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At least 253 records · Page 14Linked to original sources

PubMed2006

DNA-damage response pathways triggered by viral replication.

Many viruses, with distinct replication strategies, activate DNA-damage response pathways, including the lentivirus human immunodeficiency virus (HIV) and the DNA viruses Epstein-Barr virus (EBV), herpes simplex virus 1, adenovirus and SV40. DNA-damage response pathways involving DNA-dependent protein kinase, ataxia-telengiectasia mutated (ATM) and 'ataxia-telengiectasia and Rad3-related' (ATR) have all been implicated. This review focuses on the effects of HIV and EBV replication on DNA repair pathways. It has been suggested that activation of cellular DNA repair and recombination enzymes is beneficial for viral replication, as illustrated by the ability of suppressors of the ATM and ATR family to inhibit HIV replication. However, activation of DNA-damage response pathways can also promote apoptosis. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. New small-molecule inhibitors of the DNA-damage response pathways could therefore be of value to treat viral infections.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1992

Effects of transforming growth factor-beta 1 against the inhibitory action of interferon on DNA synthesis and viral replication in hepatitis B virus DNA-transfected cell.

Studies were undertaken to examine the effects of recombinant human transforming growth factor beta 1 (TGF-beta 1) on DNA synthesis and antiviral actions of interferons (IFNs) in HepG2 cell, a hepatoma cell line, transfected with hepatitis B virus (HBV) DNA. The inhibitory effects of IFN-alpha and -gamma on DNA synthesis of HepG2 cells were enhanced in a dose-dependent manner by a simultaneous addition of TGF-beta. The degree of suppression by the reagents was greater in HBV-nontransfected cells than in transfected cells. Inhibition of DNA synthesis was not due to direct cytotoxic effects of the additives, since the viability of HepG2 cells was comparable in the control and treated cultures as determined by trypan blue exclusion. Treatment of HBV DNA-transfected HepG2 cells with IFNs resulted in decrease in production of HB surface and e antigens, and in the level of HBV DNA, but TGF-beta reversed the IFN-induced antiviral state in HBV DNA-transfected HepG2 cells. TGF-beta had no direct effect on HBV replication. These results indicate that rTGF-beta 1 exerts a differential effect against the inhibitory actions of IFN on DNA synthesis and viral replication in HepG2 cells.

DNA, Viral↗

PubMed2001

The role of adenovirus E4orf4 protein in viral replication and cell killing.

It has only been within the last few years that insights have been gained into the remarkable diversity of functions of the adenovirus early transcription region 4 (E4) products. The polypeptide encoded by E4 open reading frame 4 (E4orf4) has emerged as an enigmatic product. Although it accomplishes certain functions that propel viral replication, it has also been shown to be highly toxic, an effect that could dampen the infectious cycle, but that also might serve to facilitate release of viral progeny. When expressed alone, E4orf4 induces a novel form of p53-independent apoptosis in cancer cells but not in normal human cells, thus making it of potential use in cancer gene therapy. In addition, knowledge of its mechanism of action, especially with regard to its interaction with protein phosphatase 2A (PP2A), could provide insights to develop new small molecule anti-cancer drugs. Thus future studies on E4orf4 should be both informative and potentially valuable therapeutically. In this study we review the current status of knowledge on E4orf4.

Adenoviridae↗

PubMed2002

Avian and Mammalian hepadnaviruses have distinct transcription factor requirements for viral replication.

Hepadnavirus replication occurs in hepatocytes in vivo and in hepatoma cell lines in cell culture. Hepatitis B virus (HBV) replication can occur in nonhepatoma cells when pregenomic RNA synthesis from viral DNA is activated by the expression of the nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X receptor alpha (RXR alpha) plus peroxisome proliferator-activated receptor alpha (PPAR alpha) heterodimer. Nuclear hormone receptor-dependent HBV replication is inhibited by hepatocyte nuclear factor 3 (HNF3). In contrast, HNF3 and HNF4 support duck hepatitis B virus (DHBV) replication in nonhepatoma cells, whereas the RXR alpha-PPAR alpha heterodimer inhibits HNF4-dependent DHBV replication. HNF3 and HNF4 synergistically activate DHBV pregenomic RNA synthesis and viral replication. The conditions that support HBV or DHBV replication in nonhepatoma cells are not able to support woodchuck hepatitis virus replication. These observations indicate that avian and mammalian hepadnaviruses have distinct transcription factor requirements for viral replication.

3T3 Cells↗

PubMed1981

Radioimmunoassay for hepatitis B 'e' antigen and antibody: correlations with viral replication and prognostic value.

The presence of hepatitis B 'e' antigen (HBeAg) and its antibody (anti-HBe was evaluated by radioimmunoassay (RIA) in various groups of HBsAg-positive patients. HBeAg was present in the majority of the sera from patients with acute viral hepatitis at onset of clinical symptoms and disappeared after 1 year. Almost all hemodialysis patients had HBeAg in their sera. 40% of the patients who had chronic active hepatitis and 50% with chronic persistent hepatitis had HBeAg with no relationship to the inflammatory activity of the disease evaluated by the presence of mononuclear infiltration in liver biopsy. The comparison between the presence of HBeAg and Dane particle-associated DNA-polymerase activity showed that HBeAg was consistently found in almost all the sera which presented DNA-polymerase activity. HBeAg, as determined by RIA, may therefore be useful in the screening of highly infective patients with elevated viral replication.

Antibodies, Viral↗

PubMed2005

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication.

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.

Base Sequence↗

PubMed2003

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines.

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.

Animals↗

PubMed2006

The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication.

The results presented here demonstrate that the MHV and SARS-CoV nsp2 proteins are not required for the production of infectious virus, for polyprotein expression or processing, or for viral replication complex formation in cell culture. The nsp2 protein domain resides in a region of the coronavirus replicase that is relatively nonconserved across coronaviruses. In fact, the size and amino acid sequence variability of nsp2 across the different coronaviruses has led some investigators to speculate that the nsp2 protein, along with the nsp1 and nsp3 proteins, may play host- and/or cell-specific roles in the virus life cycle. While this may be the case, it should be noted that nsp2, in some form, exists in all coronaviruses studied to date and likely plays a pivotal role in the viral life cycle. A previous study from our laboratory identified a coronavirus replicase protein that plays an important role in viral pathogenesis. Such may prove to be the case for nsp2, as well. Alternatively, beacuse nsp2 exists as a detectable precursor protein nsp2-3 prior to processing of nsp2 and nsp3 into mature proteins, nsp2 may play a critical adaptor/regulatory role for nsp3 function. Importantly, the viruses produced in this study provide a system by which the role of the nsp2 protein in viral infection can be characterized.

Animals↗

PubMed2006

Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication.

A recent report sought to demonstrate that acetylation of specific lysines within integrase (IN) by the histone acetyltransferase (HAT) p300 regulates human immunodeficiency virus type 1 (HIV-1) integration and is essential for viral replication (A. Cereseto, L. Manganaro, M. I. Gutierrez, M. Terreni, A. Fittipaldi, M. Lusic, A. Marcello, and M. Giacca, EMBO J. 24:3070-3081, 2005). We can corroborate the efficient and specific acetylation of the IN carboxyl-terminal domain (CTD) (amino acids 212 to 288) by p300 using purified recombinant components. Although arginine substitution mutagenesis of the isolated CTD confirms that the majority of p300 acetylation occurs at lysine residues 264, 266, and 273, the pattern of acetylation is not uniform and a hierarchy of reactivity can be established. Several combinatorial mutations of the CTD lysines modified by p300 in vitro were reconstructed into an otherwise infectious proviral plasmid clone and examined for viral growth and frequency of productive chromosomal integration. In contrast to the findings of Cereseto and coworkers, who used epitope-tagged viruses for their experiments, we find that an untagged mutant virus, IN K(264/266/273)R, is fully replication competent. This discrepancy may be explained by the use of an acidic epitope tag placed at the extreme carboxyl terminus of integrase, near the target site for acetylation. Although the tagged, wild-type virus is viable, the combination of this epitope tag with the RRR substitution mutation results in a replication-defective phenotype. Although IN belongs to the very small set of nonhistone proteins modified by HAT-mediated activity, an obligate role for acetylation at the reactive CTD lysines in HIV-1 IN cannot be confirmed.

Acetylation↗

PubMed2001

Increased drug susceptibility of HIV-1 reverse transcriptase mutants containing M184V and zidovudine-associated mutations: analysis of enzyme processivity, chain-terminator removal and viral replication.

The presence of the HIV reverse transcriptase (RT) resistance mutation, M184V, induced by lamivudine and abacavir treatment results in increased tenofovir, adefovir and zidovudine susceptibility for HIV-1 with zidovudine-associated RT mutations in vitro. Treatment with oral prodrugs of tenofovir and adefovir has resulted in substantial HIV-1 RNA reductions in antiretroviral-experienced patient populations who have lamivudine- and zidovudine-resistant HIV-1. An enzymatic analysis was undertaken to elucidate the mechanisms of altered drug susceptibilities of HIV-1 containing zidovudine-associated mutations in the presence or absence of M184V. The inhibition constants (Ki) for the active metabolites of tenofovir, adefovir and zidovudine did not vary significantly between recombinant mutant and wild-type RT enzymes. Although increased removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent unblocking correlated with reduced susceptibility of viruses with zidovudine-associated mutations, a reduction in the removal of chain-terminators was not observed, which would explain the increased drug susceptibility of mutants containing M184V plus zidovudine-associated mutations. However, analyses of single-cycle processivity of the mutant RT enzymes on heteropolymeric RNA templates showed that all M184V-containing mutant RT enzymes were less processive than wild-type RT, most notably for mutants expressing both zidovudine-associated mutations and M184V. Similarly, the in vitro replication capacity of a mutant virus expressing a zidovudine-associated mutation and M184V was significantly reduced compared with wild-type virus. The observed decrease in enzymatic processivity of the M184V-expressing RT enzymes might result in decreased viral replication, which then might contribute to the increased drug susceptibility of HIV-1 expressing these RT mutations.

Anti-HIV Agents↗

PubMed1999

No association of HIV type 1 long terminal repeat sequence pattern with long-term nonprogression and in vivo viral replication levels in European subjects.

The HIV-1 long terminal repeat (LTR) promotes and modulates proviral transcription in the infected cell. It has been suggested that truncations and even point mutations in functional sites of the LTR are associated with low viral replication and attenuated pathogenesis in HIV-1-infected long-term nonprogressors (LTNPs). We performed a detailed analysis of LTR sequences from proviral DNA of 21 Italian and Swedish, well-characterized LTNPs and of 15 progressor patients. No truncation was found and no correlation was identified between specific LTR mutations and disease progression. We also failed to find a significant correlation between phylogenetic distance and clinical status. Although HIV-1 LTR interpatient heterogeneity among LTNPs and subjects with HIV-1 RNA levels <500 copies/ml tended to be lower, no sequence mutation was correlated with in vivo viral loads. Our results suggest that HIV-1 LTR defects are rare among Italian and Swedish LTNPs.

Adolescent↗

PubMed2003

Requirement of varicella-zoster virus immediate-early 4 protein for viral replication.

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZV open reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.

Dimerization↗

PubMed2006

The expression of p16INK4a tumor suppressor is upregulated by human cytomegalovirus infection and required for optimal viral replication.

The human cytomegalovirus (HCMV) induces a replicative senescence program after arresting host cell cycle progression so as to create a favorable environment for its replication. Here, we report that HCMV infection stimulates the expression of p16(INK4a), a direct effector of the senescence phenotype. The increase in p16(INK4a) gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the p16(INK4a)-encoding CDKN2A gene promoter was strongly induced by HCMV infection. The results of deletion and mutational analysis of the CDKN2A promoter further suggest the involvement of Ets transcription factors in HCMV-mediated stimulation of p16(INK4a) gene expression. The significance of p16(INK4a) upregulation during the HCMV replicative cycle is underscored by the finding that virus replication was severely impaired in fibroblasts homozygous for an intragenic deletion in CDKN2A locus and devoid of functional p16(INK4a). Moreover, a retrovirus-mediated p16(INK4a) small interfering RNA (p16-siRNA) effectively reduced viral replication, thus providing direct evidence that p16(INK4a) upregulation plays a positive role for HCMV replication.

Artificial Gene Fusion↗

PubMed1990

Effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (HIV) long terminal repeat-directed transcription in cells chronically infected with HIV-1.

The protein kinase C (PKC) activator phorbol myristate acetate (PMA) was used to upregulate viral replication in a clone of promonocytic cells chronically infected with human immunodeficiency virus (HIV)-1. Induction of virus could be inhibited by the triphenylethylene anti-estrogen tamoxifen at concentrations that had minimal effects on cellular DNA synthetic responses and cell cycle kinetics. This effect correlated with tamoxifen's ability to block PMA-mediated enhancement of HIV-promoter-driven transactivation in cells of monocyte and CD4+ T-lymphocyte lineages. No interference with a primary infection was noted. Tamoxifen's mechanism of action may relate both to its capacity to inhibit PKC and to consensus sequences for gonadal steroid responsive elements in the HIV long terminal repeat, as it was able to partially inhibit another HIV activator, 5-azacytidine, which does not modulate PKC function. The finding that regulation of HIV in a model for low-level chronic or latent infection is amenable to a nonimmunosuppressive steroid antagonist may suggest approaches to pharmacologic intervention early in HIV infection.

Cell Cycle↗

PubMed1991

Molecular characterization of two bipartite geminiviruses causing squash leaf curl disease: role of viral replication and movement functions in determining host range.

The genomes of two distinct, but highly homologous, bipartite geminiviruses have been identified in and cloned from extracts of squash leaf curl diseased field squash. These two squash leaf curl viruses (SqLCVs) have covalently closed, circular single-stranded DNA genomes with the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Infectivity studies using virus preparations or cloned viral genomic components on different potential host plants demonstrated that these two SqLCVs have different host range phenotypes which can be explained by specific interactions among the different viral genomic components that act to influence viral replication and systemic movement in the plant. Analysis of Agrobacterium-inoculated leaf discs demonstrated that replication of the restricted virus was rescued in trans by the nonrestricted virus, providing an explanation for the mixtures of viral DNA components often found in particular hosts in the field. Sequence analysis of the common regions of these two SqLCVs identified a 13-base deletion in the restricted virus as compared to the nonrestricted virus, suggesting a potential sequence alteration likely to be involved in their host range phenotypic differences and strengthening the conclusion based on hybridization studies of their close evolutionary relationship. Also identified in the original field squash was a defective viral component which appeared to interfere with movement of the restricted SqLCV in its normally permissive hosts and accounted for another aspect of host range variation observed for this virus.

Base Sequence↗

PubMed2002

The HLA class II transcriptional activator blocks the function of HIV-1 Tat and inhibits viral replication.

The expression of HLA class II genes is under the control of a transcriptional activator, CIITA, encoded by the AIR-1 locus. Here we show that CIITA inhibits HIV-1 LTR transactivation mediated by Tat. The inhibition occurred when CIITA and Tat were transiently expressed in cells after transfection and, most importantly, when tat cDNA was transfected in cells expressing CIITA in a constitutive fashion and at physiological levels. Furthermore, CIITA inhibited the HIV-1 LTR transactivation mediated by extracellular Tat protein. CIITA inhibition of Tat function could be reversed by overexpression of Cyclin T1, the cellular cofactor used by Tat to facilitate elongation of viral transcripts. CIITA inhibition of Tat function had a dramatic effect on HIV-1 productive infection of human T cells because CIITA(+) T cells supported very poorly, if any, viral replication. These results indicate that sustained expression of CIITA in HIV-1-susceptible targets may down-regulate viral expression both in cells actively replicating the virus and in silently infected cells requiring exogenous Tat to reactivate virus from latency.

Gene Products, tat↗

PubMed2002

Critical role of human T-lymphotropic virus type 1 accessory proteins in viral replication and pathogenesis.

Human T-cell lymphotropic virus type 1 (HTLV-1) infection is associated with a diverse range of lymphoproliferative and neurodegenerative diseases, yet pathogenic mechanisms induced by the virus remain obscure. This complex retrovirus contains typical structural and enzymatic genes but also unique regulatory and accessory genes in four open reading frames (ORFs) of the pX region of the viral genome (pX ORFs I to IV). The regulatory proteins encoded by pX ORFs III and IV, Tax and Rex, respectively, have been extensively characterized. In contrast the contribution of the four accessory proteins p12(I), p27(I), p13(II), and p30(II), encoded by pX ORFs I and II, to viral replication and pathogenesis remained unclear. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. Emerging evidence indicates that the HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T-lymphocyte activation, and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes, suggesting a role for p12(I) in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12(I), encoded from pX ORF I, activates NFAT, a key T-cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30(II) localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related coactivators of transcription. p13(II) targets mitochondrial proteins, where it alters the organelle morphology and may influence energy metabolism. Collectively, studies of the molecular functions of the HTLV-1 accessory proteins provide insight into strategies used by retroviruses that are associated with lymphoproliferative diseases.

Animals↗

PubMed2004

Insertion of an exogenous promoter in the E1A regulatory region of adenovirus does not disturb viral replication despite reduced E1A transcription.

Insertion of an exogenous promoter into adenoviral regulatory regions may result in altered specificity and activity of the integrated promoter-mediated transcription in the context of recombinant adenoviruses (Ad). The alteration is due to the influence of the viral regulatory elements. Specificity of oncolytic Ad, in which the E1A expression is designed to be controlled by a tumor-specific promoter, could thus be modulated by the Ad E1A enhancer/promoters. We prepared recombinant Ad bearing a midkine (MK) promoter region in the 3'-side of the E1A promoter and investigated the relationship between transcriptional activity by the E1A promoter-fused MK fragment and the viral replication. Reporter assays revealed that the transcriptional activity of the fused E1A-MK fragment was significantly lower than that of respective E1A and the MK promoters. However, the replication of the Ad bearing the MK promoter was greater than or comparable to that of wild-type Ad. The present study suggests that the replication of oncolytic Ad in tumors is not directly correlated with the promoter activity to transcribe the E1A gene and that insertion of an exogenous promoter downstream to the E1A regulatory region may not always disturb Ad replication.

Adenoviridae↗