Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein

Abstract

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.

FIG. 1

Schematic representation of the expression plasmids used in this study. The organization of the nonstructural region of the viral polyprotein is shown at the top. Each numbers above the bar indicates the position of the N-terminal amino acid of the following protein within the viral polyprotein. The horizontal line indicates the 3′ UTR. R, ribozyme sequence. The vertical bars indicate the boundaries between the different proteins. The HCV polyprotein portions expressed by the different constructs are shown below. myc, epitope with the amino acid sequence EQKLISEEDL; A, the mutation S1165A in NS3.

FIG. 2

Characterization of minimal requirements for NS5A hyperphosphorylation. Hep3B cells were transfected with the indicated constructs and labelled with either [³⁵S]methionine (A and B) or [³²P]orthophosphate (C) for 3 h. NS5A was immunoprecipitated with NS5A antiserum, and protein was loaded onto an SDS–7.5% polyacrylamide gel. (B and C) Immunoprecipitated proteins were incubated with (+) or without (−) λ-phosphatase as described in Materials and Methods prior to loading for SDS-PAGE. NS5A p56 and p58 are indicated by arrows on the right; the sizes of molecular weight marker proteins (M) are indicated on the left. BK, HCV proteins derived from the BK strain; H, HCV proteins derived from the H strain.

FIG. 3

Time course of NS5A synthesis and phosphorylation. (A) Pulse-labelling of NS5A expressed from the construct pcD3-5A. Hep3B cells were transfected with pcD3-5A and labelled with [³⁵S]methionine for the indicated times. (B) Pulse-chase analysis of NS5A phosphorylation. Cells were transfected as described above, labelled for 15 min with [³⁵S]methionine, and chased for the indicated times as described in Materials and Methods. NS5A was immunoprecipitated with NS5A antiserum, and proteins were loaded onto an SDS–7.5% polyacrylamide gel. NS5A p56 and p58 proteins as well as the precursor proteins NS3-5A, NS4A-5A, and NS4B-5A are indicated by arrows on the right. M, molecular weight marker proteins.

FIG. 4

Analysis of NS5A hyperphosphorylation with NS5A and NS3 expressed in trans. Cells were cotransfected with the following plasmids: lane 2, pcD3-5Amyc alone; lanes 3, 4, and 9, pcD3-5A plus pcD5Amyc; lanes 5 and 10, pcD4A-5A plus pcD3-4A; lanes 6 and 11, pcD4B-5A plus pcD3-4A; lanes 7 and 12, pcD3-4B plus pcD5A. Proteins were immunoprecipitated with Myc-specific monoclonal antibodies (α-myc) NS5A antiserum (α-NS5A), or with NS3 antiserum (α-NS3) and loaded onto an SDS–7.5% polyacrylamide gel. NS5A p56, p58, and NS3 are indicated by arrows on the right; p56-Myc and p58-Myc are indicated by arrows on the left. M, molecular weight marker proteins.

FIG. 5

NS4A/B is required in cis for the formation of p58. Cells were cotransfected with the indicated constructs. (A) Proteins were immunoprecipitated with NS5A antiserum (α-NS5A) and loaded onto an SDS–7.5% polyacrylamide gel. (B) Proteins were immunoprecipitated with NS4 antiserum (α-NS4) and loaded onto an SDS–15% polyacrylamide gel. Arrows on the right indicate HCV-specific proteins. 3/5A, pcD3/5A, 3-4A/5A, pcD3-4A/5A; 3/4B-5A, pcD3/4B-5A; 4A, pCITE4A; 4B, pCITE4B; M, molecular weight marker proteins.

FIG. 6

NS5A hyperphosphorylation requires an active NS3 protease. Cells were transfected with the indicated plasmids in the presence (+) or absence (−) of plasmid pcD3-4A. Proteins were immunoprecipitated with NS5A (α-5) or NS3 (α-3) antiserum and loaded onto an SDS–7.5% polyacrylamide gel. HCV-specific proteins are indicated by arrows. The positions of NS5A p56 and p58 expressed from the control plasmid pcD3-5A are indicated on the left. M, molecular weight marker proteins.

FIG. 7

Comparison of NS3/NS4A complex stability. Cells were transfected with the indicated plasmids, and proteins were immunoprecipitated under native conditions as described in Materials and Methods with either NS3 (α-NS3) or NS4 (α-NS4) antiserum. Immunoprecipitated proteins were then loaded onto an SDS–13.5% polyacrylamide gel. NS4, NS3 expressed from pCITE(SX), and NS3 expressed from pcD3-5A are indicated by arrows on the right. M, molecular weight marker proteins.