Characterization of the Interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19)

J Biol Chem. 2011 Apr 1;286(13):11814-24. doi: 10.1074/jbc.M110.210179. Epub 2011 Feb 4.

Abstract

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 5' Untranslated Regions / physiology
  • Animals
  • Hantavirus / physiology*
  • Hantavirus Infections / genetics
  • Hantavirus Infections / metabolism
  • Hantavirus Infections / therapy
  • HeLa Cells
  • Humans
  • Nucleocapsid Proteins / genetics
  • Nucleocapsid Proteins / metabolism*
  • Peptide Chain Initiation, Translational / physiology*
  • Protein Binding
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • Rabbits
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism*
  • Ribosome Subunits, Small, Eukaryotic / genetics
  • Ribosome Subunits, Small, Eukaryotic / metabolism
  • Virus Replication / physiology*

Substances

  • 5' Untranslated Regions
  • Nucleocapsid Proteins
  • RNA, Viral
  • Ribosomal Proteins
  • ribosomal protein S19
  • RNA-Dependent RNA Polymerase