The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly

Abstract

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.

FIG. 1.

Phenotypes of NS4A mutants. (A) Jc1/GLuc2A reporter construct used in the present study. The sequence of the NS4A C-terminal acidic domain is shown in single-letter amino acid code. Closed circles represent signal peptidase cleavages, the open circle represents a signal peptide peptidase cleavage, the open arrowhead represents NS2-3 cysteine autoprotease cleavage, and closed arrowheads represent NS3-4A serine protease cleavage sites. (B) Replication of NS4A mutants. Cells were transfected with each indicated mutant, and media were collected at 24, 48, 72, and 96 h posttransfection. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation of the mean. (C) Released infectivity of NS4A mutants. Relative infectivity levels in the media from panel B were quantified by infecting naive cells and monitoring secreted GLuc activity. (D) RNA release of NS4A mutants. The amount of HCV RNA present in the media at 72 h posttransfection was determined by quantitative RT-PCR as described in Materials and Methods. Values represent average RNA quantities from at least three independent transfections. Error bars represent the standard deviation of the mean. (E) Intracellular infectivity of NS4A mutants. Cells were lysed at 48 h posttransfection and used to infect naive cells as described in Materials and Methods. Values represent average relative infectivity measurements from at least three independent transfections. Error bars represent the standard deviation of the mean.

FIG. 2.

Polyprotein processing of NS4A mutants. Parallel cultures of Huh-7.5 cells were infected with vaccinia virus vTF7-3 for 1 h, transfected with the indicated Jc1/GLuc2A cDNA clones, and lysed at 24 h postinfection. Proteins were separated by SDS-PAGE and detected by Western blotting as described in Materials and Methods.

FIG. 3.

NS4B I7F partially suppresses the replication defect of subgenomic replicons containing the NS4A F48A mutation. (A) The pYSGR-JFH construct used in the present study and the workflow to identify revertants or suppressor mutants. HCV polyprotein processing sites are annotated as in Fig. 1. (B) pYSGR-JFH/GLuc reporter construct used in this study and workflow of our reporter virus assay. RNA was electroporated into Huh-7.5 cells, and culture medium was harvested at various times posttransfection. Secreted GLuc activity was assayed as detailed in Materials and Methods. (C) Stable replication phenotypes of NS4A F48A and its suppressor NS4B I7F. Colony-forming activity was measured for NS4A F48A and the suppressor NS4B I7F in the context of pYSGR-JFH. Values represent CFU calculated from three independent transfections. Error bars represent the standard deviation from the mean. (D) Transient replication phenotypes of NS4A F48A and its suppressor NS4B I7F. Luciferase activity was measured for NS4A F48A and the suppressor NS4B I7F in the context of pYSGR-JFH/GLuc. Huh 7.5 cells were transfected with the indicated mutants, and media were collected at 6, 12, 24, 48, 72, and 96 h posttransfection. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation from the mean.

FIG. 4.

NS4B I7F partially suppresses the replication defect of infectious genomes containing the NS4A F48A mutation. (A) Replication phenotypes of NS4A F48A and its suppressor NS4B I7F. Huh7.5 cells were transfected with each indicated mutant, and medium was collected at 24, 48, 72, and 96 h posttransfection. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation of the mean. (B) Infectivity phenotypes of NS4A F48A and its suppressor NS4B I7F. The infectivity levels in the medium from panel A were quantified by infecting naive cells and monitoring secreted GLuc activity.

FIG. 5.

NS3 Q221L suppresses the assembly defect of subgenomic replicons containing the NS4A K41A mutation. (A) Workflow used to identify revertants or suppressor mutants via trans-packaging. RNAs were electroporated into Huh-7.5[core-NS2] cells, and the cell culture media were collected at each cell passage. The media were used to infect naive cells and productively infected cells were selected with G418. (B) trans-Packaging infectivity phenotypes of NS4A K41A and variants containing NS3 Q221L or NS4B S230P. Colony-forming activity was measured for NS4A K41A and the suppressors NS3 Q221L and NS4B S230P in the context of pYSGR-JFH. Values represent the CFU calculated from three independent transfections. Error bars represent the standard deviation from the mean. (C) Replication phenotypes of NS4A K41A and variants containing NS3 Q221L or NS4B S230P. The luciferase activity was measured for NS4A K41A and the suppressor NS3 Q221L and NS4B S230P in the context of pYSGR-JFH/GLuc. Huh-7.5[core-NS2] cells were electroporated with each indicated mutant genome and media were collected at 6, 12, 24, 48, 72, and 96 h posttransfection. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation of the mean. (D) trans-Packaging infectivity phenotypes of NS4A K41A and variants containing NS3 Q221L or NS4B S230P. The infectivity levels in the media from panel C were quantified by infecting naive cells and measuring secreted GLuc activity.

FIG. 6.

NS3 Q221L suppresses the assembly defect of full-length genomes containing the NS4A K41A mutation. (A) Replication phenotypes of NS4A K41A and variants containing NS3 Q221L or NS4B S230P. Huh-7.5 cells were transfected with each indicated mutant, and media were collected at 24, 48, 72, and 96 h posttransfection. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation of the mean. (B) Relative infectivity phenotypes of NS4A K41A and variants containing NS3 Q221L or NS4B S230P. The levels of infectivity levels in the media from panel A were quantified by infecting naive cells and monitoring secreted GLuc activity. Values represent the time course of secreted GLuc activity for each transfection, expressed as an average of at least three independent transfections. Error bars represent the standard deviation of the mean.

FIG. 7.

Enzymatic activities of WT and mutant forms of NS3-4A. (A) Serine protease activities of WT (black), NS4A K41A (red), NS3 Q221L (blue), and NS4A K41A + NS3 Q221L (purple). The data were collected at 1-s intervals as described in Materials and Methods. Values represent averages from three independent experiments, and a buffer control (no NS3-4A) is shown in green. (B) RNA unwinding activities of WT (black), NS4A K41A (red), NS3 Q221L (blue), and NS4A K41A + NS3 Q221L (purple) forms of NS3-4A. Values represent the average of three independent experiments. Error bars represent the standard deviation from the mean. (C) RNA-stimulated ATPase activities of WT (black), NS4A K41A (red), NS3 Q221L (blue), and NS4A K41A + NS3 Q221L (purple) forms of NS3-4A. Values represent the average of three independent experiments. Error bars represent standard deviation from the mean.