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 segregated in two somewhat independent domains. The essential machinery of a serine 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 expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease 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 activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.
Morgenstern
K A
KA
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4242, USA.
Landro
J A
JA
Hsiao
K
K
Lin
C
C
Gu
Y
Y
Su
M S
MS
Thomson
J A
JA
D017766:Acid Anhydride Hydrolases / Q000187:drug effects*; D000818:Animals; D019556:COS Cells; D004265:DNA Helicases / Q000187:drug effects*; D010450:Endopeptidases / Q000187:drug effects*; D043583:Nucleoside-Triphosphatase; D011072:Poly U / Q000494:pharmacology; D011119:Polynucleotides / Q000494:pharmacology*; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000187:drug effects*; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017766:Acid Anhydride Hydrolases; D004265:DNA Helicases; D010450:Endopeptidases; D043583:Nucleoside-Triphosphatase; D011072:Poly U; D011119:Polynucleotides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus