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.
Highlights
Structural studies of RNA-dependent RNA-polymerase (RdRp) from the human infecting influenza A strains in contrast, have been limited to date, partly due to difficulties to produce recombinant H3N2 or H5N1 polymerases
Using this strategy17, soluble and active heterotrimeric RNAdependent RNA-polymerases of influenza B virus (IBV) and bat influenza A virus could be produced, crystallized and the structure determined at high resolution5,6
The three genes of each heterotrimeric complex were combined with Tobacco Etch Virus (TEV) protease and Cyan Fluorescent Protein (CFP) in a single large open reading frame (ORF)
Summary
Structural studies of RdRp from the human infecting influenza A (human-IAV) strains in contrast, have been limited to date, partly due to difficulties to produce recombinant H3N2 or H5N1 polymerases. By applying our co-expression strategy, we show that the previously proposed PA-PB1-RanBP5 import complex can be purified, which we characterise using small angle X-ray scattering (SAXS). Biochemical analysis of this complex reveals a role for RanBP5 in hindering 5′-vRNA binding. Taken together, these data provide evidence for a cellular RdRp assembly pathway following a sequential and conditional process of assembly
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