Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro

Abstract

The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5B⌬21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5B⌬21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5B⌬21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A (S25-C447) , but not NS5A (S25-K215) , enabled the NS5B⌬21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCEThe molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A (S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5B⌬21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors. Hepatitis C virus (HCV) is a blood-borne pathogen that infects an estimated 130 million to 200 million people worldwide and constitutes a global health problem (1). A majority of people exposed to HCV (ϳ85%) go on to develop chronic hepatitis, which can result in liver cirrhosis, liver failure, or hepatocellular carcinoma (2).HCV is a positive-stranded RNA virus belonging to the Flaviviridae family. It has an ϳ9.6-kb genome which encodes a single polyprotein that is co-and posttranslationally processed by cellular and viral proteases to give rise to structural proteins C, E1, and E2 and nonstructural (NS) proteins P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which are involved in polyprotein processing, genome replication, or virion packaging (3). HCV replication is carried out by a membrane-associated ribonucleoprotein complex which is comprised of HCV nonstructural proteins and host factors (4, 5). While the functions of some of the HCV proteins, such as NS3 protease, NS3 helicase, and NS5B RNA-dependent RNA polymerase (RdRp), are well understood, the functions of others, such as NS4B and NS5A, are less clear (3). In addition, it is unknown how the various viral and host proteins assemble into a replication complex and participate in the replication process (6).NS3 is a bifunctional protein with an N-termi...

Full-dose sofosbuvir plus low-dose ribavirin for hepatitis C virus genotype 2-infected patients on hemodialysis

“…This drug is phosphorylated by the liver into the active metabolite GS-461203. Dephosphorylation of GS-461203 results in the formation of the inactive metabolite GS-331007 [12]. Approximately 78% of the inactive metabolite is eliminated renally.…”

Bioorthogonal dissection of the replicase assembly of hepatitis C virus

“…We cannot exclude the possibility that, in the presence of viral RNA, multiple dimeric NS5As bind to viral RNA to form multimers ( Figure 7 C). In an in vitro replication system that uses an artifact rolling-circle RNA as a template, NS5A has been demonstrated to be a processivity factor for viral RNA synthesis ( Mani et al., 2015 ), which is probably attributed to the binding of viral RNA by dimeric NS5A ( Figure 7 C).…”

Hepatitis C Viral Replication Complex

“…In addition to NS5B, other viral and cellular proteins also contribute substantially to HCV RNA synthesis. NS5B recruits NS3 to facilitate processive elongation of RNA synthesis [ 114 ]. NS3 contains a carboxy-terminal DExD-box helicase domain (NS3h) and an amino-terminal protease domain that functions in conjunction with the cofactor, NS4A ( Figure 4 ).…”

“…Domain III (a.a. 356–447) functions primarily in virion assembly [ 138 ]. NS5A has been reported to help NS5B bind to the HCV RNA template [ 114 ].…”

“…DDX3 [ 180 ], Y-box binding protein 1 (YB-1) [ 180 ], FKBP6 [ 181 ], and human choline kinase-α (hCKα) [ 182 , 183 ] could interact with NS5A to facilitate HCV RNA replication. Cellular Cyp A [ 137 , 184 ] and human replication protein A (RPA) [ 114 ] could bind to NS5A and stimulate the binding of NS5A to NS5B and viral RNA to facilitate HCV RNA replication.…”