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.

FIG. 1.

Localization of HCV nonstructural proteins and plus-strand RNA in Huh-7 cells harboring a subgenomic replicon. (a) Huh-7-derived 9-13 replicon cells were examined by IF with HCV NS3-specific MAb 1B6 (33). NS3 was located in dot-like structures and in a reticular staining pattern. (b) HCV plus-strand RNA was detected by FISH with a directly FITC-labeled riboprobe of negative polarity. HCV plus-strand RNA accumulated in dot-like structures. Bar, 10 μm. Naive Huh-7 cells stained in parallel showed no specific staining.

FIG. 2.

Membrane alterations in Huh-7 cells harboring a subgenomic replicon. (a) A naive Huh-7 cell displays unaltered organelles. Bar, 1 μm. (b) Low-power overview showing the membranous web (arrows) in a 9-13 replicon cell. Bar, 1 μm. (c) Higher magnification of the membranous web (arrows). A second membrane alteration that closely resembles the contiguous vesicles found in HCV-inducible cell lines and that did not harbor HCV proteins (9) was less abundant in replicon cells (arrowhead). Bar, 500 nm. G, Golgi apparatus; M, mitochondria; N, nucleus.

FIG. 3.

Immunogold detection of HCV nonstructural proteins on the membranous web in replicon cells. (a) 9-13 replicon cells were labeled with NS5A-specific MAb 11H (; kindly provided by Jan Albert Hellings, Organon Teknika B.V., Boxtel, The Netherlands). Gold particles accumulated on a membranous structure (surrounded by arrowheads). In addition, some label was found on the ER (arrows). Bar, 500 nm. (b) Identically treated naive Huh-7 cells were virtually devoid of gold particles. Bar, 500 nm. (c and d) At higher magnification, 9-13 replicon cells labeled with NS5A-specific MAb 11H (c) and NS3-specific MAb 1B6 (d). Both antigens are found almost exclusively on the membranous web. Bar, 200 nm.

FIG. 4.

EM-in situ hybridization of Huh-7 cells harboring a subgenomic HCV replicon. (a) HCV plus-strand RNA in 9-13 replicon cells, detected with a digoxigenin-labeled riboprobe of negative polarity and a gold-conjugated anti-digoxigenin antibody, is associated with the membranous web. (b) An adjacent area of the same cell shows no label accumulation above the background. Bar, 200 nm.

FIG. 5.

Detection of nascent HCV RNA in Huh-7 cells harboring a subgenomic replicon. 9-13 replicon cells were metabolically labeled with 5-bromouridine 5′-triphosphate in the presence of actinomycin D and then subjected to double-labeling confocal laser scanning microscopy. (a) Detection of NS5A with the polyclonal antiserum WU144 and an FITC-conjugated secondary antibody. (b) Detection of newly synthesized, bromouridine-labeled viral RNA with a MAb against bromodeoxyuridine and a Texas Red-conjugated secondary antibody. (c) The overlay demonstrates colocalization (yellow) of NS5A and nascent viral RNA. Each image covers an area of 37 by 37 μm. Negative controls included identically treated naive Huh-7 cells.