A second hepatitis C virus-encoded proteinase.

Abstract

Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.Hepatitis C virus (HCV), a recently identified agent of parenterally transmitted non-A non-B hepatitis, causes the vast majority of transfusion-associated cases of hepatitis and a significant proportion of community-acquired hepatitis (for review, see refs. 1 and 2). HCV infection results in varied clinical outcomes, and chronic infections are common and have been associated with an increased incidence of hepatocellular carcinoma. Although a interferon has been partially successful in inhibiting HCV replication, reliable therapeutic agents for controlling or eradicating HCV infection are currently lacking.

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells

“…The expression of vv/C-E1-E2-p71 809 gave rise to two proteins that reacted with the anti-E1 serum (Fig. lb, lane 2): a 31 kDa E1 product as described before (Grakoui et al, 1993 c) and a 73 kDa protein. The latter co-migrated with the unprocessed E2-p7 polypeptide ( Fig.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…[40][41][42] NS3 is composed of an N-terminal serine-type protease domain and a C-terminally located domain endowed with RNA helicase and NTPase activities. 43,44 The NS3 protease domain forms a very stable heterodimer with NS4A, which acts as a protease cofactor and tethers NS3 to intracellular membranes ( Fig. 1).…”

Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells

“…NS2 is a membrane-associated cysteine protease (Grakoui et al, 1993;Hijikata et al, 1993b;Lorenz et al, 2006). The N terminus of NS2 consists of one or more transmembrane domains, whilst the C-terminal domain of NS2, together with the N-terminal one-third of NS3, forms the NS2-3 protease, an enzyme that catalyses a single cleavage at the NS2/NS3 boundary.…”

“…The approximately 9.6 kb HCV genome encodes one large open reading frame (ORF) that is flanked at the 59 and 39 ends by untranslated regions (UTRs) (Choo et al, 1991). The HCV polyprotein is processed into at least 10 proteins by viral proteases and cellular signalases (Grakoui et al, 1993;Hijikata et al, 1993a;McLauchlan et al, 2002). The structural proteins core, E1 and E2 are located in the N terminus of the polyprotein, followed by p7 and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B (Bartenschlager & Sparacio, 2007).…”

“…Another possibility is that the E2 glycoprotein with the N534H mutation gains higher affinity for other HCV receptors. Further investigation will be required to elucidate the mechanism of this adaptive mutation.Our results showed that a combination of the mutations in E2, together with four additional mutations in NS2, NS5A and NS5B, resulted in higher infectivity of HCV, suggesting that the additional four mutations possess an advantage at different steps.NS2 is a membrane-associated cysteine protease (Grakoui et al, 1993;Hijikata et al, 1993b;Lorenz et al, 2006). The N terminus of NS2 consists of one or more transmembrane domains, whilst the C-terminal domain of NS2, together with the N-terminal one-third of NS3, forms the NS2-3 protease, an enzyme that catalyses a single cleavage at the NS2/NS3 boundary.…”