Interaction between influenza A virus nucleoprotein and PB2 cap-binding domain is mediated by RNA

PLoS One. 2020 Sep 28;15(9):e0239899. doi: 10.1371/journal.pone.0239899. eCollection 2020.

Abstract

Influenza A virus controls replication and transcription of its genome through the tight regulation of interaction between the ribonucleoprotein (RNP) complex subunits. The helical scaffold of RNP is maintained by nucleoprotein (NP). Previous studies have revealed that NP interacts with both PB2 N-terminal and C-terminal regions, with both regions sharing similar affinity to NP as revealed in co-immunoprecipitation assay. Our work here suggests that the interaction between NP and PB2 N-terminal region lies in the cap-binding domain (residue 320-483). By co-immunoprecipitation assay, the interaction was found to involve RNA. On the other hand, the cap-binding activity was not essential in the interaction. As shown by the NHS pull-down assay, a specific RNA sequence was not required. Among the cap-binding domain, residues K331 and R332 of PB2 play a role in RNP function so that polymerase activity was reduced when these residues were mutated, while K331 was found to be more crucial in the NP interaction. Collectively, our findings suggest a new binding mode between NP and PB2 which was mediated by RNA, and such interaction may provide a novel interacting site for influenza drug development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • HEK293 Cells
  • Host Microbial Interactions / genetics*
  • Humans
  • Immunoprecipitation
  • Nucleocapsid Proteins
  • Plasmids / genetics
  • Protein Binding / genetics
  • Protein Domains*
  • RNA, Messenger / metabolism*
  • RNA, Viral / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • RNA-Dependent RNA Polymerase / chemistry
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Transfection
  • Viral Core Proteins / genetics
  • Viral Core Proteins / metabolism*
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virus Replication / genetics

Substances

  • NP protein, Influenza A virus
  • Nucleocapsid Proteins
  • PB2 protein, Influenzavirus A
  • RNA, Messenger
  • RNA, Viral
  • RNA-Binding Proteins
  • Viral Core Proteins
  • Viral Proteins
  • RNA-Dependent RNA Polymerase

Grants and funding

PC Shaw received a grant (Project number: 14113114) from the Research Grants Council https://www.ugc.edu.hk/eng/rgc/ and a Theme-based Research Grant (Project number: T11-705/14N) from Hong Kong SAR. https://www.ugc.edu.hk/eng/rgc/funded_research/layman/theme/themes.html The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.