Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCVinfected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCETRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.
Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP-and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.
The propagation of hepatitis C virus (HCV) is highly dependent on host cellular factors. To identify the cellular factors involved in HCV propagation, we have previously performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of ϳ9,000 host proteins immobilized in a microarray, ϳ90 cellular proteins were identified as HCV NS5A interacting partners. Of these candidates, we selected Abelson interactor 1 (Abi1) for further characterization. Binding of HCV NS5A to Abi1 was verified by both in vitro pulldown and coimmunoprecipitation assays. HCV NS5A interacted with Abi1 through regions I ؉ II of Abi1 and domain I of NS5A. We further demonstrated that Abi1 colocalized with the HCV NS5A protein in the cytoplasm. We showed that NS5A inhibited epidermal growth factor-mediated ERK and Egr1 activations and this inhibitory activity of NS5A was nullified in Abi1-knockdown cells. Moreover, silencing of Abi1 expression impaired HCV replication, whereas overexpression of Abi1 promoted HCV propagation. Collectively, these data indicate that HCV exploits host Abi1 protein via NS5A to modulate MEK/ERK signaling pathway for its own propagation. Hepatitis C virus (HCV)3 is a major etiologic agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. HCV is an enveloped virus with a positive-sense, singlestranded RNA genome. HCV belongs to the genus Hepacivirus within the Flaviviridae family (1). The HCV genome is ϳ9.6 kb in length and encodes a 3,010-amino acid protein from a single large open reading frame. This polyprotein precursor undergoes cleavage by both host cellular and viral proteases to generate 3 structural proteins (core, E1, and E2) and 7 nonstructural proteins (p7 and NS2 to NS5B) (2). The structural proteins are the components of the virion, whereas the nonstructural proteins are involved in the replication of the viral genome. Nonstructural 5A (NS5A) is a multifunctional protein and interacts with many cellular proteins to regulate cellular signaling pathways and viral propagation. NS5A contains three consensus proline-rich PXXP motifs, which bind to SH3 domains. These motifs are commonly found in many cellular signaling molecules (3). NS5A modulates the MAPK signaling pathway to regulate cell growth and activation. NS5A specifically interacts with the two SH3 domains of growth factor receptorbound protein 2 (Grb2) and inhibits the MAPK/ERK pathway (4). NS5A containing mutations within the C-terminal prolinerich motif neither interacts with Grb2 nor inhibits epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation (5-7). Furthermore, NS5A expressed in recombinant herpes simplex virus-infected cells also interacts with Grb2 and inhibits the Erk1/2 signaling pathway (8). These data indicate that NS5A regulates the MAPK pathway via Grb2. However, the exact molecular mechanisms by which NS5A interferes with function of Grb2 remains unclear.Abelson interactor 1 (Abi1), also known as E3B1, is an adaptor protein in the receptor tyrosine kinase (RTK) sig...
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