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PubMed2004

HIV-1 specific CD8+ T cells with an effector phenotype and control of viral replication.

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.

Antiretroviral Therapy, Highly Active↗

PubMed1997

The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.

Animals↗

PubMed1994

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Base Sequence↗

PubMed1997

The Epstein-Barr virus-induced Ca2+/calmodulin-dependent kinase type IV/Gr promotes a Ca(2+)-dependent switch from latency to viral replication.

The switch from latency to viral replication in Epstein-Barr virus (EBV)-transformed human B cells is mediated by Zta, the protein product of immediate-early EBV gene BZLF1. BZLF1 transcription is normally suppressed in EBV-transformed B cells but can be induced in some cell lines upon ligation of surface immunoglobulin by mechanisms that include the activation of Ca(2+)-dependent signaling pathways. The multifunctional Ca2+/calmodulin-dependent kinase type IV/Gr (CaMKIV/Gr) is normally absent in primary human B cells, but its expression is induced by the EBV oncoprotein LMP1 in the course of B-cell growth transformation by EBV. In this study, we demonstrate that activated CaMKIV/Gr induces transcription from the BZLF1 promoter and upregulates the expression of Zta in permissive cells. Transcriptional activation of the BZLF1 promoter by CaMKIV/Gr is dependent on the CREB/AP1 binding element ZII and is greatly augmented by the Ca2+/calmodulin-dependent phosphatase calcineurin. These results outline a virus-regulated mechanism involving CaMKIV/Gr which promotes transition from latency to productive viral replication in response to Ca(2+)-mobilizing extracellular signals.

Binding Sites↗

PubMedDate not supplied

Granulocyte/monocyte apheresis induces sustained increases in CD4 T cells in HIV-1 infected patients with poor CD4 T cell restoration after suppression of viral replication by HAART.

Current antiretroviral regimens (HAART) are generally effective in reducing viral replication to undetectable levels and inducing a raise in CD4 T cells. However, in approximately 5 to 15% of patients suppression of viral replication is not followed by an increase in CD4 T cells. Such patients may be at increased risk for opportunistic infections. Here we report the results from a phase II open label randomised trial on 30 patients classified as poor responders to HAART who were either subjected to eight consecutive cycles of selective monocyte apheresis or maintained under HAART alone. The results show that monocyte apheresis results in increased CD4 T cell counts which are maintained for at least 31 weeks after last apheresis. This effect was observed only on patients with complete suppression of viral replication. Other effects of monocyte apheresis included a strong reduction of TNF-alpha production in patients with high baseline levels of this cytokine and activation of resting T cells during the apheresis cycles. In two patients with high cellular HIV DNA load apheresis was followed by a 98% reduction, suggesting purging of infected cells. There was no evidence of increased viral replication during or after the apheresis cycles. The data show that monocyte apheresis is safe, well tolerated and may be indicated in patients who respond poorly to HAART.

Adult↗

PubMed2003

Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep).

Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery. Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication. It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR). By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication. We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA). Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep. We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.

Amino Acid Sequence↗

PubMed2006

COPI activity coupled with fatty acid biosynthesis is required for viral replication.

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.

Animals↗

PubMed2005

HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo.

Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

Animals↗

PubMed1995

Intraocular viral replication after systemic murine cytomegalovirus infection requires immunosuppression.

PURPOSE: Human cytomegalovirus retinitis is the most common blinding complication of acquired immune deficiency syndrome. However, the pathogenesis of the disease is poorly understood. The authors sought to characterize intraocular viral replication after systemic murine cytomegalovirus (MCMV) infection in the normal and immunosuppressed Balb/c mouse. METHODS: Normal or immunosuppressed mice (400 rads radiation plus antilymphocyte serum) were infected intravenously with a recombinant MCMV (RM408) that carries an MCMV IE1 promoter--LacZ insert. In vivo MCMV replication and its tissue distribution were monitored by beta-gal activity with x-gal staining on frozen tissue sections of multiple organs harvested from infected mice at different time points after inoculation. RESULTS: MCMV replication within the eye can be detected in the immunosuppressed Balb/c mouse but not in the normal host. Intraocular viral replication was noted first, and most frequently, in the ciliary body and was mainly restricted to the uveal tract. Intraocular viral replication coincided with the peak of systemic viral replication; however, the neurosensory retina was spared. In contrast, supraciliary inoculation of MCMV in the immunosuppressed Balb/c mouse resulted in massive viral replication and destruction of the neurosensory retina. CONCLUSIONS: This study demonstrated that intraocular MCMV replication after systemic infection requires systemic immunosuppression. Furthermore, the ciliary body is the portal of entry for the virus within the eye. MCMV can replicate in the epithelium of the uvea and retinal pigment epithelium, but it does not replicate within the neurosensory retina. The absence of MCMV replication within the neurosensory retina is not caused by either a defect in the recombinant virus or the inability of the host tissue to support viral replication.

Animals↗

PubMed1994

Flow cytometry CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication in chronic hepatitis B.

T lymphocytes have been assumed to play an essential role in tissue injury in patients with chronic hepatitis B. As hepatitis B virus (HBV) is considered as a major factor controlling liver inflammation, we assessed whether a particular T lymphocyte subset could be preferentially detected in the liver in accordance with viral replication. Liver-derived lymphocytes and peripheral blood lymphocytes were analysed by flow cytometry in 21 patients with histologically confirmed chronic hepatitis B without cirrhosis. Viral replication was quantified by hybridization of serum HBV DNA. Eleven patients exhibited an active viral replication with serum HBV DNA ranging from 10 to 388 pg/ml at the time of the liver biopsy, whereas 10 patients had no detectable serum HBV DNA. In patients exhibiting viral replication, CD4+/CD8+ ratios of liver-derived lymphocytes were significantly higher (P < 0.05) than those obtained in patients without viral replication. In contrast, the percentage of T cells expressing the gamma/delta receptor and that of CD2+/CD57+ cells were similar in both groups of patients. Furthermore, in patients exhibiting viral replication, CD4+CD8+ ratios of liver-derived lymphocytes correlated with serum HBV DNA levels (P < 0.001). No relationship between CD4+/CD8+ ratio of liver-derived and peripheral blood lymphocytes was observed. Our data indicate that, in patients with chronic hepatitis B, the CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication. This suggests that in situ helper/inducer CD4+ T lymphocytes may positively regulate the cytotoxic T cell activity in patients with HBV-related chronic hepatitis.

Adult↗

PubMed1991

Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.

Serum and liver HBsAg bear an inverse relation to each other during the evolution of chronic hepatitis B virus infection and the quantity of HBsAg in tissue rises gradually with time. In this study, intracellular and extracellular levels of HBsAg were measured by radioimmunoassay in primary culture of hepatocytes from 30 patients with chronic hepatitis B virus infection to determine a possible relationship with hepatitis B virus replication. Serum levels of HBsAg correlated with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.005, and tissue HBcAg, p less than 0.02) but inversely with tissue HBsAg (p less than 0.05). In similar fashion, in vitro export of HBsAg was also related to the presence of active viral replication markers (serum hepatitis B virus DNA, p less than 0.02, and tissue HBcAg, p less than 0.05) and negatively with tissue HBsAg (p less than 0.001). Export of HBeAg also correlated positively with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.05 and tissue HBcAg, p less than 0.05). Further experiments indicated that intrahepatic pre-S1 and pre-S2 correlated closely with intrahepatic HBsAg, indicating that a failure to export HBsAg was unlikely to be attributable to deficient intracellular expression of pre-S1 or pre-S2. These data indicate that in vitro primary hepatocyte culture of hepatitis B virus-infected cells provides an accurate reflection of in vivo export of HBsAg and that this is closely related to the presence of active viral replication.

Adult↗

PubMed2005

The cellular HIV-1 Rev cofactor hRIP is required for viral replication.

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after reintroduction of hRIP protein. Taken together, our results indicate that hRIP is an essential cellular cofactor for Rev function and HIV-1 replication. Because hRIP is not required for cell viability, it may be an attractive target for the development of new antiviral strategies.

Gene Products, rev↗

PubMed1993

The human immunodeficiency virus type 1 (HIV-1) vif protein is located in the cytoplasm of infected cells and its effect on viral replication is equivalent in HIV-2.

The human immunodeficiency virus type 1 (HIV-1) vif gene (viral infectivity gene) plays an important role in viral replication in vitro. We demonstrated that the Vif protein is membrane associated in HIV-1-infected cells and have investigated the role in viral replication of the equivalent gene in HIV-2. We constructed an HIV-2 vif minus mutant and studied its virulence and cellular tropism in vitro. Parallel experiments were also performed with an HIV-1 vif mutant to ascertain whether the two distantly related HIV-2 and HIV-1 genes might exert the same effect on viral replication. The results indicated that both HIV-1 and HIV-2 vif minus cell-free infection was not impaired when the SupT-1 cell line was used. However, differential degrees of impairment in viral replication were observed when other cell lines were used (Molt-3, U-937). Nevertheless, when viral production could not be detected, rescue experiments by coculture with the permissive cell line SupT-1 were generally positive, indicating that the viruses were still present in the inoculated cells. In contrast, when primary human cells (peripheral blood mononuclear cells and purified macrophages) were infected with HIV-1 and HIV-2 vif minus viruses no productive infection was observed and generally no virus was rescued by cocultivation. Thus, like in HIV-1, the vif gene of HIV-2 is crucial for viral infectivity in primary cells and might represent an attractive target for therapy.

Cell Compartmentation↗

PubMed2005

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication.

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.

Amino Acid Sequence↗

PubMed2003

Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection.

To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre-replication complex. Antibody to Ki-67, a conventional marker of cell division, was also studied. Eighty-seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV-infected patients and the proportion of hepatocytes expressing Mcm-2 (3-40%) always exceeded that expressing Ki-67 (1-14%) and correlated positively with increasing stage of fibrosis (P = 0.0001) and viral replication (P = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm-2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm-2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol (P = 0.0067). The proportion of Ki-67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm-2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm-2 is a more sensitive marker of hepatocyte proliferation than Ki-67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.

Adult↗

PubMed2006

HCV infection and chronic arthritis: Does viral replication matter?

BACKGROUND: HCV infection beside chronic hepatitis can induce immunological disorders with different clinical expressions such as chronic arthritis. AIM: To study the prevalence of arthritis in HCV-Ab positive patients and verify possible correlation with viral replication, hepatic damage and autoimmunity imbalance. STUDY DESIGN: Three hundred and eighty patients (196 M and 184 F) affected by HCV infection were examined and 38 (10%) were selected according to the presence of arthritis. Eight of them were excluded because arthritis raised before HCV infection. Each patient, once undergone liver biopsy, was evaluated for: clinical examination (articular evolution), Rx examination, serum expression of hepatic damage (mainly ALT), viral replication, and involvement of autoimmunity (ANA, RF, crioglobulins, AKA, CCP). RESULTS: Data from patients [Lamprecht P, Gause A, Gross WL. Cryoglobulinemic vasculitis. Arthritis Rheum 1999; 42:2507-16.] with AKA and CCP positivity were not considered for statistical examination because the clear correlation between rheumatoid arthritis and these parameters. The remaining 20 patients showed hepatic damage 47%, viral replication in 74%, RF 42%, ANA 16%, crioglobulins 42% (RF positive). No correlation was evident between ANA serum concentrations and viral replication; furthermore a significant negative correlation between RF positivity and viral replication only in a subgroup of patients with serologic expression of hepatic damage was found. CONCLUSIONS: These data support hypothesis that the onset of arthritis and presence of autoimmunity parameters ANA, RF are not related to the viral replication but others mechanism immunological induced by HCV might be considered.

Journal Article↗

PubMed1981

Chronic hepatitis B: correlation between viral replication and clinical course.

Twenty carriers of hepatitis B virus (HBV) were followed for two to seven years, and their histologic progression was correlated with HBV core-associated DNA polymerase (DNAP) activity as a marker of viral replication. Seventeen patients were divided into two groups according to their pattern of viral replication: group 1, consistently high levels of DNAP; group 2, low levels of DNAP. Chronic persistent hepatitis predominated in group 1; chronic active hepatitis predominated in group 2. The three remaining patients were consistently negative for DNAP. In two patients in group 2, prominent viral replication preceded a transient increase in transaminase levels, as in acute hepatitis. Although groups 1 and 2 were distinct in their patterns of viral replication, they did not differ significantly in histologic progression. Thus, viral replication is related, at least in part, to hepatic cell necrosis but does not correlate closely with progression to liver cirrhosis.

Adult↗

PubMed1983

The simian-virus-40 large-tumor antigen in replicating viral chromatin. A salt-resistant protein-DNA interaction.

The large tumor antigen (T antigen) is a genome regulation protein, coded by simian virus 40, that binds with high affinity to specific binding sites on viral DNA. The specifically bound T antigen is released from these sites in 0.2-0.3 M NaCl. Immunoprecipitation techniques were used to show that T antigen also dissociates in 0.2-0.3 M NaCl from mature viral chromatin but not from replicating viral chromatin. In fact, a considerable fraction of T antigen remains associated with replicating chromatin at NaCl concentrations as high as 1.2 M NaCl when most chromatin proteins, including histones, dissociate. However, T antigen binding to both replicating DNA and mature DNA is sensitive to intercalating drugs such as caffeine and ethidium bromide. We consider the possibility that the unexpectedly tight binding of T antigen to replicating DNA is related to the function that T antigen performs during viral DNA replication.

Antigens, Neoplasm↗