RESEARCH SUMMARY

Over 3% of the world population is chronically infected by the hepatitis C virus (HCV). Chronic infection is associated with liver disease that leads to fibrosis and cirrhosis and ultimately to the development of hepatocellular carcinoma (HCC). There is no vaccine for HCV and the current therapy, which is associated with severe adverse effects, only cures ~50% of the patients that complete the treatment. Consequently, HCV infection is one of the leading causes of liver transplantation worldwide. Although the number of new infections was drastically reduced since the implementation of blood screening tests and the improvement of prophylactic procedures, the burden on public health due to the long-term impact of the chronic infections is predicted to rise significantly.

Hepatitis C virus infection is also associated with alterations of the liver lipid metabolism. These alterations are manifested in more than half of the chronically infected patients by abnormal accumulation of neutral lipids in the liver parenchyma (steatosis) and low blood levels of triglyceride-rich lipoproteins (hypobeta-lipoproteinemia). These clinical observations lead to the hypothesis that chronic HCV infection interferes with cellular lipid and lipoprotein metabolism. This notion is reinforced by the fact that, in patients responding to interferon treatment, steatosis subsides as the viral titers decrease, suggesting that HCV replication and viral protein expression are a major determinant for these alterations.

The lack of appropriate cell culture infection systems has traditionally hampered the study of basic aspects of HCV infection. It was only recently that a cell culture model of HCV infection became available. We used this model to study basic aspects of HCV infection, including its relationship with lipid and lipoprotein metabolism. Many studies have now shown that HCV depends heavily on cellular lipid and lipoprotein metabolism to replicate and produce infectious particles. However, we only begin to understand the molecular mechanisms underlying these phenomena. Thus, understanding how HCV exploits the cellular lipid and lipoprotein biosynthetic machinery might contribute to understanding important aspects of the biology and pathogenesis of chronic HCV infection.

One of the first observations in cell culture that pointed at a tight relationship between HCV assembly and lipoprotein metabolism was that infectious HCV particles are assembled as intracellular precursors similar in size (~65-70 nm) but higher buoyant density (~1.15-1.20 g/ml) than the secreted particles (~1.03-1.16 g/ml). This finding suggested that the biochemical composition of HCV particles is altered during viral egress. Since it had been suggested that the low density of HCV particles in the serum of infected patients reflects their association with very low-density lipoprotein (VLDL) components, e.g. apolipoprotein B (apoB), apolipoprotein E (apoE) and triglycerides (TG) and having found that HCV undergoes a biophysical transformation similar to that of VLDL, we postulated that HCV might be assembled as a triglyceride-rich liver lipoprotein. In 2008, we showed using specific inhibitors and RNAi, that HCV assembly depends on elements of the VLDL biosynthetic machinery. Short-hairpin RNAs (shRNAs) directed against the major protein component of VLDL (apoB) suppress both intracellular and extracellular infectivity titers during single cycle infection experiments in proportion to their ability to suppress apoB secretion as shown in (Figure 1b) and 6. In addition, pharmacological inhibition of microsomal transfer protein (MTP), which controls VLDL assembly, provoked a reduction of HCV secretion that was proportional to that of VLDL. Overall these results suggest that HCV is assembled and secreted in a manner that resembles that of VLDL, a notion that is supported by data published by several other groups. Moreover, it has been recently showed that, similarly to what has been shown for virions isolated from patients, lipid and apoprotein composition of cell culture grown HCV is similar to that of VLDL and LDL.

ApoE has also been shown to be essential for HCV assembly 8 and is also a structural component of purified HCV particles by dual staining of individual HCV particles with antibodies against the viral envelope (E2) and apoE observed under the electron microscope. These studies reinforce the notion that HCV particles are similar to liver-derived lipoproteins and that associated cellular components may contribute to their survival and mediate their entry into target cells.

Our laboratory is interested in deciphering the molecular events underlying the functional dependence of HCV infection on lipid and lipoprotein metabolism and how to exploit these pathways to inhibit HCV infection. We use a cell culture infection system based on the infectious molecular clone JFH-1 and the Huh-7 hepatoma cell line and derived subclones to evaluate the impact that genetic and pharmacological manipulation of cellular lipid metabolism has on HCV infection and pathogenesis.

We are also exploiting this HCV cell culture system to find new antiviral molecules against this important pathogen. To facilitate this process we have developed a colorimetric, unbiased, miniaturized cell-based screening system that enables interrogation of chemical libraries for compounds that inhibit HCV infection at any known or unknown step of the infection, be the target viral or cellular 10. This system enabled identifying both clinical drugs for non-HCV applications and experimental compounds with potent anti-HCV activity. Studying the mode of action of these compounds, as well as their molecular targets will provide valuable information on basic aspects of the biology of HCV infection.