Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

Our study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.Formation of a membrane-associated replication complex composed of viral proteins, replicating RNA, and altered cellular membranes is a hallmark of all of the plus-strand RNA viruses investigated thus far (4,11,23,27,29,32). This strategy may offer multiple potential advantages, including (i) compartmentalization and local concentration of viral products, (ii) physical support and organization of the RNA replication complex (20), (iii) tethering of the viral RNA during unwinding, (iv) provision of lipid constituents important for replication (1, 34), and (v) protection of the viral RNA from double-stranded RNA-mediated host defenses or RNA interference.Hepatitis C virus (HCV) contains a plus-strand RNA genome of approximately 9,600 nucleotides that encodes a polyprotein precursor of about 3,000 amino acid residues (17, 21). We have recently shown that expression of the HCV polyprotein in tetracycline-regulated human osteosarcoma cell lines induces distinct membrane alterations. A prominent alteration, designated the membranous web, was found to contain all of the viral structural and nonstructural proteins and therefore was proposed to represent the HCV replication complex (9). The membranous web could be induced by NS4B alone and was very similar to the "sponge-like inclusions" previously observed by electron microscopy (EM) in the livers of HCV-infected chimpanzees (24).The recent development of replicon systems for HCV has allowed, for the first time, the study of efficient and genuine HCV RNA replication in Huh-7 human hepatoma cells in vitro (6,13,19,25). The aims of the present study were to locate the site of HCV RNA replication and to identify the HCV replication complex in Huh-7 cells harboring a prototype subgenomic HCV replicon.First, Huh-7-derived clone 9-13, harboring a bicistronic subgenomic replicon (19), was investigated by immunofluorescence microscopy (IF) to determine the subcellular distribution of the viral nonstructural proteins. Fixation, permeabilization, and immunostaining of cells were performed as previously described (10). As shown in Fig. 1a, NS3 was found in the cytoplasm as brightly fluorescing dots and in a reticular staining pattern. Very similar staining patterns have consistently been observed with monoclonal antibodies (MAbs) directed against NS4A, NS4B, NS5A, and NS5B (data not shown). By double-labeling IF, HCV nonstruct...