Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons

Abstract

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...

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

“…Previously, we have shown that HCV RNA replication occurs on speckle-like structures [29], which may be equivalent to the membranous web, as reported by Gosert et al [45]. The immunofluorescence staining study presented here showed that PTB in Huh-7 cells mainly existed in the nucleus, while PTB in the replicon cells localized in not only the nucleus but also distinct structures in the cytoplasm and colocalized with the newly synthesized HCV RNA (Figure 1).…”

Illuminating the host – How RNAi screens shed light on host‐pathogen interactions

“…(( Table 2) were found to be involved in lipid metabolism, membrane biogenesis, sorting and trafficking, supporting previous observations that HCV, like other positive-strand RNA viruses, replicates on altered host membranes, also described as "membranous webs" [68,69]. Furthermore, 6 genes were identified as being associated with the overlap between the results of these three screens and the study by Tai et al [65].…”

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

“…19,20,[27][28][29] It is largely unknown how NS4B interacts with other proteins. Recently, we have predicted a basic leucine zipper (bZIP) motif within the aminoterminal part of HCV NS4B.…”

“…13,18,19 The subcellular structure is considered to harbour the HCV replication complex, since all nonstructural proteins as well as viral genomic RNA were found to co-localize within this ER alterations. 19,20 The aminoterminal amphipathic a-helix (amino acid 1 to 26) has been described to mediate membrane association and correct localization of the replication complex within the ER. 7 Recently, Gouttenoire et al proposed the amphipathic a-helix to be located between amino acid 42 to 66.…”

“…17 In this publication no membrane association of amino acid 1 to 29 or amino acid 1 to 40 was detected. 17 Most likely, the HCV NS4B protein is anchored to the ER membrane by the four TMDs, initiating ER membrane alterations and recruiting other host and viral factors of the replication complex, 20,21 while the aminoterminal region contains an amphipathic a-helix and mediates membrane association. 7,17 Exact mechanisms how NS4B causes ER membrane alterations or interacts with other proteins are still unknown.…”