An RNA-Binding Protein, hnRNP A1, and a Scaffold Protein, Septin 6, Facilitate Hepatitis C Virus Replication

Abstract

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5-nontranslated region (5 NTR) and the 3 NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.Approximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for most cases of chronic liver disease, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (45,70).HCV has a single-stranded RNA genome of positive polarity that contains a single long open reading frame flanked by nontranslated regions (NTRs) at its 5Ј and 3Ј ends. Nearly the entire 5Ј NTR is needed for efficient RNA amplification, although the minimal replication element is within the first 120 nucleotides (20,35,58). In addition, the 5Ј NTR has been found to contain an internal ribosomal entry site (IRES), which is required for the translation of polyprotein (29,67,75). The 3Ј NTR is composed of a nonessential variable region, a poly(U)/poly(U ⅐ C) tract, and a highly conserved and essential 3Ј X domain (19,40,74). The viral proteins are translated as a single large polyprotein of 3,010 to 3,040 amino acids, which is cotranslationally and/or posttranslationally processed by cellular and viral proteases into mature structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) viral proteins (57). NS5b is an RNA-dependent RNA polymerase that produces complementary RNAs from template RNAs (3, 66).Subgenomic HCV RNA replicons have been developed to mimic the replication of a viral RNA infecting a host cell. These replicons are composed of the HCV 5Ј NTR; a selection marker, such as neomycin phosphotransferase; the IRES of encephalomyocarditis virus; HCV nonstructural proteins NS3, -4a, -4b, -5a, and -5b; and the HCV 3Ј NTR. When introduced into human hepatoma (Huh 7) cells, the HCV replicon RNA replicates autonomously (47). Using this replicon system, several groups have reported that HCV replication occurs in a distinct replication complex, which comprises viral RNA and HCV proteins (1,12,23,54). The replication complex is formed on intracellular membranes with vesic...

Differential effect of acute and persistent Junín virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B

“…A previous report showed that the arenavirus Pichinde virus causes a transient cytoplasmic translocation of hnRNP A1 (Bowick et al, 2009). Here, we demonstrated that acute JUNV infection induces the cytoplasmic accumulation of hnRNP A1 in most infected cells but, in accordance with previous results obtained for hepatitis C virus-infected cells (Kim et al, 2007), some infected cells exhibited a nuclear hnRNP A1 pattern of distribution. The m.o.i.…”

“…Since all hnRNP A/B proteins are structurally similar and are particularly homologous in their RNAbinding domains it would not be surprising if they replaced each other in their functions. 2007; Cammas et al, 2007;Kim et al, 2007;Lin et al, 2008Lin et al, , 2009Gui et al, 2010). A previous report showed that the arenavirus Pichinde virus causes a transient cytoplasmic translocation of hnRNP A1 (Bowick et al, 2009).…”

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

“…Our study using both HCV replicon and JFH1 infectious cell culture systems clearly demonstrates that depletion of HuR leads to a significant reduction in the HCV RNA and protein levels. HuR has been shown to stimulate IRES-driven translation of HCV RNA (25). Interestingly, in our assay it appears that HuR plays a more dominant role in the replication step of the viral life cycle than in translation.…”

“…La and PCBP2 proteins have been shown to interact with both of the UTRs and link the 5= and 3= ends of the viral genome, facilitating replication (22,23). Other independent studies on RNA binding proteins like hn-RNPD, hnRNPA1, HuR, Unr, hnRNPU, etc., have indicated that they interact with the viral RNA (24)(25)(26)(27). However, the molecular basis of regulation of viral replication by these RBPs remains a mystery.…”

Sept7bis essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis

“…Dysfunction of the mammalian septin family has been identified in diverse pathologies, including neoplasia (Montagna et al, 2003), neurodegenerative conditions (Kinoshita et al, 1998) and infections (Kim et al, 2007). However, the function of septins in the kidney in vivo has not been studied; such analysis is required to test the relevance of the functions of septins in ciliogenesis and cilia structure that have been identified in epithelial cells in tissue culture.…”