Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain

Abstract

Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.

FIG. 1.

Amino acid substitutions affecting highly conserved serine residues in NS5A and their impact on RNA replication and hyperphosphorylation. (A) The structure of the subgenomic HCV replicon used for transient RNA replication analyses is shown in the upper panel. The HCV 5′ and 3′ NTRs are indicated by thick lines, the PV IRES (P-I) directing the translation of the reporter gene coding for firefly luciferase (FF-luc) is depicted as an oval. The EMCV IRES (E-I) directs the translation of the HCV NS3 to -5B coding sequence. The positions of cell culture adaptive mutations in NS3 are indicated by asterisks. The lower panel shows a schematic representation of NS5A indicating the major phospho acceptor site (S2194, grey arrow) and sites required for hyperphosphorylation (S2197, S2201, and S2204, black arrows). Regions important for basal phosphorylation are shaded in grey. Serine residues highly conserved between multiple HCV isolates and genotypes were grouped into three clusters, with cluster 1 including the sites involved in hyperphosphorylation as well as the major phospho acceptor site. Amino acid substitutions generated in this study are indicated below the scheme. In the case of cluster 1, in addition to alanine substitutions, selected replacements by glutamic acid were analyzed. In the case of cluster 3, substitutions affecting 3, 4, or 7 aa residues were combined (mutants 3-3, 3-4, and 3-7, respectively). The deletion removing cluster 3 completely is indicated below (Δcl3). Numbers refer to the positions within the polyprotein of the HCV Con1 isolate (EMBL database accession no. AJ238799). (B) Result of transient replication assays. Mutants specified below the graph were transfected into Huh-7 cells. Replication was measured by determining the relative light units (RLU) at 48 h postelectroporation and normalization for transfection efficiency by using the luciferase activity measured 4 h after transfection. The value determined with the parental replicon (parent.) was set as 100% and used as a reference to normalize the replication of all other replicons. The replicon carrying an inactivating mutation in the NS5B RNA polymerase (GND) served as a negative control. Values are means and standard deviations from at least three independent experiments, each measured at least in duplicate. (C) Phosphorylation analysis of NS5A by using the vaccinia virus T7 hybrid system. NS3-to-NS5B polyprotein fragments carrying a given mutation in NS5A were transfected into Huh-7 cells, and proteins were radiolabeled by using [³²P]orthophosphate. NS5A proteins were isolated by immunoprecipitation and separated by SDS-PAGE (10% polyacrylamide). Mock-transfected cells served as a negative control (lane 18). The two phosphoprotein variants p56 and p58 are marked.

FIG. 2.

Effect of adaptive mutations in NS4B, NS5A, and NS5B on hyperphosphorylation of NS5A. T7-based plasmids encoding NS3 to -5B polyproteins and carrying the mutations specified above the lanes were transfected into Huh-7 cells that stably express T7 RNA polymerase. Proteins were radiolabeled metabolically with [³⁵S]methionine and [³⁵S]cysteine, and NS5A proteins were isolated by immunoprecipitation. One-half of the immunocomplex was treated with λ-phosphatase (+), the other half was mock treated (−), and both samples were separated by 10% polyacrylamide (A) or 8% polyacrylamide (B) SDS-PAGE. Mock-transfected cells served as a negative control. The positions of p56 and p58 are indicated to the right. Values below the lanes depict replication efficiencies of the corresponding mutants as reported by Lohmann et al. (28). The parental replicon carrying the two weakly adaptive mutations in NS3 was set as 1.

FIG. 3.

Mutations at the C terminus of NS5A reduce basal phosphorylation and have only minor impact on RNA replication. (A) Substitutions affecting single serine residues in cluster 2 or multiple serine residues in cluster 3, as well as a deletion of cluster 3 (Δcl3), were analyzed in a transient replication assay as described in the legend to Fig. 1. The parental replicon (parent.), carrying two weakly adaptive mutations in NS3 and the inactive mutant (GND) served as a positive and negative control, respectively. All values were measured 48 h after electroporation and, after correction for their transfection efficiency, normalized with respect to the parental replicon, which was set 100%. (B) NS3 to -5B constructs carrying the mutations specified above the lanes were transiently expressed in Huh-7 cells by using the vaccinia virus T7 hybrid system. Proteins were radiolabeled with [³⁵S]methionine and [³⁵S]cysteine (left panel) or [³²P]orthophosphate (right panel) for 6 h, and NS5A proteins were isolated from cell lysates by immunoprecipitation. In the case of the ³⁵S-labeled proteins, one-half of the samples was treated with phosphatase from bacteriophage λ (+) whereas the other half was mock treated (−) prior to SDS-PAGE (10% polyacrylamide).

FIG. 4.

Insertion of eGFP or dsRed, but not the luciferase from R. reniformis, into the C-terminal deletion of NS5A results in viable subgenomic replicons. (A) Schematic representation of the replicon indicating the positions of three cell culture adaptive mutations (E1202G, T1280I, and K1846T). Reporter genes were inserted into the C-terminal deletion (Δcl3). (B) Time course of a transient replication assay. Transfected Huh-7 cells were harvested 4, 24, 48, and 72 h after electroporation, and luciferase activities in the cell lysates were determined. Values obtained after 24 (light grey bars), 48 (dark grey bars), and 72 (black bars) h posttransfection after normalization for transfection efficiency by using the 4-h values are shown. The replicon carrying no insertions in NS5A and the inactive replicon (GND) served as positive and negative controls, respectively. (C) Analysis of polyprotein processing by using the vaccinia virus/T7 hybrid system. NS3 to -5B proteins carrying given insertions in NS5A were expressed in Huh-7 cells in parallel with the parental polyprotein (4B adapt), and 4 h after radiolabeling with [³⁵S]methionine and [³⁵S]cysteine, given HCV proteins were analyzed by immunoprecipitation and SDS-PAGE (10% polyacrylamide). (D) Fluorescence analysis of the NS5A-eGFP fusion protein in the context of an NS3 to -5B polyprotein. Huh-7 cells constitutively expressing T7 RNA polymerase were transfected with the expression vector only (mock), the vector containing only the eGFP gene (pTM/eGFP), or the vector containing the NS3 to -5B polyprotein with the NS5A-eGFP fusion protein. Cells were fixed 6 h after transfection with 3% paraformaldehyde and analyzed by fluorescence microscopy.