Host cell factor requirement for hepatitis C virus enzyme maturation

Abstract

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2͞3 protease of hepatitis C virus. The cleavage activity of NS2͞3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2͞3. Furthermore, these HSP90 inhibitors prevent NS2͞3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2͞3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone͞folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.A n estimated 170 million people are infected with hepatitis C virus (HCV) worldwide. The infection is usually persistent, and after an asymptomatic period often lasting years, many patients develop chronic liver disease, including cirrhosis and hepatocellular carcinoma (1, 2). The preferred existing treatment with IFN-␣ is successful in only a small fraction of cases, so an urgent need exists for the development of therapeutically effective inhibitors of HCV replication. With the precedent of numerous successful antiviral therapies based on viral enzyme inhibition, the enzymes encoded by HCV present particularly attractive targets. The recombinant HCV enzymes, NS5B RNA polymerase, NS3 protease, and NS3 helicase have been purified, their enzymatic properties are being defined in vitro, and their potential for inhibition is being explored by many laboratories (3-7).In contrast, the HCV protease activity responsible for cleavage between NS2 and the NS3 proteins, known as NS2͞3 protease, has been reported only in cell lysate translation systems and transfection experiments, despite having been described initially several years ago (8-10). Thus, NS2͞3 cleavage as a target for inhibition has been particularly refractory to detailed analysis, so that the definition of the critical components of this activity is needed. Based on mutational analyses, the protein region essential for NS2͞3 cleavage activity has been approximately mapped to amino acids 898-1207 of the HCV ORF. This region of the HCV polyprotein includes the N-terminal portion of NS3 that is known to be sufficient for NS3 protease activity, residues 1027-1207. Because of the overlap of sequence essential for the two distinct protease activities, it is worthwhile to emphasize that the NS3 protease activity can be totally eliminated through mutagenesis of the NS3 active site Ser-1165 without affecting NS2͞3 cleavage activity (10).Additional features of NS2͞3 cleavage activity known at this time include identification of Cys-993 and His-952 as essential residues (8-13). The only known cleavage site sequence, ArgLeu-Leu2Ala-Pro-Ile, is conserved across HCV strains, and the catalytic mechanism of NS2͞3 cleavage is unknown, but the enzyme is sp...

The Cellular Chaperone Heat Shock Protein 90 Facilitates Flock House Virus RNA Replication in Drosophila Cells

“…We observed no significant suppression of FHV RdRp activity in vitro with up to 50 M geldanamycin (Fig. 3A, lane 4), a concentration well above levels previously shown to suppress Hsp90 activity in vitro (20,54,57), 10-fold higher than that used in cellular assays ( Fig. 1 and 2), and approximately 3,500-fold above the IC 50 for geldanamycin and FHV RNA replication in infected cells (Fig.…”

“…We used cerulenin, a fatty acid synthetase inhibitor previously shown to suppress positivestrand RNA virus replication (16,36), as a positive control. We chose initial inhibitor concentrations of 50 M cerulenin, 5 M geldanamycin, and 5 M radicicol on the basis of published studies of cultured cells that maximized target inhibition and minimized cellular toxicity (16,20,23,36,54). None of the inhibitors significantly reduced S2 cell viability during a 24-h incubation, although both geldanamycin and cerulenin reduced cell proliferation such that by 24 h cell recovery was 50 to 60% of control levels (Fig.…”

Role of heat-shock protein 90 in hepatitis E virus capsid trafficking

“…Another study demonstrated that HSP90 is important for vaccinia virus growth in cultured cells through interaction with viral core protein 4a (Hung et al, 2002). HSP90 is required to maintain the function of hepatitis B virus reverse transcriptase (Hu et al, 2004) and the enzyme maturation of hepatitis C virus (Waxman et al, 2001). Recently, HSP90 was also found to be a component of the dengue virus receptor complex in human cells and participated in the entry of the virus (Reyes-Del Valle et al, 2005).…”

Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication

“…Although the mechanism of NS2/3 degradation has yet to be elucidated, it is likely that cellular proteins are involved. Cellular factors have also been proposed to be required for efficient NS2/3 protease activity (20,22), and the availability of these factors may modulate the degree of NS2/3 cleavage over the course of infection. In addition, the NS2/3 cleavage products themselves could potentially be involved in the regulation of these processes through their actions on host cellular proteins.…”

“…Mutagenesis studies have identified His 952 and Cys 993 within NS2 as being essential for NS2/3 protease activity (15,16), and in addition, mutations thought to perturb the local conformation of the cleavage site also inactivate the enzyme (21). Furthermore, molecular chaperones have been proposed to be required for efficient cleavage at the NS2/3 site (22).…”