Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Abstract

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.Hepatitis C virus (HCV), of the family Flaviviridae, is an enveloped, positive-stranded RNA virus. Spread mostly by blood-borne transmission, HCV infects more than 170 million people worldwide. The viral genome is composed of a single open reading frame (ORF) plus 5Ј-and 3Ј-nontranslated regions. The ORF encodes a large polyprotein that is cleaved by cellular and viral proteases into 10 viral proteins. The structural proteins, including the capsid protein (core), two glycoproteins (E1 and E2), and a small ion channel protein (p7), reside in the N-terminal half of the polyprotein. The rest of the ORF encodes six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3 through NS5B assemble into a replication complex (RC) and are necessary and sufficient for HCV RNA replication in cell culture (8, 42). NS3 is a multifunctional protein with both a serine protease and an RNA helicase activity. The protease activity is responsible for cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (5), and the helicase activity is probably required to unwind the double-stranded RNA intermediates formed during replication (38). NS4A serves as an essential cofactor for the NS3 protease and anchors the NS3 protein to intracellular membranes (25,36,39). NS4B induces the formation of a "membranous web" that is probably the site of HCV replication (16). It also contains a GTP-...

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

“…Interaction of NS5B with Cyclophilin A-As CypA is an essential host factor for HCV replication and as interaction with the NS5B RNA-dependent RNA polymerase has been repeatedly reported (28,29,36,40), we equally compared the 1 H, 15 N HSQC spectra of purified 15 N-CypA alone or in the presence of NS5B ⌬21 in a 1:1 ratio (50 M each). As for the individual domains of NS5A, we looked for chemical shift per-turbations and/or signal broadening in the CypA spectrum.…”

“…Although this apparent discrepancy may arise from the different constructs or genotypes that were used, a more plausible hypothesis is that the described CypA-NS5B interaction was indirect and mediated by a third interacting partner. Indeed, to our knowledge no studies have been done in vitro with only the two purified proteins but rather with cell lysate pulldown experiments (29,40), crude replication complex preparations pulldown experiments (28), or in in vitro translation reactions (36) that at least contain RNAs. Without interaction between NS5B and CypA, the interplay between HCV NS5B, NS5A, and host CypA, three essential viral replicase components, may be driven by the common binding site on NS5A-D2 that we identified.…”

“…In vivo, HCV RNA replication occurs in a replication complex that contains endoplasmic reticulum-derived membrane(s), NS3 to NS5B viral proteins (25), and viral RNA as well as several host factors such as hVAP-33 (26), VAP-B (27), and cyclophilin A (CypA) (28). The latter is a major host factor required for HCV replication (29).…”

“…Watashi et al (35) initially proposed that cyclophilin B would be the target of CsA and proposed a mechanism whereby CypB would be a positive regulator of NS5B by modulating its RNA binding activity. Later, several members of the cyclophilins have been proposed to be involved into the HCV replication (29,31), and NS5B interactions with CypB (35)(36)(37)(38)(39) and CypA (28,29,36,40) have been reported. However, more recent evidence points to CypA as the major key player both for HCV replication and CsA sensitivity (29,41).…”

“…However, conflicting results have been reported as for the role of CypA for the proper association of these three proteins into the replication complex (28,40). Here we report NMR spectroscopy analyses at a per residue level of the molecular interactions between NS5A domains 2 and 3, NS5B, and CypA.…”