The focus of my laboratory is to understand the mechanism of positive strand RNA virus genome replication and the transition from replication to virus particles formation. Our model system is hepatitis C virus (HCV), which remains a major public health concern despite the advent of direct-acting antivirals targeting HCV proteins. Approximately 4 million Americans are chronically infected with HCV, leading to chronic liver disease and more than 10,000 annual deaths.
HCV has evolved to co-opt host cytoplasmic membranes to build a unique replication platform called the membranous web (MW). The MW shelters and concentrates the HCV replication complex in distinct membrane domains for efficient genome replication. We have discovered that the host glycosphingolipids transport and biosynthetic proteins (e.g. four-phosphate adaptor protein 2 or FAPP2 and glucosylceramide synthase or GCS) are crucial for HCV RNA synthesis. For example, we have shown that FAPP2 depletion attenuates HCV infectivity and impedes HCV RNA synthesis. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescues replication, implying a requirement for glycosphingolipids in HCV RNA synthesis. Additionally, we have evidence that FAPP2 oligomerizes but the significance of FAPP2 quaternary structure in HCV RNA synthesis is unknown. Questions of interest include:
1. How does FAPP2 quaternary structure contribute to its function in HCV replication?
2. What are the role of FAPP2 domains in HCV replication?
3. How is FAPP2 recruited to the HCV replication complex?
4. Roles of sphingolipid biosynthetic proteins in HCV replication and virus-induced liver disease?
5. Can we utilize in vitro reconstitution to dissect the roles of FAPP2, and sphingolipids, in HCV replication?
In a complementary line of investigation, my laboratory is studying the mechanism whereby HCV nonstructural 4B (NS4B) protein organizes the virus replication complex and facilitates virus assembly. We have shown that HCV NS4B transmembrane domains (MSDs) are engaged in intra- and intermolecular interactions that are crucial for HCV replication. We have identified two protein-protein interaction motifs in these MSDs. These results raise the possibility that many NS4B protein-protein interactions could occur in the membrane bilayer and might be ideal targets for pharmacological intervention. Additionally, we have shown that NS4B facility HCV genome encapsidation by regulating the stability and/or phosphorylation state of HCV NS5A protein. Uncovering the host kinases or proteases as well as viral factors involved in HCV assembly will likely lead to better insight into the replication of HCV and related viruses.