Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System

Abstract

Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in the United States, with sequelae including liver fibrosis, cirrhosis, and hepatocellular carcinoma (reviewed in reference 19). Identified in 1989 as a plus-strand RNA virus (4), HCV causes an infection that was originally distinguished by its characteristic induction of microtubule (MT) aggregates in infected hepatocytes, implicating MTs in HCV-associated disease (2,11,23,28,29,32,36). Studies with a related flavivirus (Kunjin virus) have demonstrated that MT aggregates similar to those induced by HCV are also important for Kunjin RNA synthesis (15,22). Moreover, MT paracrystals have recently been detected in cultured cells transfected with an HCV subgenomic replicon (21). Taken together, these studies suggest that cytoskeletal elements may be required for HCV replication. Yet, despite the historical emphasis on MTs and HCV from a clinical perspective, the role of MTs in the HCV life cycle at a molecular level remains poorly understood.To determine if HCV RNA synthesis requires functional actin or MT networks, we examined the effects of cytoskeleton inhibitors on the efficiency of HCV RNA synthesis in the HCV replicon cell system ( Fig. 1) (18). In this system, Huh7 cells stably transfected with an HCV replicon RNA are used to mimic the RNA synthesis that occurs in an ongoing, persistent infection with HCV. Because the replicon construct encodes a neomycin resistance gene for G418 (Geneticin) selection, HCV RNA synthesis in the replicon cells can be detected by quantitative PCR using a primer-probe set specific for the neomycin sequence. We have validated this approach by demonstrating a dose-dependent decrease in replicon RNA levels upon alpha interferon treatment (data not shown), using alpha interferon concentrations similar to those cited previously by other laboratories (1, 10, 18). In addition, we have confirmed that transcription of the replicon in our HCV replicon cells is resistant to actinomycin D, as originally demonstrated by Lohmann et al. (18), verifying that transcription of replicon RNA is specific to the RNA-dependent RNA polymerase activity of the HCV replication complex. A single HCV replicon cell line-a serially passaged line originally generated by stable transfection of Huh7 cells with a replicon with a sequence identical to that used by Lohmann et al. (18) (Fig. 1)-was used for all experiments.Because vinblastine sulfate (VS) is a well-characterized inhibitor of MT polymerization and has been shown to alter Kunjin virus replication (15, 22), we first determined the effect of VS on HCV replicon RNA synthesis. One day prior to addition of the inhib...

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer, Productive Infection, and Replication and Their Requirement for HCV Receptors

“…7d). Contrary to our studies, other studies have found that nocodazole treatment inhibits microtubule-dependent transport of the HCV replication complex, initiation of productive HCV infection, and, as a result, HCV RNA replication (75,77,78). Moreover, two recent reports have shown that postassembly the vesicular HCV core protein puncta traffic via microtubules and that trafficking is inhibited by nocodazole treatment (28,50).…”

“…Thus, we determined the roles of actin and/or microtubule cytoskeletons in CCCM HCV infection. The actin cytoskeleton has been shown to be involved in interaction with multiple steps of cell-free HCV infection, including viral entry (44,74) and replication (75,76). In agreement with these findings, our studies have shown that an intact actin network was required for CCCM HCV transfer, as treatment with actin polymerization inhibitor cytochalasin D in the coculturing assay prevented CCCM HCV transfer ( Fig.…”

Role of Annexin A2 in the Production of Infectious Hepatitis C Virus Particles

“…Once present, ANXA2 participates in the formation of higher-order structures of the membranous web via its membrane organization functions (67), possibly stabilizing membrane microdomains involved in virion morphogenesis. Alternatively, it also could provide contacts to the actin cytoskeleton (23,48), which has been shown to be linked to HCV replication complexes (10,28). Interestingly, ANXA2 has been identified in a pulldown of NS3 from HEK 293T cells expressing NS3/4A together with components of the cytoskeleton, providing some evidence for this concept (28).…”

Hepatitis C virus F protein: A double-edged sword in the potential contribution of chronic inflammation to carcinogenesis

“…However, this does not mean that F protein has no correlation with HCV replication. It was reported that HCV replication complex-mediated RNA synthesis requires intact microtubule and actin polymerization (28). Therefore, interruption of normal microtubule formation likely results in decreased HCV replication.…”