The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus

Abstract

Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro. Thus, we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than for modulating the enzymatic functions of NS3-4A.

FIG 1

Amino acid alignment of the linker regions from several HCV isolates. Based on the structure of NS3, we define the linker region as the residues between NS3 P182 and P194 that connect the HCV NS3 N-terminal protease and C-terminal helicase domains (see PDB 3O8C) (14). Positions targeted for mutagenesis are indicated with stars. Conserved residues are represented as dots.

FIG 2

Replication and infectivity of HCV NS3 linker mutants in Huh-7.5 cells expressing core-NS2. (A) Diagram of the GLuc replicon construct for monitoring HCV replication and design of the replication and released-infectivity experiments. Triangles indicate sites cleaved by NS3-4A, and the circle indicates a signal peptidase cleavage site at the N terminus of the GLuc gene. IRES, internal ribosome entry site; EMCV, encephalomyocarditis virus; HCVcc, cell culture–derived HCV. (B) Replication of NS3 linker mutants in the GLuc-JFH1 replicon context in Huh-7.5[core-NS2] cells, monitored as previously described (25). GNN is a mutation that disrupts the active site of the NS5B RNA-dependent RNA polymerase. (C) Released infectivity measurements of the media collected at each time point. The media tested for replication as described for panel B were used to infect naive cells, and then fresh media were assayed for secreted luciferase 48 h postinfection. For panels B and C, the luciferase activity data (y axis) at each of the time points—24, 48, 72, and 96 h—are plotted in succession from left to right (x axis) for the NS3 mutants. RLU, relative light units. (D) Intracellular infectivity of lysate from infected cells. Cell lysates were used to infect naive cells, and then fresh media were assayed for secreted luciferase 48 h postinfection. “Double” refers to P190G/P191G, “1xGins” to “4xGins” refer to 1 to 4 glycines inserted after P182, and “1xDel” to “4xDel” refer to deletion of 1 to 4 residues between F184 and N187 (starting with F184). WT, wild type. Each data point represents the average of the results of three experiments, and the error bars represent the standard deviations.

FIG 3

NS3 linker mutations do not disrupt polyprotein processing. (A) The vaccinia virus T7 RNA polymerase expression system described by Fuerst et al. (26) was used to drive HCV polyprotein expression to test for levels of NS3 expression and proper polyprotein processing of each NS3-4A linker mutant replicon construct. NS3 P190/P191 was the only replicon construct to display lower NS3 expression levels. The image is a composite of two gels. (B) The RET-S1 peptide cleavage assay described by Taliani et al. (27) was used to evaluate the serine protease activity of recombinantly expressed NS3-4A linker mutants and three NS3 controls—NS3, NS3hel[ΔN166] (amino acids [aa] 167 to 631), and NS3hel[ΔN188] (aa 189 to 631). Steady-state protease rates were calculated between the first 10 and 40 s of the reaction. Each column represents the average of the results of three experiments, and the error bars represent the standard deviations.