Establishment of infectious HCV virion-producing cells with newly designed full-genome replicon RNA

Abstract

Hepatitis C virus (HCV) replicon systems enable in-depth analysis of the life cycle of HCV. However, the previously reported full-genome replicon system is unable to produce authentic virions. On the basis of these results, we constructed newly designed full-genomic replicon RNA, which is composed of the intact 5'-terminal-half RNA extending to the NS2 region flanked by an extra selection marker gene. Huh-7 cells harboring this full-genomic RNA proliferated well under G418 selection and secreted virion-like particles into the supernatant. These particles, which were round and 50 nm in diameter when analyzed by electron microscopy, had a buoyant density of 1.08 g/mL that shifted to 1.19 g/mL after NP-40 treatment; these figures match the putative densities of intact virions and nucleocapsids without envelope. The particles also showed infectivity in a colony-forming assay. This system may offer another option for investigating the life cycle of HCV.

Fig. 1

Confirmation of “divided open reading frame carrying” (dORF) replicon cells. (A) Schematic representations of replicon RNAs used in this study. All the replicon constructs contained inserts just after the T7 promotor. UTR, untranslated region; NS, non-structural protein; neo, neomycin phosphotransferase II; EMCV, encephalomyocarditis virus; IRES, internal ribosomal entry site; FMDV, foot-and-mouth disease virus; bla, beta-lactamase. (B) Northern blot analysis. A 10-μg amount of total RNA from each cell sample was loaded. Subgenomic replicon RNA: 10⁸ copies of in vitro-generated subgenomic RNA. Numbers below the lanes are the HCV copy number per microgram of total RNA. Huh-7 cell, subgenomic replicon cell, dORF replicon cell #1, #2, dORF bla replicon cell #1, #2. (C) Western blot analysis. A 10-μg amount of each cell lysate was loaded. Huh-7 cell, Huh-7-JFH1: Huh-7 cell transfected with JFH1 viral RNA, subgenomic replicon cell, full-genome replicon cell, dORF replicon cell #1, #2, dORF bla replicon cell #1, #2

Fig. 2

Density gradient analysis of supernatants. Culture supernatants were treated with RNaseA and loaded directly onto a sucrose density gradient without treatment (open square) or after NP-40 treatment (filled triangle). Quantification of HCV RNA in each fraction of supernatant from the subgenomic replicon (A) and dORF replicon (B). Analysis of concentrated culture supernatant from the subgenomic replicon (C) and dORF replicon (D). Concentrated culture supernatant from the full-genome replicon NNC#2 was also analyzed (E). Quantification of HCV core protein in each fraction of supernatant from the dORF replicon (F)

Fig. 3

Electron microscopy analysis of virus-like particles. The core-protein-rich fraction collected from the density gradient was further concentrated by ultracentrifugation and observed by scanning electron microscopy (A). The same fraction attached to formvar-coated grids was incubated with rabbit anti-E2 RR6 antibody, treated with goat anti-rabbit IgG coupled to 10-nm colloidal gold, negatively stained with uranyl acetate, and then examined by transmission electron microscopy (B)

Fig. 4

Infectivity of supernatants from various replicon cells. Colonies of cells infected with the indicated supernatant. Numbers shown below the plates are the average of a total of four plates per condition (A). Inhibition of infection by anti-CD81 antibody. Cured cell K4 cells (No.2 in Fig. 4A) were treated with mouse IgG1 as the negative control or anti-CD81 before infection (B)

Fig. 5

Northern blot analysis of colonies formed after infection. 10⁷, 10⁹: amounts of in vitro-generated subgenomic replicon RNA loaded. Numbers below the lanes are the HCV copy number per μg of total RNA (A). Huh-7 cells, subgenomic replicon cells, dORF replicon cell #2, dORF bla replicon cell #2, subgenomic replicon sup: colony from cells transduced with subgenomic replicon supernatant, colony No.1, 2 of dORF replicon #2 sup: colonies from cells infected with dORF replicon #2 supernatant, colony No.1, 2, and 3 of dORF bla replicon #2 sup: colonies from cells infected with dORF bla replicon #2 supernatant. Western blot analysis of colonies formed after infection (B). The order of the lanes is identical to that for the northern blot, except for the dORF and dORF bla replicons, which represent two clones in this figure

Fig. 6

Detection of beta-lactamase activity in dORF replicon cells. Parental dORF bla replicon #2 cell (A) and colony no. 3 cloned from cells infected with dORF bla replicon #2 cell supernatant (B). Blue fluorescence shows high beta-lactamase activity, indicating that the reporter gene functioned normally after infection