Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein

Abstract

The NS5A protein of hepatitis C virus (HCV) plays an important but undefined role in viral RNA replication. NS5A has been proposed to be a three-domain protein, and the crystal structure of the well-conserved amino-terminal domain I has been determined. The remaining two domains of NS5A, designated domains II and III, and their corresponding interdomain regions are poorly understood. We have conducted a detailed mutagenesis analysis of NS5A domains II and III using the genotype 1b HCV replicon system. The majority of the mutants containing 15 small (8- to 15-amino-acid) deletions analyzed were capable of efficient RNA replication. Only five deletion mutations yielded lethal phenotypes, and these were colinear, spanning a 56-amino-acid region within domain II. This region was further analyzed by combining triple and single alanine scanning mutagenesis to identify individual residues required for RNA replication. Based upon this analysis, 23 amino acids were identified that were found to be essential. In addition, two residues were identified that yielded a small colony phenotype while possessing only a moderate defect in RNA replication. These results indicate that the entire domain III region and large portions of domain II of the NS5A protein are not required for the function of NS5A in HCV RNA replication.

FIG. 1.

(A) Schematic representation of the hepatitis C virus NS5A protein. NS5A has been proposed to consist of three domains (labeled domains I, II, and III) with domains separated by low-complexity sequences (labeled LCS I and II). The position of the amino-terminal amphipathic helix membrane anchor is shown (labeled helix). The four cysteine residues corresponding to the NS5A zinc coordination site are indicated by gray bars in the domain I region of the protein. The location of the previously described Δ47 (Delta 47) deletion is shown. The amino (N) and carboxyl (C) termini of NS5A are shown. All numbers refer to the amino acid number of the Con1 isolate of NS5A 1b, with the amino terminus of the mature protein designated as amino acid one. (B) Schematic of deletions within domains II and III of NS5A. The domain II and III regions of NS5A are shown in greater detail from panel A. Deletions are labeled A through O. Numbers above each deletion letter indicate the amino acids deleted, and the black bars approximate the locations of the deletions on the domain organization model of NS5A. Deletions indicated with solid black letters are deletions that are dispensable for HCV RNA replication. Deletions labeled with outline block white letters are not viable in the HCV replicon system. The six carboxyl-terminal amino acids of NS5A are indicated by a black block, indicating that these residues were not tested for their role in RNA replication in this work. The designations for domains II and III and Δ47 are as described above for panel A. (C) RNA replication fitness of replicons bearing deletions within NS5A domains II and III. Graph presenting transduction efficiency of replicon RNAs in units of CFU/μg of RNA. The pol⁻ designation refers to a Con1/SG-neo replicon RNA bearing a nonfunctional RNA-dependent RNA polymerase sequence that serves as a negative control for replication experiments. The transduction efficiency of the parental Con1/SG-neo GIT replicon (labeled GIT) is shown. The letters A through O on the abscissa refer to the analysis of the individual deletion mutants shown in panel B. The dashed line indicates the limit of detection of the assay.

FIG. 2.

(A) Sequence of the B deletion from Fig. 1B showing additional alanine scanning mutagenesis performed. Black letters within the gray shaded box correspond to the single-letter code amino acid sequence of deletion B. Numbers above the shaded box refer to the amino acid number of the NS5A protein. White outline block letters indicate the amino acid substitutions introduced at the positions indicated by the black horizontal brackets. Bold black letters under the horizontal brackets refer to the designation for the clone containing the amino acid substitution above the brackets. For example, the boldface letter B1 refers to a triple alanine mutation of R294, E295, and V296. (B) Graph of transduction efficiency for each replicon construct shown in panel A. Graph labeling and controls are as described in the legend to Fig. 1C. The letters B1 through B4 correspond to the designations in panel A.

FIG. 3.

(A) Sequence of the C deletion from Fig. 1B showing additional alanine scanning mutagenesis performed. Numbering and clone designation conventions are as described in the legend to Fig. 2A. C1, C2, and C3 refer to triple alanine mutation of the designated residues. C3-1 through C3-4 designate individual alanine or glycine mutations of residues above the black brackets. (B) Graph of transduction efficiency for each replicon construct shown in panel A. Graph labeling and controls are as described in the legend to Fig. 1C. The letters C1 through C3 and C3-1 through C3-4 correspond to the designations in panel A.

FIG. 4.

(A) Sequence of the D deletion from Fig. 1B showing additional alanine scanning mutagenesis performed. Numbering and clone designations are as described in the legends to Fig. 2A and 3A. (B) Graph of transduction efficiency for each replicon construct shown in panel A. Graph labeling and controls are as described in the legend to Fig. 1C. Asterisks above column bars indicate replicons that showed a small colony phenotype in the replication assay. (C) Representative colony phenotypes for normal and small colony mutants. Crystal violet-stained colonies following G418 selection. GIT represents 10⁴ cells plated in a 100-mm dish for the parental strain GIT replicon. D2-1 represents 10⁶ cells plated in a 100-mm dish of a small colony phenotype mutant to give approximately the same number of colonies as the GIT control.

FIG. 5.

(A) Sequence of the E deletion from Fig. 1B showing additional alanine scanning mutagenesis performed. Numbering and clone designations are as described in the legends to Fig. 2A and 3A. (B) Graph of transduction efficiency for each replicon construct shown in panel A. Graph labeling and controls are as described in the legend to Fig. 1C.

FIG. 6.

(A) Sequence of the F deletion from Fig. 1B showing additional alanine scanning mutagenesis performed. Numbering and clone designations are as described in the legends to Fig. 2A and 3A. (B) Graph of transduction efficiency for each replicon construct shown in panel A. Graph labeling and controls are as described in the legend to Fig. 1C.

FIG. 7.

Secondary analysis of selected mutants. (A) Transient RNA replication analysis of selected mutants by real-time reverse transcriptase PCR assay of HCV RNA. Values shown are normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA and expressed as the number of HCV RNA copies per 100 ng of total RNA. Only the replication-competent GIT RNA was capable of producing detectable RNA replication in this experiment. (B) Transient protein expression of various NS5A mutants analyzed by Western blotting with an anti-NS5A monoclonal antibody. The two phosphoforms of NS5A are clearly visible.

FIG. 8.

(A) Schematic representation of the hepatitis C virus NS5A protein. Labeling is essentially as described in the legend to Fig. 1A. (B) Enlargement of the domain II region of NS5A indicating the locations of residues determined to be important in RNA replication. The large dark gray box from 294 to 303 indicates the position of the lethal deletion B1. Additional small dark gray boxes indicate the locations of other residues that produced lethal or near lethal phenotypes in the replicon system assays. Light gray boxes designate residues that produced a small colony phenotype in the replicon system assay yet still produced significant levels of colony formation. Asterisks indicate positions that yielded small colony phenotypes independent of G418 transduction efficiency. Numbers labeling these boxes refer to the amino acid number of the highlighted residue in the NS5A protein. (C) Enlargement of the region in domain II containing residues analyzed in the context of the S2204I replicon. Residues shaded in dark gray or light gray designate replication phenotypes as described above for panel B. Asterisks indicate replicons producing a small colony phenotype. Regions not highlighted in gray were not analyzed in the S204I replicon.