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Biomedical subjects

Francis V Chisari

Publications and source records attributed to Francis V Chisari.

At least 37 records · Page 2Linked to original sources
PubMed2005

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously demonstrated that high-level HCV replication during acute infection of chimpanzees is associated with the modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we demonstrate that Huh-7 cells harboring replicating, full-length HCV RNAs express elevated levels of ATP citrate lyase and acetyl-CoA synthetase genes, both of which are involved in cholesterol and fatty acid biosynthesis. Further, we confirm that the cholesterol-biosynthetic pathway controls HCV RNA replication by regulating the cellular levels of geranylgeranyl pyrophosphate, we demonstrate that the impact of geranylgeranylation depends on the fatty acid content of the cell, and we show that fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. These results illustrate a complex cellular-regulatory network that controls HCV RNA replication, presumably by modulating the trafficking and association of cellular and/or viral proteins with cellular membranes, suggesting that pharmacologic manipulation of these pathways may have a therapeutic effect in chronic HCV infection.

ATP Citrate (pro-S)-Lyase↗
PubMed2005

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs.

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBV-specific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.

Adenoviridae↗
PubMed2005

Oscillating CD8(+) T cell effector functions after antigen recognition in the liver.

When hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are adoptively transferred into HBV transgenic mice, they enter the liver, recognize antigen, secrete interferon gamma (IFNgamma), inhibit viral replication, and kill their target cells, causing hepatitis. In the current study, we examined the impact of antigen recognition on the evolution of the activation phenotype, antiviral effector functions, expansion and contraction kinetics, and compartmentalization of the transferred CTLs. The results reveal that noncytolytic and cytolytic effector functions and expansion-contraction kinetics of the CTLs are regulated asynchronously and in an oscillatory manner as a consequence of antigen recognition in the liver and in association with PD-1 upregulation. We suggest that such oscillations maximize viral clearance and minimize tissue injury during HBV infection and that poor coordination of these events could lead to viral persistence and chronic liver disease.

Adoptive Transfer↗
PubMed2005

Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.

Toll-like receptors (TLR) play a key role in innate immunity. To examine the ability of diverse TLRs to modulate hepatitis B virus (HBV) replication, HBV transgenic mice received a single intravenous injection of ligands specific for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. All of the ligands except for TLR2 inhibited HBV replication in the liver noncytopathically within 24 h in a alpha/beta interferon-dependent manner. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation could represent a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.

Animals↗
PubMed2005

Immune tolerance split between hepatitis B virus precore and core proteins.

The function of the hepatitis B virus (HBV) precore or HBeAg is largely unknown because it is not required for viral assembly, infection, or replication. However, the HBeAg does appear to play a role in viral persistence. It has been suggested that the HBeAg may promote HBV chronicity by functioning as an immunoregulatory protein. As a model of chronic HBeAg exposure and to examine the tolerogenic potential of the HBV precore and core (HBcAg) proteins, HBc/HBeAg-transgenic (Tg) mice crossed with T cell receptor (TCR)-Tg mice expressing receptors for the HBc/HBeAgs (i.e., TCR-antigen double-Tg pairs) were produced. This study revealed three phenotypes of HBe/HBcAg-specific T-cell tolerance: (i) profound T-cell tolerance most likely mediated by clonal deletion, (ii) T-cell clonal ignorance, and (iii) nondeletional T-cell tolerance mediated by clonal anergy and dependent on the structure, location, and concentration of the tolerogen. The secreted HBeAg is significantly more efficient than the intracellular HBcAg at eliciting T-cell tolerance. The split T-cell tolerance between the HBeAg and the HBcAg and the clonal heterogeneity of HBc/HBeAg-specific T-cell tolerance may have significant implications for natural HBV infection and especially for precore-negative chronic hepatitis.

Animals↗
PubMed2005

Lambda interferon inhibits hepatitis B and C virus replication.

Lambda interferon (IFN-lambda) induces an intracellular IFN-alpha/beta-like antiviral response through a receptor complex distinct from the IFN-alpha/beta receptor. We therefore determined the ability of IFN-lambda to inhibit hepatitis B virus (HBV) and hepatitis C virus (HCV) replication. IFN-lambda inhibits HBV replication in a differentiated murine hepatocyte cell line with kinetics and efficiency similar to IFN-alpha/beta and does not require the expression of IFN-alpha/beta or IFN-gamma. Furthermore, IFN-lambda blocked the replication of a subgenomic and a full-length genomic HCV replicon in human hepatocyte Huh7 cells. These results suggest the possibility that IFN-lambda may be therapeutically useful in the treatment of chronic HBV or HCV infection.

Animals↗
PubMed2004

A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen.

A unique characteristic of the hepatitis B virus is the production of a secreted form (precore or HBeAg) of the structural nucleocapsid (core or HBcAg). By using T cell receptor (TCR) transgenic (Tg) and TCR x HBc/HBeAg double- and triple-Tg pairs, we demonstrate that HBeAg elicits T cell tolerance, whereas HBcAg is nontolerogenic in this system. In fact, TCR x HBc double-Tg mice spontaneously seroconvert to IgG anti-HBc positivity at an early age. However, the presence of HBeAg in the serum of TCR x HBc x HBe triple-Tg mice prevents anti-HBc seroconversion. HBeAg mediates its immunoregulatory effect by eliciting tolerance in HBc/HBeAg-specific T cells. The results suggest that hepadnaviruses have retained a secretory form of the nucleoprotein because it functions as a T cell tolerogen and regulates the immune response to the intracellular nucleocapsid. This HBeAg-mediated immune regulation may predispose to chronicity during perinatal infections and prevent severe liver injury during adult infections.

Adoptive Transfer↗
PubMed2004

Genomic analysis of the host response to hepatitis B virus infection.

Previous studies in hepatitis B virus (HBV)-infected humans and chimpanzees suggest that control of HBV infection involves the cells, effector functions, and molecular mediators of the immune response. The objective of the current study was to identify, in the liver of acutely HBV-infected chimpanzees, the spectrum of virus-induced and immune response-related genes that regulate the infection. The results demonstrate that HBV does not induce any genes during entry and expansion, suggesting it is a stealth virus early in the infection. In contrast, a large number of T cell-derived IFN-gamma-regulated genes are induced in the liver during viral clearance, reflecting the impact of an adaptive T cell response that inhibits viral replication and kills infected cells, thereby terminating the infection.

Animals↗
PubMed2004

Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees.

We have previously shown that hepatitis B virus (HBV) replication is controlled by noncytolytic mechanisms that depend primarily on the effector functions of the CD8(+) T cell response, especially the production of IFN-gamma in the liver. The mechanisms that control the nuclear pool of viral covalently closed circular DNA (cccDNA) transcriptional template of HBV, which must be eliminated to eradicate infection, have been difficult to resolve. To examine those mechanisms, we quantitated intrahepatic HBV cccDNA levels in acutely infected chimpanzees whose virological, immunological, and pathological features were previously described. Our results demonstrate that the elimination kinetics of the cccDNA are more rapid than the elimination of HBV antigen-positive hepatocytes during the early phase of viral clearance, and they coincide with the influx of small numbers of IFN-gamma producing CD8(+) T cells into the liver. In contrast, terminal clearance of the cccDNA is associated with the peak of liver disease and hepatocellular turnover and with a surge of IFN-gamma producing CD8(+) T cells in the liver. Collectively, these results suggest that cccDNA clearance is a two-step process mediated by the cellular immune response. The first step reduces the pool of cccDNA molecules noncytolytically, probably by eliminating their relaxed circular DNA precursors and perhaps by destabilizing them. The second step enhances this process by destroying infected hepatocytes and triggering their turnover. Surprisingly, despite this multipronged response, traces of cccDNA persist indefinitely in the liver, likely providing a continuous antigenic stimulus that confers lifelong immunity.

Acute Disease↗
PubMed2004

Signal transduction pathways that inhibit hepatitis B virus replication.

The replication of hepatitis B virus (HBV) in hepatocytes is strongly inhibited in response to IFN-alpha/beta and IFN-gamma. Although it has been previously demonstrated that IFN-alpha/beta eliminates HBV RNA-containing capsids from the cell in a proteasome-dependent manner, the precise cellular pathway that mediates this antiviral effect has not been identified. Because IFN-induced signal transduction involves kinase-mediated activation of gene expression, we used an immortalized hepatocyte cell line that replicates HBV in an IFN-sensitive manner to investigate the role of cellular kinase activity and the cellular transcription and translation machinery in the antiviral effect. Our results indicate that Janus kinase activity is required for the antiviral effect of IFN against HBV, but that phosphatidylinositol 3-kinase, cyclin-dependent kinase, mitogen-activated protein kinase, and NF-kappaB activity are not. Additionally, we found that inhibitors of cellular transcription and translation completely abolish the antiviral effect, which also appears to require cellular kinase activity downstream of signal transduction and gene expression. Collectively, these results identify IFN-regulated pathways that interrupt the HBV replication cycle by eliminating viral RNA-containing capsids from the cell, and they provide direction for discovery of the terminal effector molecules that ultimately mediate this antiviral effect.

Animals↗
PubMed2004

MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs.

We recently showed that antigen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1(+) cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all antigen-nonspecific Gr-1(-) mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1(+) cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of antigen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Animals↗
PubMed2003

Interference of hepatitis C virus RNA replication by short interfering RNAs.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFNalpha in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

Base Sequence↗
PubMed2003

Transcriptional and posttranscriptional control of hepatitis B virus gene expression.

Hepatitis B virus (HBV) infects humans and certain nonhuman primates. Viral clearance and acute disease are associated with a strong, polyclonal, multispecific cytotoxic T lymphocyte response. Infiltrating T cells, as well as other activated inflammatory cells, produce cytokines that can regulate hepatocellular gene expression. Using an HBV transgenic mouse model, our laboratory has previously demonstrated that adoptive transfer of HBV-specific cytotoxic T lymphocytes or injection of IL-2 can noncytopathically inhibit HBV gene expression by a posttranscriptional IFN-gamma- and/or tumor necrosis factor alpha-dependent mechanism. Here, we report that HBV gene expression can also be controlled at the posttranscriptional level during persistent lymphocytic choriomeningitis virus infection. In contrast, it is controlled at the transcriptional level during acute murine cytomegalovirus infection or after repetitive polyinosinic-polycytidylic acid injection. Finally, we show that transcriptional inhibition of HBV is associated with changes in liver-specific gene expression. These results elucidate pathways that regulate the viral life cycle and suggest additional approaches for the treatment of chronic HBV infection.

Animals↗
PubMed2003

CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection.

Although the CD4(+)- and CD8(+)-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8(+) cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8(+)-T-cell response.

Acute Disease↗
PubMed2003

Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma inhibit hepatitis B virus (HBV) replication noncytopathically in the livers of HBV transgenic mice and in hepatocyte cell lines derived from these mice. The present study was designed to identify transcriptionally controlled hepatocellular genes that are tightly associated with the inhibition of HBV replication and that might, therefore, mediate the antiviral effect of these cytokines. Twenty-nine genes were identified, many of which have known or potential antiviral activity. Notably, multiple components of the immunoproteasome and ubiquitin-like proteins were strongly induced by both IFN-alpha/beta and IFN-gamma, as were a number of GTP-binding proteins, including GTPases with known antiviral activity, chemokines, signaling molecules, and miscellaneous genes associated with antigen processing, DNA-binding, or cochaperone activity and several expressed sequence tags. The results suggest that one or more members of this relatively small subset of genes may mediate the antiviral effect of IFN-alpha/beta and IFN-gamma against HBV. We have already exploited this information by demonstrating that the antiviral activity of IFN-alpha/beta and IFN-gamma is proteasome dependent.

Animals↗
PubMed2002

Genomic analysis of the host response to hepatitis C virus infection.

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-alpha response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-gamma-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

Animals↗
PubMed2002

Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease.

To define the early events that determine the outcome of acute hepatitis C virus (HCV) infection, we compared the course of viremia with the peripheral and intrahepatic T cell response and intrahepatic cytokine profile in six acutely infected chimpanzees. Three different outcomes were observed after peak viral titers were reached: sustained viral clearance, transient viral clearance followed by chronic infection, and chronic infection that persisted at initial peak titers. The results indicate that HCV spread outpaces the T cell response and that HCV rapidly induces but is not controlled by IFN-alphabeta; that viral clearance follows the entry and accumulation of HCV-specific IFN-gamma-producing T cells in the liver; and that it may not require the destruction of infected cells.

Acute Disease↗