Genetic interactions between hepatitis C virus replicons

Abstract

To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

FIG. 1.

HCV genome and replicon organization. (A) Schematic representation of HCV genomic RNA. The amino-terminal one third of the polyprotein, translated through the action of an IRES, encodes the HCV structural proteins, including the capsid protein, core, and the envelope glycoproteins E1 and E2. The remainder of the polyprotein encodes the viral nonstructural proteins NS2, -3, -4A, -4B, -5A, and -5B. (B) Organization of the prototype HCV subgenomic replicon. The majority of the structural region of the Con1 HCV polyprotein was replaced with the neomycin phosphotransferase gene (neo), cloned as a fusion with the first 16 amino acids of the core protein, such that translation is driven by the HCV IRES. Translation of the remaining NS proteins is mediated by the encephalomyocarditis virus IRES element (EMCV). (C) Organization of alternatively selectable subgenomic replicons tested in this study. Each drug resistance gene was cloned as fusions to the core protein, as in the neo replicon. (D) Location and description of replicon mutations utilized in this study.

FIG. 2.

(A) Comparison of neo- and bsd-resistant replicon efficiencies. Huh7 cells were electroporated with 1 μg of indicated neo- or bsd-resistant replicon RNA. Equal numbers of cells (10⁶) were plated and selected in media containing the pertinent drug. Shown are Coomassie-stained plates following 2 to 3 weeks of selection. (B) Schematic representation of sequential replicon transfections. Huh7 cells previously transfected and selected for maintenance of A2199T neo replicons were transfected with 1 μg of S2204I bsd or, as a negative control, the replication-incompetent Con1 S2204I Pol-fs. (C) Following transfection, 10⁶ cells were plated and selected with the indicated drugs. Shown are Coomassie-stained plates following 2 to 3 weeks of selection.

FIG. 3.

Simultaneous replicon transfections into naïve Huh7 and Huh7.5 cells. (A) Schematic representation of simultaneous transfection of naïve Huh7 cells with mixtures of neo and bsd replicons. (B) Naïve Huh7 cells were electroporated with 1 μg each of indicated combinations of neo and bsd replicons, and 10⁶ cells were plated and selected in media containing the indicated drug(s). Shown are Coomassie-stained plates following 2 to 3 weeks of selection. (C) Example of transcomplementation assays evaluated in Huh7.5 cells. Naïve Huh7.5 cells were electroporated with 1 μg each of indicated combinations of neo and bsd replicons. Cells (10⁶) were plated and selected in media containing the indicated drug(s).

FIG. 4.

Replicon competition experiments. (A) G418-resistant colonies derived following cotransfection of naïve Huh7 cells with a fixed amount (0.5 μg) of the Con1 A2199T neo replicon and, as competitor, increasing amounts of the Con1 A2199I bsd replicon. To maintain a constant amount of RNA (total of 8.5 μg) per transfection, the replication-incompetent Con1 S2204I Pol⁻ bsd RNA was added as filler. Transfections were plated (10⁶ cells) and selected in media containing G418. Following selection, plates were stained and counted. Error bars represent the standard deviations across three separate transfections. (B) Various bsd replicons were cotransfected in 10-fold excess as competitor against a fixed amount (0.5 μg) of A2199T neo replicon RNA. G418-resistant colony numbers derived from these transfections are reported. Error bars represent the standard deviation across three separate transfections.