The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4°C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.Hepatitis C virus (HCV) is an important human pathogen associated with non-A, non-B hepatitis and is the leading cause of chronic hepatitis and liver cirrhosis. As a member of the Flaviviridae family, HCV contains a positive-sense, singlestranded RNA genome of approximately 9.6 kb. The viral genome encodes a single polyprotein of about 3,010 amino acids, which is proteolytically processed by a combination of host-and virus-encoded proteases into 10 viral structural and nonstructural (NS) proteins arranged in the following order:The establishment of the HCV subgenomic replicon and the subsequent analysis of the adaptive mutations revealed that most of the HCV NS proteins, with the probable exception of NS2, are involved in HCV RNA replication (6,29,37). NS3 is a helicase and a serine protease, whose function is dependent on NS4A. It is conceivable that the enzymatic activities of these proteins are key components of the HCV replication complex. The function of NS4B is thus far unknown, although it has been implicated in inducing transformation (34) and intracellular membrane alterations (13); the latter may play a significant role in the formation of the HCV RNA replication complex. NS5A is known to be a multifunctional protein implicated in the pathogenesis and interferon resistance of HCV infection. However, it has become evident that NS5A also plays an indispensable role in the replication of the HCV subgenomic replicon (6), but the underlying mechanism has yet to be identified. HCV NS5B is an RNA-dependent RNA polymerase (RdRp). All of these NS proteins, together with host prote...
Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.
The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.
Hepatitis C virus (HCV) core protein, a component of viral nucleocapsid, has been shown to modulate cellular and viral promoter activities. To identify potential cellular targets for HCV core protein, a human liver cDNA library was screened for core-interacting proteins using the yeast two-hybrid system. Among the proteins identified was heterogeneous nuclear ribonucleoprotein K (hnRNP K), which has been demonstrated to be a transcriptional regulator. The interaction of HCV core protein with hnRNP K was confirmed by glutathione Stransferase fusion protein binding assay, proteinprotein blotting assay, and coimmunoprecipitation in vitro and in vivo. Additionally, these two proteins were shown to be partially colocalized in the nucleus. The hnRNP K-binding site in HCV core protein was mapped to the region from amino acid residues 25-91, a hydrophilic area near the N terminus. The HCV core proteinbinding domain was located within amino acid residues 250 to 392, which contain the three proline-rich domains, of hnRNP K. Furthermore, HCV core protein relieved the suppression effect of hnRNP K on the activity of the human thymidine kinase gene promoter. The specific binding of HCV core protein to hnRNP K suggests that multiple functions of hnRNP K may be disrupted by the core protein during HCV infection and thus explains, in part, the pathogenesis of HCV.
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