Human Eukaryotic Initiation Factor 4AII Associates with Hepatitis C Virus NS5B Protein in Vitro

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...

Section: Resultsmentioning

confidence: 67%

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

Kim

Seol

Song

et al. 2007

“…These nonstructural viral proteins, together with host factors, form the viral replicase, the complex in which viral replication is thought to take place. NS5B has been shown to interact with a SNARElike protein (38) and with human eIF4AII, an RNA-dependent ATPase/helicase and a component of the translation initiation complex (19). Interactions of RdRp's of other viruses with host proteins have also been reported.…”

mentioning

confidence: 99%

Cellular RNA Helicase p68 Relocalization and Interaction with the Hepatitis C Virus (HCV) NS5B Protein and the Potential Role of p68 in HCV RNA Replication

Goh

Tan

Lim

et al. 2004

108

Chronic infection by hepatitis C virus (HCV) can lead to severe hepatitis and cirrhosis and is closely associated with hepatocellular carcinoma. The replication cycle of HCV is poorly understood but is likely to involve interaction with host factors. In this report, we show that NS5B, the HCV RNA-dependent RNA polymerase (RdRp), interacts with a human RNA helicase, p68. Transient expression of NS5B alone, as well as the stable expression of all the nonstructural proteins in a HCV replicon-bearing cell line (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113), causes the redistribution of endogenous p68 from the nucleus to the cytoplasm. Deletion of the C-terminal two-thirds of NS5B (NS5B⌬C) dramatically reduces its coimmunoprecipitation (co-IP) with endogenous p68, while the deletion of the Nterminal region (NS5B⌬N1 and NS5B⌬N2) does not affect its interaction with p68. In consistency with the co-IP results, NS5B⌬C does not cause the relocalization of p68 whereas NS5B⌬N1 does. With a replicon cell line, we were not able to detect a change in positive-and negative-strand synthesis when p68 levels were reduced using small interfering RNA (siRNA). In cells transiently transfected with a full-length HCV construct, however, the depletion (using specific p68 siRNA) of endogenous p68 correlated with a reduction in the transcription of negative-strand from positive-strand HCV RNA. Overexpression of NS5B and NS5B⌬N1, but not that of NS5B⌬C, causes a reduction in the negative-strand synthesis, indicating that overexpressed NS5B and NS5B⌬N1 sequesters p68 from the replication complexes (thus reducing their replication activity levels). Identification of p68 as a cellular factor involved in HCV replication, at least for cells transiently transfected with a HCV expression construct, is a step towards understanding HCV replication.Hepatitis C virus (HCV) is a positive-stranded RNA virus belonging to the family Flaviviridae. It infects more than 170 million people worldwide and poses an important medical problem in developed and underdeveloped countries. HCV replicates in the liver, and chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. Practically all processes in the life cycle of viruses, including viral entry, translation, processing and modification of viral proteins, maturation, and release of viral particles from host cells, are dependent on the host cell machinery and involve intimate interaction between viral and host proteins. The role of host proteins in the replication cycle of HCV and the effect of virus-host interaction have remained poorly understood. This is due to a lack of cell culture and animal model systems for studying the biology of this virus. The study of the interaction of HCV with cellular proteins has been limited to the identification of host proteins interacting with individual viral proteins.The proteins encoded by the HCV genome are three structural proteins (core protein and envelope proteins E1 and E2) and se...

“…For example, hepatitis C virus (HCV) RdRp is known to bind to nucleolin (an RNA binding protein) and initiation factor 4A (11,15,20). The HCV NS5A and NS5B replication proteins interacted with hVAP-33, a SNARE-like protein bound to cellular membranes (47).…”

mentioning

confidence: 99%

Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

Serva

Nagy

2006

172

219

Viral replicases are the key enzymes in the replication of plus-stranded RNA viruses (1, 7). The viral replicase complexes, which are assembled on intracellular membranes, consist of virus-encoded proteins, such as the RNA-dependent RNA polymerase (RdRp) and auxiliary viral protein(s), as well as host-derived proteins and the viral RNA template (2, 19). Studies with a small number of plant RNA viruses identified several host factors which are likely involved in the targeting of the viral replication proteins to intracellular compartments and replicase assembly and/or function. For example, subunit 45 of eukaryotic initiation factor 3 was found in association with brome mosaic virus (BMV) RdRp (35), while subunit 56 of eukaryotic initiation factor 3 was detected in a tobacco mosaic virus replicase preparation (25). Moreover, a molecular chaperone (Ydj1p) is known to be essential for the activation of the BMV replicase (45). TOM1 and TOM3 integral membrane proteins of Arabidopsis were found to interact with the tobacco mosaic virus replicase proteins and cofractionated with the RdRp activity (46, 53).Host factors interacting with and/or affecting viral replicase activities have also been identified in animal plus-stranded RNA viruses. For example, hepatitis C virus (HCV) RdRp is known to bind to nucleolin (an RNA binding protein) and initiation factor 4A (11,15,20). The HCV NS5A and NS5B replication proteins interacted with hVAP-33, a SNARE-like protein bound to cellular membranes (47). hVAP-33 has been suggested to serve as a docking site to anchor the HCV RdRp to the membrane during assembly of the replicase complex (12, 47). Also, host chaperones are involved in proteolytic processing of the HCV nonstructural proteins (44). The Hsp90 molecular chaperone has been shown to enhance Flock House virus replication (18). Poly(C) and poly(A) binding proteins were found to interact with both the poliovirus RNA and the viral polymerase precursor 3CD (14, 49). Nucleolin and Sam68, which are involved in virus replication, are relocalized in the cell during poliovirus infection (21, 48). In addition, the cellular COPII proteins are involved in the assembly of the poliovirus replication complex (39). hnRNP A1 was found to be part of the mouse hepatitis virus transcription/replication complex, and it is involved in subgenomic RNA synthesis and genome replication (42,50). Overall, the above examples illustrate that host factors play complex and significant roles in the replication of many plus-stranded RNA viruses.Tomato bushy stunt virus (TBSV) and the closely related cucumber necrosis virus (CNV) are nonsegmented plusstranded viruses of plants. Among the five TBSV-encoded proteins, only p33 and p92 are required for replication (26,30,40,52), whereas the other proteins are involved in cell-to-cell movement, encapsidation, and suppression of gene silencing (52). The p92 replication protein has the RdRp signature motifs in its unique C terminus, whereas the auxiliary p33, which overlaps with the N-terminal sequence of p92, play...