP kinase pathway.

DNA, Viral↗
PubMed2006

Heat shock enhances the susceptibility of BHK cells to rotavirus infection through the facilitation of entry and post-entry virus replication steps.

Rotavirus infection is known to induce several cellular stress proteins, although their possible involvement in the replication cycle of the virus has not been studied. In addition, the heat shock cognate protein hsc70 has been shown to function as a post-attachment receptor during virus entry. In this work we have studied the effect of heat shock on the susceptibility of cells to rotavirus infection. BHK cells, which are largely refractory to the virus, became about 100-fold more susceptible when heat-treated, while the rotavirus highly susceptible MA104 cells did not significantly modified their susceptibility upon heat stress, suggesting that heat shock induces factors that are rate-limiting the replication of rotaviruses in BHK but not in MA104 cells. The heat treatment was shown to facilitate the rotavirus infection of BHK cells at the penetration and post-penetration levels, and each of these stages seems to contribute comparably to the overall observed 100-fold increase in infectivity. Since the binding of the virus to the cell surface was not affected, the caloric stress probably facilitates the penetration and/or uncoating of the virus. The pathway of virus entry into heat-shocked BHK cells seems to be similar to that used in MA104 cells, since treatments that affect MA104 cell infection also affected rotavirus infectivity in heat-treated BHK cells.

Animals↗
PubMed2002

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement.

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.

Cucumovirus↗
PubMed1994

Identification of the bovine herpesvirus 1 circ protein, a myristylated and virion-associated polypeptide which is not essential for virus replication in cell culture.

We have recently reported immediate-early (IE) transcription over covalently joined genome ends of bovine herpesvirus 1 (BHV-1). A spliced 1.5-kb IE RNA (IER1.5) is coterminal with an unspliced 1.1-kb late RNA (LR1.1) which is transcribed from the left end of the genome. Sequence analysis reveals an open reading frame common to IER1.5 and LR1.1 predicted to encode the 247-amino-acid circ polypeptide. This paper reports on the identification of circ as a protein. Using a rabbit antiserum raised against a synthetic oligopeptide representing the carboxy terminus of the predicted circ polypeptide for Western blot (immunoblot) analyses and immunofluorescence assays, we identified a 34-kDa virion-associated protein which accumulated in the cytoplasm of infected cells. To confirm that LR1.1 indeed encoded the 34-kDa polypeptide, we inserted a DNA fragment containing circ coding sequences into the Autographa californica baculovirus genome. A group of recombinant polypeptides with sizes of 32, 34, and 35 kDa were identified by their reactivity with the antipeptide serum. Chicken egg yolk antibodies raised against total proteins of insect cells infected with the recombinant baculovirus identified the 34-kDa circ protein specified by BHV-1. The recombinant circ polypeptides and the circ protein specified by BHV-1 were both myristylated, as determined by radiolabeling with [3H]myristic acid. It was noted that the circ gene could be deleted from the BHV-1 genome without impairing virus replication in cell culture.

Amino Acid Sequence↗