J Biol Chem 282:37158C37169. life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to Pyrroloquinoline quinone facilitate virion assembly. IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates numerous intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV contamination 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 Pyrroloquinoline quinone required for the assembly of HCV. Collectively, our study identifies that CDC46 Rab32 is usually a novel host factor essential for HCV particle assembly. melanophores, Rab32 controls melanosome transport in a cyclic AMP (cAMP)-dependent protein kinase A (PKA)-dependent manner (11). Despite the ubiquitous expression of Rab32 in most human tissues (12, 13), the precise functions of Rab32 in nonmelanogenic cells and tissues are poorly characterized. In cell types other than melanocytes, such as COS7 and Pyrroloquinoline quinone WI-38 fibroblasts, Rab32 was found to colocalize with mitochondria. In addition, Rab32 modulates targeting of PKA to mitochondrial and endoplasmic reticulum (ER) membranes and determines mitochondrial dynamics and apoptosis onset (13, 14). Furthermore, Rab32 has been demonstrated to be essential for the autophagic response in HeLa and COS7 cells (15). Recently, it has been reported that Rab32 increases lipid biosynthesis and autophagosome formation during the reprogramming process (16). Rab32 has also been involved in acute brain inflammation in mice (17). Moreover, Rab32 interacts with leucine-rich repeat kinase 2 (LRRK2) and regulates LRRK2 transport, implicated in Parkinson’s disease (18). To date, the functional involvement of Rab32 in the HCV life cycle or HCV-induced pathogenesis Pyrroloquinoline quinone has not been demonstrated. In the present study, we demonstrate that HCV concomitantly upregulated Rab32 expression and induced conversion of the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, which resulted in the aggregation of Rab32 protein and thus made it less susceptible to cellular degradation machinery. We further show that GDP-bound Rab32 selectively interacts with HCV core protein and deposits core in ER-associated Pyrroloquinoline quinone Rab32-derived aggregated structures in the perinuclear region that are likely to be viral assembly sites. Moreover, we demonstrate that Rab32 is usually specifically required for HCV particle assembly. Collectively, these data suggest that HCV may modulate Rab32 activity to generate the core protein-containing structures necessary for HCV virion assembly. RESULTS Rab32 level is usually increased in the context of HCV contamination. In an attempt to identify host factors that play essential functions in HCV propagation, we previously employed high-throughput RNA sequencing (RNA-Seq) technology to characterize the genome-wide transcriptomic changes in cell culture-grown (HCVcc)-infected cells. By performing quantitative real-time PCR (qRT-PCR analysis), we ultimately verified that 30 host genes were markedly increased in the context of HCV contamination (19). In the present study, we selected Rab32 for more sophisticated characterization in order to delineate its possible functional involvement in regulating HCV propagation. To confirm the increase in Rab32 expression in HCVcc-infected cells, we measured Rab32 mRNA levels in Jc1-infected Huh7.5 cells at different time points. As expected, Rab32 mRNA was noticeably increased at day 2, and its level was doubled at day 6 in HCV-infected cells compared with the level in mock-infected cells (Fig. 1A). To investigate if the transcriptional level of Rab32 was also regulated by HCV contamination, Huh7.5 cells were either mock infected or infected with Jc1. At 4 h postinfection, cells were further transfected with a luciferase (Luc) reporter plasmid consisting of nucleotides (nt) ?643 to +260 of the Rab32 promoter, and then luciferase activity was analyzed at 2 days postinfection. Physique 1B shows that Rab32 promoter activity was significantly increased in HCV-infected cells. Consistently, the protein level of Rab32 was proportionally elevated during the course of HCV contamination (Fig. 1C). We further verified that this Rab32 mRNA level in HCV-replicating main human hepatocytes significantly increased compared with the level in the replication-defective control (Fig. 1D). Additionally, we also examined the Rab32 level in HCV subgenomic replicon cells derived from genotype 1b. We showed that both the mRNA level (Fig. 1E) and the protein expression level (Fig. 1F) of Rab32 in.
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