William B. Lott, PhD New Mexico State University wlott@nmsu.edu Phone:(505) 646-3218 Personal Website

Title: The Role of the N-Terminus in HCV Capsid Formation

Project Description:

The Hepatitis C virus (HCV) is an important and deadly human pathogen chronically infecting over 170 million people worldwide. The lack of a cell culture system or small animal model makes HCV a difficult virus to study, and therefore a difficult viral infection to combat. HCV's well-established inefficient nucleocapsid assembly might contribute to the low numbers of infectious virions observed in HCV carriers. Sequences in the 5' end of the HCV ORF that code for the N terminus of the nucleocapsid protein (core) are believed to be required at the RNA level for translation initiation or regulation, but it is not known if the peptide sequence expressed from this coding region is a functional part of the core protein or a nonsense sequence that is simply tolerated by the virus. A nonsense fusion product could potentially interfere with proper nucleocapsid assembly leading to inefficient assembly and unusual nucleocapsid morphology. The broad and long range goal of this research is to increase the efficiency of HCV nucleocapsid formation thereby increasing the likelihood of establishing a viable cell culture system for future research. As a first step the hypothesis that the RNA required for translational control encodes for a nonsense peptide sequence that is detrimental to nucleocapsid formation must be evaluated. Inserting the pesti-virus Npro autoprotease into the core coding region at variable positions will create N terminal deletions from the HCV core protein. These core truncations will be assayed by in vitro assembly of nucleocapsid-like particles using well-established methodologies. Electron microscopy will be used to characterize the efficiency of assembly and the morphology of the resulting particles. If the central hypothesis is supported by these data, a chimeric HCV virus functionally incorporating Npro at the position defined by this research can be constructed as part of a future funding proposal and evaluated for viability in cell culture and small animal systems.