The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus

Abstract

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.

FIG. 1.

Identification of amino acids in the NS4B C terminus that are critical for HCV RNA replication. (A) Schematic representation of the predicted topological arrangement of NS4B bound to the ER membrane. NS4B is predicted to contain four TMDs (numbered 1 to 4) that span the ER membrane, with the flanking N- and C-terminal ends located in the cytosol. Previously predicted features (α-helix between amino acids 6 and 29 and TMX) in the N-terminal region and the location of a nucleotide binding motif (NBM) are shown. Helices (H1 and H2), predicted by PSIPRED, that are located in the C-terminal domain are indicated. Below the cartoon, the amino acid sequence for the C-terminal region of NS4B encoded by strain JFH1 is presented. Amino acid residues that were replaced with alanine are underlined and numbered M1 to M15. Amino acids predicted to lie within helices H1 and H2 are overlined and shown in red. Asterisks denote conserved amino acids, and residues are numbered according to the N-terminal end of NS4B following cleavage from the polyprotein by the NS3/4A protease. (B) Schematic representation showing the Luc-JFH1_GFP SGR encoding NS5A-GFP and the position of the C-terminal end of NS4B containing mutations M1 to M15. (C) Huh-7 cells were electroporated with in vitro-transcribed RNA derived from wt Luc-JFH1_GFP, mutants M1_luc to M15_luc, and Luc-JFH1_GND. Extracts were prepared at 4, 24, 48, and 72 h to determine luciferase activity at each time point. Assays were performed in duplicate. RLU, relative light units.

FIG. 2.

The NS4B C terminus contains determinants for focus formation. (A) Schematic representations of the HCV polyproteins encoded by pCMV-JFH1_Poly and pCMV-JFH1_Poly-Δ4B, both of which express NS5A-GFP. (B) Huh-7 cells were transfected with the indicated pCMV-JFH1_Poly plasmids. At 20 h after transfection, cells were fixed and probed with NS4B antiserum R1063 by indirect immunofluorescence. Subcellular localization patterns for NS4B (red) and NS5A-GFP (green) are shown. Localization patterns for both proteins were separated into three phenotypic categories: proteins predominantly in foci (+), proteins either in foci or with a distribution consistent with an ER-like pattern (+/−), and proteins with a distribution consistent with an ER-like pattern (−). (C and D) Huh-7 cells were transfected with the pCMV-JFH1_Poly plasmids and either fixed (C) or used to prepare extracts (D) at 20 h posttransfection. In panel C, distribution patterns for NS5A-GFP were assessed as either predominantly in foci (left image) or at the ER membrane (right image); the graph shows the percentage of NS5A-GFP in both patterns for each construct. In panel D, cell extracts were probed with NS4B antiserum R1063 by Western blot analysis. The species corresponding to NS4B is indicated by an arrow. Scale bars in panels B and C represent 10 μm.

FIG. 3.

The NS4B C terminus influences mobility and hyperphosphorylation of NS5A. Huh-7 cells were transfected with the pCMV-JFH1_Poly plasmid series as well as constructs expressing DNase X and NS5A-GFP. At 24 h posttransfection, cells were either examined by FRAP analysis (A and C) or extracts were prepared for Western blot analysis (B and D). (A and C) Fluorescence recovery curves for NS5A-GFP expressed by the indicated constructs. The time at which regions of interest were photobleached is indicated by an arrow. Numbers in parentheses correspond to the percent fluorescence recovery and were calculated by dividing the fluorescence intensity measured at 120 s after bleaching by prebleach values. (B and D) Cell extracts were probed by Western blot analysis with NS5A antiserum. The positions of bands corresponding to NS5A-GFP are indicated by arrows. In panel B, the asterisk denotes hyperphosphorylated NS5A-GFP. In panel D, the upper and lower arrows indicate the positions of hyper- and hypophosphorylated NS5A-GFP, respectively.

FIG. 4.

trans-complementation of SGRs with defective NS4B in cells expressing a wt HCV replicon. (A) Schematic representation of mutant and wt (helper) SGRs. The positions of mutations at the C terminus of NS4B are indicated by arrows, and the location of GFP in the NS5A coding region for the mutant SGRs is also indicated. The helper SGR was constitutively expressed in 2/1 cells. (B) Huh-7 (i) and 2/1 (ii) cells were electroporated with in vitro-transcribed RNAs derived from wt Luc-JFH1_GFP, the indicated M_luc mutants, and Luc-JFH1_GND. Extracts were prepared at 4, 24, 48, and 72 h to determine luciferase activity at each time point. Assays were performed in duplicate. RLU, relative light units. (C and D) 2/1 cells were electroporated with in vitro-transcribed RNAs derived from wt Luc-JFH1_GFP, the indicated M_luc mutants, and Luc-JFH1_GND. At 72 h after electroporation, cells were fixed and probed with NS5A antiserum (red). Cells containing NS5A alone (indicated by arrowheads) represent expression from the wt SGR in 2/1 cells; cells containing both NS5A and NS5A-GFP (indicated by arrows) represent expression from both mutant and wt SGRs. In panel D, merged and unmerged images from cells expressing both NS5A (red) and NS5A-GFP (green) are shown. Nuclei were stained with DAPI. Scale bars represent 20 μm (C) and 10 μm (D).

FIG. 5.

trans-complementation of a mutant NS5A SGR in cells expressing a wt HCV replicon. (A) Schematic representation of mutant (Luc-JFH1_S232I) and wt (helper) SGRs. The position of the S232I mutation in NS5A is indicated. The helper SGR was constitutively expressed in 2/1 cells. (B) Huh-7 and 2/1 cells were electroporated with in vitro-transcribed RNA derived from Luc-JFH1_S232I. For controls, Huh-7 and 2/1 cells were electroporated with Luc-JFH1_GND and Luc-JFH1_GFP RNAs, respectively. Extracts were prepared at 4, 24, 48, and 72 h to determine luciferase activity at each time point. Assays were performed in duplicate. RLU, relative light units.

FIG. 6.

Reconstitution of active replication by combining defective SGRs in NS4B and NS5A. (A) Schematic representation of mutant SGRs in NS4B (M2_luc, M4_luc, etc.) and NS5A (Neo-JFH1_S232I). Positions of mutations at the C terminus of NS4B in the M_luc SGRs and in NS5A in Neo-JFH1_S232I are indicated. (B and C). Huh-7 cells were electroporated with combinations of in vitro-transcribed RNAs from the M_luc SGRs and from Neo-JFH1_S232I. For control purposes, Huh-7 cells were electroporated with Luc-JFH1_GFP and Neo-JFH1_S232I RNAs. For panel B, extracts were prepared at 4, 24, 48, and 72 h to determine luciferase activity at each time point. RLU, relative light units. Assays were performed in duplicate. For panel C, cells were fixed at 72 h after electroporation and probed with monoclonal antibody J2 to detect dsRNA (red). Cells were examined also for the presence of NS5A-GFP (green). Nuclei were stained with DAPI, and the scale bar represents 10 μm.

FIG. 7.

NS4B influences production of infectious HCV virions. (A) Schematic representation of JFH1 and J6-JFH1 constructs containing mutations at the C terminus of NS4B. For JFH1, the positions of 10 inserted mutations in NS4B are indicated by arrows. For J6-JFH1, the location of the M6 mutation in NS4B is shown. The gray region (core-NS2) indicates the coding sequences from strain HC-J6, which replaced the corresponding segment in strain JFH1. (B) Huh-7 cells were electroporated with wt and mutant (M_JFH1) JFH1 RNAs, and medium was removed from cells at 24, 48, and 72 h after electroporation. Naïve Huh-7 cells were inoculated with medium to determine TCID₅₀ values at 24, 48, and 72 h. Error bars indicate standard deviations. (C) Huh-7 cells were either electroporated with JFH1 and M6_JFH1 RNAs or infected with 1 ml of medium removed at 24, 48, and 72 h after electroporation. For electroporated samples, extracts were prepared at 24, 48, and 72 h, and for infected samples, extracts were prepared at 72 h after infection. Extracts were probed with NS5A antiserum by Western blot analysis, and bands corresponding to NS5A are indicated by arrows. (D) Huh-7 cells were electroporated with J6-JFH1 and M6_J6-JFH1 RNAs, and medium was removed from cells at 24, 48 and 72 h after electroporation. Naïve Huh-7 cells were inoculated with medium to determine TCID₅₀ values at 24, 48, and 72 h. (E) Huh-7 cells were either electroporated with J6-JFH1 and M6_J6-JFH1 RNAs or infected with 1 ml of medium removed at 24, 48, and 72 h after electroporation. Samples were prepared and extracts were analyzed as described for panel C.

FIG. 8.

Summary of the mutations that affect NS4B function and model for reconstitution of active replication from defective replicons. (A) Schematic representation of the C terminus of NS4B. The position and properties of each mutation that alters the behavior of NS4B are shown. NT, not tested. (B). Model for generation of sites with active RCs from SGRs with defects in NS4B and NS5A.