The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication

J Virol. 2007 Sep;81(17):8905-18. doi: 10.1128/JVI.00937-07. Epub 2007 Jun 20.


Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / physiology*
  • Cell Line, Tumor
  • Circular Dichroism
  • DNA Mutational Analysis
  • Hepacivirus / genetics
  • Hepacivirus / physiology*
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Protein Folding
  • Protein Interaction Mapping
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Suppression, Genetic
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / physiology*
  • Virus Replication / genetics
  • Virus Replication / physiology*


  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • NS3 protein, hepatitis C virus
  • NS4A cofactor peptide, Hepatitis C virus
  • Viral Nonstructural Proteins
  • Viral Proteins
  • NS-5 protein, hepatitis C virus