Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5

Sci Rep. 2016 Apr 20;6:24727. doi: 10.1038/srep24727.

Abstract

The genome of influenza A virus (IAV) comprises eight RNA segments (vRNA) which are transcribed and replicated by the heterotrimeric IAV RNA-dependent RNA-polymerase (RdRp). RdRp consists of three subunits (PA, PB1 and PB2) and binds both the highly conserved 3'- and 5'-ends of the vRNA segment. The IAV RdRp is an important antiviral target, but its structural mechanism has remained largely elusive to date. By applying a polyprotein strategy, we produced RdRp complexes and define a minimal human IAV RdRp core complex. We show that PA-PB1 forms a stable heterodimeric submodule that can strongly interact with 5'-vRNA. In contrast, 3'-vRNA recognition critically depends on the PB2 N-terminal domain. Moreover, we demonstrate that PA-PB1 forms a stable and stoichiometric complex with host nuclear import factor RanBP5 that can be modelled using SAXS and we show that the PA-PB1-RanPB5 complex is no longer capable of 5'-vRNA binding. Our results provide further evidence for a step-wise assembly of IAV structural components, regulated by nuclear transport mechanisms and host factor binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation, Viral
  • Humans
  • Influenza A virus / genetics*
  • Influenza A virus / metabolism*
  • Influenza, Human / metabolism*
  • Influenza, Human / virology*
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Multimerization
  • Protein Subunits / metabolism*
  • RNA, Viral / genetics
  • RNA-Dependent RNA Polymerase / chemistry
  • RNA-Dependent RNA Polymerase / genetics*
  • RNA-Dependent RNA Polymerase / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • beta Karyopherins / metabolism*

Substances

  • IPO5 protein, human
  • Protein Subunits
  • RNA, Viral
  • Viral Proteins
  • beta Karyopherins
  • RNA-Dependent RNA Polymerase