O.196 Interferon response against HCV

Oral presentations: PL05: Late Breakers/Controversies and Perspectives anchor. Such a conformational change would liberate the RNAbinding groove and move the NS5B catalytic domain away from the membrane, while the connecting segment might be involved in binding of viral or cellular factors required for RC formation. NS5A is a phosphoprotein thought to be an essential component of the HCV replication complex. It is composed of a N-terminal amphipathic alpha-helix and 3 distinct domains. The N-terminal alpha-helix constitute an in-plane membrane anchor ensuring the membrane attachment of domain I involved in RNA binding. This in-plane alpha-helix exhibits a hydrophobic side embedded in the cytosolic leaflet of the phospholipid bilayer and a fully conserved polar side that may serve as a platform for the recruitment of proteins involved in RNA replication. HCV core protein is targeted to lipid droplets (LDs) by its C-terminal domain. The major structural elements of core attachment to LDs consist of two amphipathic alpha-helices separated by a hydrophobic loop (HL). Structural and mutational studies demonstrated that the combination of Helix I, HL and Helix II is essential for efficient LD association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from LD association, membrane interaction of this structural motif is necessary for folding and stability of core following maturation at the ER membrane by signal peptide peptidase. The membrane domains of these proteins are essential for HCV life cycle and thus constitute possible targets for antiviral interventions.

I0_•

Production of infectious HCV particles in three-dimensional liver cultures

T. Miyamura*, T. Suzuki. Department of Virology II, National

Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan In order to reproduce the full life cycle of HCV in cells, we have been trying to establish an efficient in vitro system to support the full replication of HCV. Here, we show that a dicistronic hepatitis C virus genome (genotype lb) supports the production and secretion of infectious HCV particles into the medium. Efficient production is only observed in two independent three-dimensional (3D) culture systems, (i) the radial-flow bioreactor and (ii) the thermoreversible gelation polymer (TGP). It was not observed in monolayer cultures of the same Huh-7 cells. Immunoreactive enveloped particles, which are 50-60 nm in diameter and are surrounded by membranelike structures, are observed in the culture medium as well as at the endoplasmic reticulum membranes and in dilated cytoplasmic cisternae in spheroids of Huh-7 cells. Infection of HCV particles is neutralized by anti-E2 antibody or patient sera which interfere with E2 binding to human cells. The 3D/TGP culture system is further useful to screen and evaluate antiviral drugs. The replicon-based 3D culture system allows the production of infectious HCV particles and provides a valuable tool in studies of HCV morphogenesis in a natural host cell environment. I-0_~

Interferon response against HCV

C. Seeger 1, D. Stiffler 1, R. Coudry 1, C. Spittle 1, C. Liu 2. 1Fox

Chase Cancer Center, Philadelphia; 2University of Florida, Gainesville, USA Investigations on the interferon (IFN) response against HCV in tissue culture cells demonstrated that IFN-? is a potent inhibitor of viral replication that can cure replicon-expressing cells. A comparison of HCV- and West Nile virus (WNV) replicating cells revealed that WNV evades the antiviral activity of IFN-,Y, by inhibiting the JAK-Stat signal transduction pathway. The discrepancy between the two viruses raises the question about how HCV can persist in the absence of a mechanism that inhibits the IFN-? response. DNA microarray analyses with liver RNA from infected chimpanzees demonstrated that HCV could induce an IFN response (Bigger et al., J. Virol. 75:7059, 2001; S u e t al., PNAS 99:15669, 2002). Based on these and other observations, we proposed that HCV and ISG RNA levels in infected hepatocytes oscillate in phases that are offset with respect to each other (Seeger, Trends Microbiol. 13:528, 2005). This hypothesis is consistent with the low average HCV RNA levels that are generally

$59 observed in HCV infected livers. Moreover, it predicts that ISGs are induced in hepatocytes. To investigate this possibility we have begun an analysis of HCV RNA and ISG mRNA profiles in liver cells from HCV infected patients. Consistent with previous results, we found that HCV infections induced ISG expression in the liver. Experiments that were performed with laser capture microdissection technology indicated that ISGs were expressed in hepatocytes. Our results are consistent with a model predicting that HCV replication directly, or indirectly induces an IFN response that reduces intracellular viral replication.

P S 3 2 - Late B r e a k e r s

(Tuesday, July 4, 2006, 14:00-15:30)

P L 0 5 - Late B r e a k e r s / C o n t r o v e r s i e s Perspectives

and

(Tuesday, July 4, 2006, 16:00-18:00) [-~--~

Shall we treat acute hepatitis B virus infection?

H.L. Tillmann. University Leipzig, Philipp-Rosenthal Str. 27, 04105 Leipzig, Germany Introduction: HBV infection is the leading viral cause of hepatitis world wide. While chronicity is a concern in HCV infection, this is of minor relevance in adults with acute hepatitis B, being as low as less than 1% in a Greece study. Thus, could there be any reason for treating acute Hepatitis B? Treatment in case of fulminant hepatitis B: A couple of investigators have reported very good results for using lamivudine in patients with fulminant hepatitis B, which is presumed to develop in 0.5% to 1% of cases of acute symptomatic hepatitis B. In our own initial attempt, we treated patients with severe acute or fulminant hepatitis B with lamivudine in order to prevent hepatitis B virus re-infection after potential liver transplantation. However, while 16 of 20 "historic" patients required a liver transplantation, only 3 of 17 patients with severe acute to fulminant hepatitis B virus treated with 100 or 150 mg lamivudine daily required a liver transplant. The remaining 14 recovered completely. Further confirmation was achieved by acquiring information on another 20 patients from different site, of whom also only 5 required a transplant, while two of three patients treated without lamivudine in the same time frame required a transplant. Similar good results have been reported from different sites such as UK, Israel, and Japan. In contrast patients with fulminant hepatitis B were reported to die in case of no initiation of lamivudine therapy. Conclusion: Based in these promising results of fast recovery with the potential to prevent liver failure and liver transplantation when lamivudine is administered early enough, one would be reluctant to perform a placebo controlled trial in patients with fulminant hepatitis. Such a trial is well feasible at least ethically in patients with less severe acute hepatitis B. In addition, in the absence of fulminant hepatitis acute HBV still leads to morbidity, and influenza likewise is not treated for its risk of chronicity. The present data on treatment of acute to fulminant hepatitis B will be reviewed. ~

Treatment of chronic HBV - optimization of the mean results by individualized viral kinetics analysis

A.U. Neumann *. Faculty of Life Sciences, Bar-I/an University,

Ramat-Gan 52900, Israel The results of viral kinetics (VK) during treatment of chronic hepatitis B infection are traditionally analyzed by the mean viral decline over time, together with the fraction of undetectable HBV-DNA or HBeAg loss at specific times. However, this is a very misleading approach since during treatment with both classes of therapy, either polymerase-inhibitors or IFN-alfa, as well as in non-treated patients, there are distinctively different patterns of viral kinetics in different