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The Chisari Lab

Laboratory of Experimental Virology / Scientific Overview

IMPACT OF INTRAHEPATIC ANTIGEN RECOGNITION ON PRIMING OF THE CD8+ T-CELL RESPONSE TO THE HEPATITIS B VIRUS.
Masanori Isogawa and Francis V. Chisari

The CD8 T cell response contributes to the pathogenesis of liver disease and viral clearance during hepatitis B virus (HBV) infection, and failure to induce and/or sustain that response results in viral persistence, chronic hepatitis, and hepatocellular carcinoma. In these studies we are delineating the mechanisms that regulate the CD8 T cell response to HBV using HBV core- and envelope-specific T cell receptor (TCR) transgenic mice. When adoptively transferred into HBV transgenic mice, naïve CD8 T cells proliferate vigorously intrahepatically before they appear in lymphoid tissues but do not develop antiviral effector functions, suggesting that intrahepatic T cell priming induces functionally defective T cell responses. In contrast, when transferred into HBV transgenic mice whose professional antigen presenting cells are activated by agonistic anti-CD40 antibodies, naïve T cells differentiate into fully functional cytotoxic T cells, inhibit HBV antigen expression, and cause liver disease. Current experiments focus on delineating the precise mechanism that underlies the anti-CD40 mediated induction of functional CD8 T cell responses. These studies provide insight into the generation of functional CD8 T cell responses to intrahepatically expressed viral antigens, and shed light on previously unknown early immunological events that occur in response to HBV infection.

PLATELET INTERACTIONS WITH CYTOTOXIC T LYMPHOCYTES IN THE HEPATIC MICROCIRCULATION.
M. Iannacone, G. Sitia, M. Isogawa, F.V. Chisari, Z.M. Ruggeri* and L.G.Guidotti
*Department of Molecular and Experimental Medicine, TSRI

Using transgenic mice that replicate HBV at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), along with normal inbred mice infected with hepatotropic, replication-deficient adenoviruses, we have shown that, upon activation, platelets contribute to liver disease and viral clearance by promoting the recruitment of virus-specific CTLs into the liver. As indicated by ex vivo experiments under flow, this effect is most likely dependent on specific interactions between platelets and CTLs. Thus, it is conceivable that platelet/CTL interactions occurring within the hepatic microcirculation may direct CTLs to extravasate, reach parenchymal cells (i.e. hepatocytes) and perform pathogenetic and/or antiviral effector functions. The intrahepatic visualization and mechanistic understanding of platelet/CTL interactions represent major goals of this research program. Through the use of virus-specific CTLs, HBV transgenic mice, and mice genetically deficient for various platelet molecules we are defining the molecular basis of platelet/CTL interactions in vivo by intravital image analysis of the liver utilizing epifluorescence microscopy for the simultaneous detection of platelets and CTLs labeled with different fluorochromes. Preliminary results indicate that platelets and CTLs interact within sinusoids, specialized capilllaries of the liver characterized by the lack of a basal membrane and very low-flow conditions.

THE ROLE OF OF IFN- IN THE PATHOGENSIS OF VIRUS-INDUCED HEMORRHAGE.
M. Iannacone, I. Brunetta, G. Sitia, M. Isogawa, F. V. Chisari, Z. M. Ruggeri* and L. G. Guidotti.
*Department of Molecular and Experimental Medicine, TSRI

Mice infected with lymphocytic choriomeningitis virus (LCMV) exhibit an IFN?/?-dependent platelet dysfunction that, when associated with thrombocytopenia, results in severe hemorrhage, anemia, and death. The role of IFN-?/? in this process is demonstrated by the protection afforded by absence of the corresponding receptor and by induction of the same hemorrhagic animia by the infusion of the IFN-?–inducer poly(I:C) in uninfected, platelet-depleted mice. Thus, IFN-?/? is required and sufficient to cause the platelet dysfunction seen in the course of LCMV infection. The mechanism of this action is currently under investigation. Although preliminary results indicate that platelets do express the IFN-?/?? receptor, we have found that in vitro incubation with recombinant IFN-? has no impact on platelet aggregation, possibly owing to the absence of the transcription machinery (i.e. platelets are anucleated cells) necessary to decode signals emanating from the receptor. The functional inhibition in vivo, therefore, could be mediated by IFN-?/?-dependent regulation of endothelial cell-derived platelet inhibitors, e.g. nitric oxide and prostacyclin, and experiments to test this hypothesis are underway.

PLASMACYTOID DENDRITIC CELLS SENSE HEPATITIS C VIRUS-INFECTED CELLS AND PRODUCE INTERFERON
Ken Takahashi, Marlene Dreux, Shinichi Asabe, Stefan F. Wieland and Francis V. Chisari

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects >170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and the mechanism whereby it is induced are currently undefined. We recently discovered that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 (TLR7)-signaling. Importantly, the same events are triggered by HCV subgenomic replicon cells and by transfected HCV RNA, but not by free virus particles. These results suggest the existence of a novel cell-cell RNA transfer process whereby HCV infected cells activate pDCs to produce IFN. These results not only explain how HCV induces IFN when it infects the liver, they also reveal a heretofore unsuspected aspect of the innate host-response to viruses like HCV that can subvert the classical sensing machinery in the cells they infect and do not infect or directly activate pDCs. Current experiments focus on discovery of the HCV component and the mechanism responsible for pDC activation by HCV-positive hepatocytes.

HEPATITIS C VIRUS SUPPRESES THE EXPRESSION OF INTERFERON INDUCIBLE PROTEINS.
Urtzi Garaigorta and Francis V. Chisari

We recently discovered that HCV infection strongly triggers the phosphorylation of PKR and eIF2? proteins and a decrease in de novo cellular protein synthesis in infected cells while cellular HCV protein content increases, suggesting that cellular protein synthesis is more sensitive to the inhibitory effects of activated PKR than HCV IRES-driven protein synthesis. When HCV-infected and -uninfected cells are treated with IFN?, interferon-stimulated gene (ISG) mRNA expression is highly induced in both populations but ISG protein synthesis is significantly attenuated in HCV-infected cells. These results suggest that the ability of HCV to activate PKR may be advantageous for the virus during an interferon response by preferentially suppressing the translation of antiviral effector proteins (e.g. ISGs) and thus contribute to HCV persistence. Current experiments address the mechanisms whereby HCV triggers PKR phosphorylation and the consequences of PKR phosphorylation on proviral and antiviral signaling pathways in HCV-infected cells.

THE AUTOPHAGY MACHINERY IS REQUIRED TO INITIATE HEPATITIS C VIRUS REPLICATION.
Marlène Dreux, Pablo Gastaminza, Stefan F. Wieland and Francis V. Chisari

In addition to its cellular homeostasis function, autophagy is emerging as a central component of antimicrobial host defense against diverse infections. To counteract this mechanism, many pathogens have evolved to evade, subvert, or exploit autophagy. We recently discovered that autophagy proteins (i.e. Beclin-1, Atg4B, Atg5 and Atg12) are proviral factors required for translation of incoming hepatitis C virus (HCV) RNA and, thereby, for initiation of HCV replication, but they are not required once infection is established. These results illustrate a previously unappreciated role for autophagy in the establishment of a viral infection and they suggest that different host factors regulate the translation of incoming HCV RNA and its translation once replication is established. Current experiments address the mechanisms whereby autophagy proteins and autophagosomes regulate the translation of incoming HCV RNA.

A NOVEL NS3/4A-INDEPENDENT MECHANISM ALLOWS HCV EVASION FROM THE INNATE HOST REPONSE
Marlène Dreux, Guofeng Cheng and Francis V. Chisari

Viral infections often trigger an immediate cellular response, a central event of which is the secretion of type I interferons (IFN? and IFN?), resulting in establishment of an antiviral state. To counteract this host innate immune response, many pathogenic viruses have evolved distinct counteracting mechanisms that inhibit the early signaling events leading to IFN production. For HCV, we and others have previously shown that the NS3/4A protease blocks the dsRNA-mediated signaling pathway by cleaving MAVS and blocking downstream IRF-3 phosphorylation and IFN? activation. Importantly, we have discovered a novel NS3/4A-independent mechanism whereby HCV evades the innate host response in infected cells. In recent studies, we showed that the HCV NS4B protein inhibits HCV RNA-induced IFN? promoter activation and that the RIG-I helicase/repressor domain function(s) is(are) targeted by this novel inhibitory mechanism. These results suggest that a second HCV protein, NS4B, targets the RIG-I signaling function to ensure HCV evasion from the early innate host response in infected cells. Current experiments focus on the mechanism whereby HCV inhibits RIG-I signaling and the consequences of those interactions on HCV infection in vivo.

STRUCTURE-ACTIVITY RELATIONSHIP (SAR) STUDIES ON SMALL MOLECULE INHIBITORS OF HCV INFECTION GENERATED BY CLICK CHEMISTRY
Pablo Gastaminza, Suresh Pitram, Larissa Krasnova, Valery Fokin, Barry K. Sharpless and Francis V. Chisari.

Screening of a click chemistry library for inhibitors of HCV infection infection identified a new class of small molecule inhibitors with antiviral capacity at low concentrations (EC50 ~1µM) in the absence of apparent toxicity but with a low therapeutic index (TI ~20). Exploiting the modular nature of click chemistry we performed SAR analysis to optimize the therapeutic index of these compounds and identified the minimum structural determinants for antiviral activity and determined that the compounds display a high degree of stereospecificity. Based on these findings, optimized versions of this class yielded an inhibitor with greatly increased potency (EC50~1nM) and therapeutic index (TI ~10,000). Analysis of the mode of action of this new class of inhibitors revealed that they target early steps of infection downstream of viral entry and upstream of viral replication, suggesting that they target a hitherto undefined step in HCV infection. Current experiments focussing on the mode of action and molecular target of these compounds will provide novel information on basic aspects of HCV infection that cannot be studied in surrogate models and that may lead to the development of novel antiviral drugs.

CRYOELECTRON MICROSCOPY STUDIES OF HIGHLY PURIFIED INFECTIOUS HCV PARTICLES.
Pablo Gastaminza, Bryan Boyd, Kelly Dryden, Malcolm Wood, Marc Yeager and Francis V. Chisari.

The development of a robust cell culture system for HCV infection created the opportunity to produce monoclonal infectious virus in amounts sufficient for structural analysis. Having adapted HCV to cell culture and having produced hyperpermissive cell clones, we have achieved the production of high titer virus stocks suitable for cryoEM analysis after extensive purification and concentration. Analysis of the virus preparations has identified a pleomorphic population of enveloped and nonenveloped particles with heterogenous diameter and morphology. These results, obtained with a monoclonal virus population, demonstrate that particle heterogeneity is intrinsic to HCV and that enveloped and non-enveloped particles co-exist in the supernatant of infected cells. Future experiments will address the origin, structural and functional properties of each of these populations to determine their relative contribution to the viral pathogenesis.