Kouacou Konan Assistant Professor of Biochemistry and Molecular Biology 308 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863-8254 Fax: (814) 863-7024 E-mail: kvk10@psu.edu M.A. in Microbiology & Molecular Biology from Indiana University Ph.D. in Microbiology & Molecular Biology from Indiana University Postdoctoral, Stanford University of California Konan Lab Web Site

Hepatitis C and related viruses: replication complex formation and genome replication

Hepatitis C virus (HCV) is a positive strand RNA virus of the family Flaviviridae, including Dengue virus, West Nile virus, and bovine viral diarrhea virus. HCV is a leading cause of liver cancer worldwide. The long-term goal of this laboratory is to understand how HCV interaction with host proteins results in pathogenesis and ultimately in liver cancer. Specifically, my lab is interested in understanding the role of nonstructural 4B protein, or NS4B, in HCV pathogenesis and biogenesis of liver cancer. Like many positive stranded RNA viruses, HCV replicates in the cytoplasm of infected cells, resulting in the formation of rearranged membranes called the membranous webs. The webs contain HCV nonstructural proteins (NS3, NS4A, NS4B, NS5A and NS5A) and viral RNA, suggesting that these novel membranes contain active HCV replication complex.

Role of NS4B protein in the recruitment of viral replication complex

One project in the lab seeks to understand how 1) all the viral proteins and 2) HCV RNA are assembled into the replication complex (Fig. 1). Expression of HCV NS4B protein alone induces the web structure but how all the replication complex is assembled into it still remains a mystery. For example, NS4A, NS5A and NS5B have membrane-anchoring domains, suggesting that they may not need NS4B to be recruited into the replication complex. On the other hand, NS3 protein is not a membrane-associated protein unless it is bound to NS4A and perhaps the other nonstructural proteins. It is therefore possible that the replication complex components are incorporated into the web structure in a random fashion. However, it is likely that NS4B and host factors play a key role in that process.

Structure-function analysis of HCV NS4B and associated host factors   

Another project focuses on uncovering all the possible functions associated with NS4B expression in the context of infectious virus. The molecular mechanism whereby NS4B promotes HCV replication complex formation still remains unknown nor do we know NS4B domains, NS4B residues as well as host partners crucial for forming this complex. For example, NS4B interacts with viral proteins, but we do not know how these interactions affect the function of NS4B and the other viral proteins. Similarly, we do not know much about NS4B host partners and how its interactions with these factors affect each other’s activity. We have shown that Rab5 and Rab7, both endosome proteins, colocalize with NS4B protein and play a role in HCV RNA synthesis. These findings are interesting given that HCV proteins are translated on the endoplasmic reticulum membranes. Studies are under way to understand the significance of Rab proteins in infectious HCV (Fig. 2) life cycle.

HCV Cytopathogenicity and Persistence

Examining whether NS4B function is conserved among flaviviruses

Another project in the lab relies on the use of bovine viral diarrhea virus (BVDV) as another model system to understand HCV replication. BVDV causes serious mucosal disease that is often fatal in infected cattle. Unlike HCV, BVDV is easier to propagate in cell culture; it can therefore provide a useful model for understanding HCV replication in infected cells. Very little is known about BVDV NS4B protein and whether this protein is key for the formation of BVDV replication complex. However, like HCV, BVDV NS4B protein is highly hydrophobic, with both the N-terminal and C-terminal domains predicted to be on the cytosolic side of the ER membrane. My lab is interested in defining the role of NS4B protein in BVDV replication and cypathogenicity, examining whether this function is conserved between HCV and BVDV and how this information can be used in the design of antivirals.

Representative Publications:

• Manna, D., J. Aligo, W.D. Heo, H. Koc, C. Xu, and K.V. Konan. Endocytic Rab proteins are required for hepatitis C virus replication complex formation. Virology (2009). In press.

• Weiskircher, E., J. Aligo, G. Ning, and K.V. Konan. Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes. Virol. J. (2009) Nov 3;6(1):185. [Epub ahead of print] PMID: 19887001.

• Aligo, J., S. Jia, D. Manna, and Konan, K.V. Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein. Virology 393 (2009) 68-83.

• Stone, M.,  Jia, S., Heo, W.D., Meyer, T., and Konan, K.V. Participation of Rab5, an early endosome protein, in hepatitis C virus RNA replication machinery. J. Virol. 81 (2007) 4551-4563.

• Turpin, E., Luke, K., Jones, J., Tumpey, T., Konan, K., and S. Schultz-Cherry. Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication. J. Virol. 79 (2005) 8802-8811.

• Konan, K.V., Giddings, Jr., T.H., Ikeda, M., Li, K., Lemon, S.M., and Kirkegaard, K. Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J. Virol. 77 (2003) 7843-7855.

• Konan, K.V. and Yanofsky, C. Rho-dependent termination in the tna operon of Escherichia coli: roles of boxA sequence and the rut site. J. Bacteriol. 182 (2000) 3981-3988.

• Konan, K.V. and Yanofsky, C. Ribosome release regulates basal level expression of the tna operon in Escherichia coli. J. Bacteriol. 181 (1999) 1530-1536.

• Konan, K.V. and Yanofsky, C. Regulation of the Escherichia coli tna operon: nascent leader      peptide control at the tnaC stop codon. J. Bacteriol. 179 (1997) 1774-1779.

• Konan, K.V. and Taylor, M.W. Importance of the two interferon-stimulated response element (ISRE) sequences in the regulation of the human indoleamine 2,3-dioxygenase gene. J. Biol. Chem. 271 (1996) 19140-19145.

• Konan, K.V. and Taylor, M.W. Treatment of ME180 cells with interferon-g causes apoptosis as a result of tryptophan starvation. J. Interf. Cytok. Res. 16 (1996) 751-756.

• Konan, K.V., Sahota, A., Graham, F.L., and Taylor, M.W. Transduction of the CHO aprt gene into mouse L cells using an adeno-5/APRT recombinant virus. Som. Cell Mol. Gen. 17 (1991) 359-368.

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