Heat-shock Protein 90 Is Essential for Stabilization of the Hepatitis C Virus Nonstructural Protein NS3

Abstract

The hepatitis C virus (HCV) is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using 17-allylaminogeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (Hsp90). Hsp90 is a molecular chaperone with a key role in stabilizing the conformation of many oncogenic signaling proteins. We examined the inhibitory effects of 17-AAG on HCV replication in an HCV replicon cell culture system. In HCV replicon cells treated with 17-AAG, we found that HCV RNA replication was suppressed in a dose-dependent manner, and interestingly, the only HCV protein degraded in these cells was NS3 (nonstructural protein 3). Immunoprecipitation experiments showed that NS3 directly interacted with Hsp90, as did proteins expressed from ⌬NS3 protease expression vectors. These results suggest that the suppression of HCV RNA replication is due to the destabilization of NS3 in disruption of the Hsp90 chaperone complex by 17-AAG. Infection by the hepatitis C virus (HCV)2 is a major public health problem, with 170 million chronically infected people worldwide (1, 2). The current treatment by combined interferon-ribavirin therapy fails to cure the infection in 30 -50% of cases (3, 4), particularly those with HCV genotypes 1 and 2. Chronic infection with HCV results in liver cirrhosis and can lead to hepatocellular carcinoma (5, 6). Although an effective combined interferon-␣-ribavirin therapy is available for about 50% of the patients with HCV, better therapies are needed, and preventative vaccines have not yet been developed.HCV is a member of the Flaviviridae family and has a positive strand RNA genome (7, 8) that encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, E1 (envelope 1), E2, p7, NS2 (nonstructural protein 2), NS3, NS4A, NS4B, NS5A, and NS5B (9, 10). NS2 and the amino terminus of NS3 comprise the NS2-3 protease responsible for cleavage between NS2 and NS3 (9, 11), whereas NS3 is a multifunctional protein consisting of an amino-terminal protease domain required for processing NS3 to NS5B (12, 13). NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer (14 -17), and the hydrophobic protein NS4B induces the formation of a cytoplasmic vesicular structure, designated the membranous web, which is likely to contain the replication complex of HCV (18,19). NS5A is a phosphoprotein that appears to play an important role in viral replication (20 -23), and NS5B is the RNA-dependent RNA polymerase of HCV (24, 25). The 3Ј-untranslated region consists of a short variable sequence, a poly(U)-poly(UC) tract, and a highly conserved X region and is critical for HCV RNA replication and HCV infection (26 -29).Hsp90 (heat-shock protein 90) is a molecular chaperone that plays a key role in the conformational maturation of many cellular proteins. Hsp90 normally functions in association with other co-chaperone proteins, which together play an important role in folding newly ...

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

“…Among chaperone proteins, Hsp90 has been implicated in HCV replication. Hsp90 is thought to enhance the maturation of the NS2/NS3 (40,49) and to form a complex with FKBP8 and NS5A to participate in HCV replication (44). This study demonstrated that Hsp72 chaperone activity could also increase levels of the replicase complex, leading to enhanced viral RNA relication.…”

“…Some of these proteins, such as hVAP-33 (41), FKBP8 (44), Hsp90 (40,44), and cyclophilin A (39), may participate directly in the replicase complex. Of these proteins, Hsp90 is a chaperone protein and cyclophilin A is a co-chaperone protein.…”

Combination Therapy for Hepatitis C Virus with Heat-Shock Protein 90 Inhibitor 17-AAG and Proteasome Inhibitor MG132

“…Treatment of the #50-1 HCV replicon cells with the Hsp90 inhibitor 17-AAG [8,16,17] suppresses HCV RNA replication and NS3 is the only HCV protein degraded in these cells [15]. This finding led us to suggest a crucial role for Hsp90-NS3 complexes in the HCV life cycle.…”

“…Western blotting NNC#2 cells were seeded at 1.5×10 5 cells in 24-well plates, treated with 17-AAG alone, MG132 alone, or combined 17-AAG and MG132, and cultured for 48 h. Western blotting analysis was performed using a previously described method [15]. The primary antibodies were monoclonal or polyclonal antibodies against PSMA7 (Abcam, Cambridge, MA, USA) and β-actine (SIGMA, Sigma-Aldrich).…”

“…The HCV non-structural protein 3 (NS3) forms a complex with Hsp90 [8,10,11] that is critical for HCV replication [15]. Treatment of the #50-1 HCV replicon cells with the Hsp90 inhibitor 17-AAG [8,16,17] suppresses HCV RNA replication and NS3 is the only HCV protein degraded in these cells [15].…”