Scientific Report 2007

Molecular and Experimental Medicine

Division of Experimental Pathology

Molecular Biology of Hepatitis B and C Viruses and the Immune Response to Their Antigens

Hepatitis B and hepatitis C viruses are noncytopathic DNA and RNA viruses that cause acute and chronic hepatitis and hepatocellular carcinoma. More than 500 million people worldwide are chronically infected, and more than 2 million people die of these infections every year. The focus of our research is to unravel the life cycle of these viruses, discover the roles played by the innate and adaptive immune responses in the control of the infections, and elucidate the mechanisms responsible for viral clearance and disease pathogenesis. Our goal is to devise novel strategies to prevent and cure these infections.

Impact of Intrahepatic Antigen Recognition on Priming of the CD8⁺ T-Cell Response

M. Isogawa, F.V. Chisari

By adoptively transferring memory CD8⁺ T cells specific for hepatitis B virus (HBV) into HBV transgenic mice, we showed that the effector functions of memory T cells are induced in an oscillatory manner as a consequence of intrahepatic recognition of antigen. To define the immunologic events that occur during priming of HBV-specific T cells, we generated T-cell receptor transgenic mice that have CD8+ T cells specific for the HBV core and envelope proteins.

Naive T cells from the T-cell receptor transgenic mice were rapidly activated in the liver when transferred into HBV transgenic mice. The intrahepatically activated T cells vigorously proliferated in situ but did not develop antiviral effector functions such as secretion of IFN-γ and cytolytic ability. The results suggest that intrahepatic T-cell priming triggers the expansion of functionally defective T cells. If similar priming events occur during HBV infections in humans, the events could account for the characteristically weak CD8⁺ T-cell responses in patients with chronic HBV infections.

Immunologic Priming by the Inoculum and Outcome of Hepatitis B Virus Infection

S. Asabe, S.F. Wieland, R.H. Purcell,* F.V. Chisari

* National Institutes of Health, Bethesda, Maryland

We previously showed that low-dose viral inocula lead to persistent hepatitis B virus infection in chimpanzees. To determine the mechanism responsible for persistent infection, we examined the peripheral CD4⁺ T-cell response at multiple times after infection. Animals that received high-dose inocula and resolved the infection produced early T-cell responses before the appearance of viral antigens in the liver or serum, suggesting that the T cells were primed by noninfectious viral antigens in the inoculum. In contrast, animals that received low-dose inocula and became persistently infected did not have early T-cell responses, suggesting that the T cells had not been primed by the inocula. Analysis of the CD8⁺ T-cell response in these animals indicated that the lack of early priming results in weak CD8⁺ T-cell responses. These results suggest that immunologic priming by input antigen during low-dose viral infection determines the outcome of hepatitis B virus infection.

Impact of Chronic Hepatitis C Virus Infection on the Course of Hepatitis B Virus Superinfection

S.F. Wieland, R.H. Purcell,* F.V. Chisari

* National Institutes of Health, Bethesda, Maryland

Hepatitis C virus (HCV) infection induces an innate immune response that is reflected by the induction of many interferon-stimulated genes (ISGs) in the liver, yet the virus persists, presumably by defeating ISG-mediated antiviral functions in infected cells. In contrast, ISGs are not induced in the liver during initiation of hepatitis B virus (HBV) infection. To determine if the HCV-induced ISGs induce an antiviral state in the liver, we compared the kinetics and magnitude of HBV infection in HCV-naive and chronically HCV-infected chimpanzees.

As expected, the HCV-naive animals had typical resolving HBV infections. In contrast, HBV infection was strongly attenuated (4 logs) and delayed (6–15 weeks) in the chronically HCV-infected animals. These results suggest that the HBV replication space is severely limited to the small fraction of HCV-infected (i.e., ISG-resistant) hepatocytes in chronically infected liver.

Hepatitis C Virus Infection and Very Low-Density Lipoprotein

P. Gastaminza, F.V. Chisari

Intracellular infectious particles of hepatitis C virus (HCV) and precursors of very low-density lipoprotein have higher buoyant density than their secreted counterparts outside the cell. These biophysical differences probably reflect different biochemical compositions and suggest that both kinds of intracellular particles acquire lipids while leaving the cell. Synthesis of very low-density lipoprotein involves the acquisition of triglycerides and cholesteryl esters by intracellular apolipoprotein B in a process catalyzed by microsomal transfer protein.

We found that an inhibitor of microsomal transfer protein and apolipoprotein B–specific short hairpin RNAs prevented the assembly and secretion of low-density HCV particles in infected cells. These findings suggest that assembly and secretion of HCV particles are tightly regulated by the very low-density lipoprotein metabolic machinery and that only mature, low-density HCV particles are secreted. Most newly assembled high-density infectious particles are degraded, suggesting that acquisition of apolipoprotein B and cellular lipids by HCV imparts a selective advantage as the virus adapts to its natural host.

Antiviral Activity of an Amphipathic α-Helical Peptide Derived From Nonstructural Protein 5A of Hepatitis C Virus

G. Cheng, A. Montero,* P. Gastaminza, C. Whitten-Bauer, S.F. Wieland, M. Isogawa, B. Fredericksen,** S. Selvarajah,*** P. Gallay,*** M.R. Ghadiri,* F.V. Chisari

* Department of Chemistry, Scripps Research
** University of Maryland, College Park, Maryland
*** Department of Immunology, Scripps Research

We recently identified a virucidal peptide derived from the membrane anchor domain of nonstructural protein 5A of hepatitis virus C (HCV) that efficiently inhibits both the initiation and the persistence of HCV infection in vitro. Additional studies indicated that the peptide blocks HCV infection by destabilizing HCV virions both extracellularly and intracellularly. The D-amino acid form of the peptide is fully active, and the D and L forms of the peptide have amphipathic α-helical structure and permeabilize artificial liposomes.

Mutational analysis indicated that the antiviral activity of the peptide depends on its membranolytic activity and α-helical structure, that amphipathicity is necessary but not sufficient for antiviral activity, and that antiviral activity depends on the amino acid composition but not the primary sequence of the peptide. Importantly, the antiviral activity of the peptide extends to other members of the Flaviviridae (West Nile virus and Dengue virus) and HIV, but not to numerous other RNA and DNA viruses. Collectively, these data indicate that the peptide may represent a novel therapeutic strategy for HCV, HIV, and other flavivirus infections.

Induction of a Common Antiviral Signaling Pathway in Human Cells by Double-Stranded DNA and Double-Stranded RNA

G. Cheng, J. Zhang, J. Chung, F.V. Chisari

Recent studies in murine systems showed that cytosolic double-stranded DNA triggers a potent type I interferon response that requires downstream components of the double-stranded RNA signaling pathway. The mechanism of cytosolic double-stranded DNA recognition is currently unknown. We found that cytosolic double-stranded DNA is a potent inducer of IFN-β promoter activation in human hepatoma Huh-7 cells and that this activation requires both the intracellular double-stranded RNA sensor retinoic acid–induced gene I and its adaptor molecule mitochondrial antiviral signaling protein, in addition to the downstream mediators TBK-1, IKK-ε, and IRF-3. These findings indicate that the innate immune signaling pathways induced by double-stranded DNA and double-stranded RNA share more components in human cells than originally thought, although double-stranded DNA appears to trigger that pathway upstream of the innate double-stranded RNA–interacting sensor retinoic acid–induced gene I.

Publications

Cheng, G., Zhong, J., Chung, J., Chisari, F.V. Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells. Proc. Natl. Acad. Sci. U. S. A. 104:9035, 2007.

Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Gerin, J.L., Chisari, F.V., Yeager, M. Native hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol. Cell 22:843, 2006.

Gastaminza, P., Kapadia, S.B., Chisari, F.V. Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J. Virol. 80:11074, 2006.

Kapadia, S.B., Barth, H., Baumert, T., McKeating, J.A., Chisari, F.V. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J. Virol. 81:374, 2007.

Maier, H., Isogawa, M., Freeman, G.J., Chisari, F.V. PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8⁺ T lymphocytes in the liver. J. Immunol. 178:2714, 2007.

Robek, M.D., Garcia, M.L., Boyd, B.S., Chisari, F.V. Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus. J. Virol. 81:483, 2007.

Sainz, B., Jr., Chisari, F.V. Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J. Virol. 80:10253, 2006.

Sakai, A., Takikawa, S., Thimme, R., Meunier J.-C, Spangenberg, H.C., Govindarajan, S., Farci, P., Emerson, S.U., Chisari, F.V., Purcell, R.H., Bukh, J. In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells. J. Virol. 81:7208, 2007.

Sidney, J., Asabe, S., Peters, B., Purton, K.A., Chung, J., Pencille, T.J., Purcell, R., Walker, C.M., Chisari, F.V., Sette, A. Detailed characterization of the peptide binding specificity of five common Patr class I MHC molecules. Immunogenetics 58:559, 2006.

Uprichard, S.L., Chung, J., Chisari, F.V., Wakita, T. Replication of a hepatitis C virus replicon clone in mouse cells. Virol. J. 3:89, 2006.

Zhong, J., Gastaminza, P., Chung, J., Stamataki, J., Isogawa, M., Cheng, G., McKeating, J.A., Chisari, F.V. Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J. Virol. 80:11082, 2006.