Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

Abstract

Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-␤-cyclodextrin (M␤CD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by M␤CD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. IMPORTANCEThe biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-␤-cyclodextrin (M␤CD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process. H epatitis C virus (HCV) is a small, enveloped, positive-strand RNA virus belonging to the Flaviviridae family (1, 2). Globally, nearly 200 million people are infected with this virus, which causes severe morbidity and mortality due to its persistent replication in the liver (3, 4). HCV encodes a single open reading frame that is processed by host and viral proteases into three structural proteins, including Core and two envelope proteins (E1 and E2), and seven nonstructural proteins, including p7, NS2 and NS3-5B (NS3, NS4A, NS4B, NS5A, and NS5B) (5)...

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly

“…Interestingly, in the present study, we showed that the GDP-but not GTP-bound form of Rab32 specifically interacted with HCV core protein and enriched core in Rab32-derived perinuclear aggregated structures and resulted in a high level of core protein expression. Our hypothesis also fits well with the study of Shanmugam et al showing that HCV core protein-associated detergent-resistant membranes may serve as platforms for virion assembly (24). Importantly, it has been previously reported that Rab32 localizes at the mitochondrion-associated membrane (MAM) and regulates MAM properties (14).…”

“…Rab32 is involved in the assembly step of the HCV life cycle. HCV core protein has been shown to be localized in the detergent-resistant membranes which are putative platforms for HCV particle assembly (23,24). In addition, HCV core protein is localized on the surface of lipid droplets (LDs) in virus-infected cells, and core proteincoated LDs aggregate in the perinuclear region which has been proven to be essential for virion assembly (30)(31)(32).…”

“…To test this hypothesis, we first examined the solubility of various forms of Rab32 protein in 1% Triton X-100 as the aggregated proteins are largely detergent resistant. Calnexin was used as the marker for the soluble fraction as reported previously (23,24). As shown in Fig.…”

Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging

“…We assessed the delivery of TopFluor-cholesterol (TF-cholesterol), to ‘new’ versus ‘old’ NS5A foci as depicted in Figure 5A. TF-cholesterol is a fluorescent cholesterol analog that closely mimics membrane partitioning and trafficking of native cholesterol [31] and has been used to monitor cholesterol trafficking to HCV replication organelles [12, 32]. While TF-cholesterol trafficked to both ‘old’ and ‘new’ NS5A foci, TF-cholesterol colocalized preferentially with ‘old’ NS5A foci (Figures 5B and C), suggesting that replication organelles become progressively enriched in cholesterol over time.…”

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication

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