POLYNUCLEOTIDE MODULATION OF THE PROTEASE, NUCLEOSIDE TRIPHOSPHATASE,AND HELICASE ACTIVITIES OF A HEPATITIS-C VIRUS NS3-NS4A COMPLEX ISOLATED FROM TRANSFECTED COS CELLS

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregate d in two somewhat independent domains. The essential machinery of a se rine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expr essed immediately downstream of NS3 in the viral polyprotein, and a ce ntral stretch of hydrophobic residues in NS4A form an integral structu ral component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic pr ocessing in vivo. This may reflect economical packaging of essential v iral replicative components, but it could also mean that there is func tional interdependence between the two domains. In this study, a full- length NS3-NS4A complex was isolated after expression and autoprocessi ng in transiently transfected COS cells. The protein was used to exami ne the effects of polynucleotides on the NTPase, helicase, and proteas e activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A act ivities were modulated by polynucleotides, with poly(U) having the mos t remarkable effect. These findings suggest that the domains within NS 3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this comple x HCV replicase component.