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. 2020 Apr 30;525(2):259-264.
doi: 10.1016/j.bbrc.2020.02.059. Epub 2020 Feb 19.

Cellular mRNA export factor UAP56 recognizes nucleic acid binding site of influenza virus NP protein

Andrew K Morris  1 , Zhen Wang  2 , Austin L Ivey  1 , Yihu Xie  1 , Pate S Hill  1 , Kevin L Schey  2 , Yi Ren  3
Affiliations

Cellular mRNA export factor UAP56 recognizes nucleic acid binding site of influenza virus NP protein

Andrew K Morris et al. Biochem Biophys Res Commun. .

Abstract

Influenza A virus nucleoprotein (NP) is a structural component that encapsulates the viral genome into the form of ribonucleoprotein complexes (vRNPs). Efficient assembly of vRNPs is critical for the virus life cycle. The assembly route from RNA-free NP to the NP-RNA polymer in vRNPs has been suggested to require a cellular factor UAP56, but the mechanism is poorly understood. Here, we characterized the interaction between NP and UAP56 using recombinant proteins and showed that UAP56 features two NP binding sites. In addition to the UAP56 core comprised of two RecA domains, we identified the N-terminal extension (NTE) of UAP56 as a previously unknown NP binding site. In particular, UAP56-NTE recognizes the nucleic acid binding region of NP. This corroborates our observation that binding of UAP56-NTE and RNA to NP is mutually exclusive. Collectively, our results reveal the molecular basis for how UAP56 acts on RNA-free NP, and provide new insights into NP-mediated influenza genome packaging.

Keywords: DEAD-Box ATPase; Host-pathogen interaction; Influenza A virus; RNA-Binding protein; mRNA nuclear export.

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Conflict of interest statement

Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
UAP56 features two binding sites for influenza NP protein. (A) Schematic representation of UAP56. Recombinant protein fragments that were used for GST pull-down assays are indicated. (B) In vitro GST pull-down assays with various GST-tagged UAP56 proteins and influenza A/PR/8 NP protein. (C) In vitro GST pull-down assays with GST-UAP56-NTEs and the monomeric NP-R416A mutant (NP*). All experiments (B-C) were repeated three times independently.
Figure 2.
Figure 2.
XL-MS analysis of the interaction between NP and UAP56-NTE. (A) Identified intermolecular cross-links between UAP56-NTE1-19 and NP* by the EDC crosslinker. Each line represents a unique crosslinking site. (B) Selected ion chromatograms for crosslinked peptides involving K214 of NP*. (C) Tandem mass spectrum for crosslinked peptide K*TR-DVDNE*LL.
Figure 3.
Figure 3.
UAP56-NTE binds to the basic groove of NP. (A) Distribution of lysine residues on the surface of a single NP molecule. Lysine residues, based on the sequence of influenza A/PR/8 NP, are labeled on a surface representation of the highly homologous influenza A/WSN/33 NP protein (PDB 2IQH). Figure was generated by PyMOL [37]. (B-E) EMSA assay of FITC-labeled UAP56-NTE1-19 binding to NP* (B), NP*-K214D (C), NP*-Δloop (D), and NP*-R174D/R175D (E). All experiments (B-E) were repeated three times independently.
Figure 4.
Figure 4.
Binding of UAP56-NTE and RNA to NP* is mutually exclusive. (A-B) EMSA assay of Alexa 488-labeled U15 RNA binding to NP* (A) and NP*-R174D/R175D (B). (C) U15 RNA displaced FITC-UAP56-NTE1-19 from the NP*•UAP56-NTE1-19 complex in a concentration dependent manner. All experiments (A-C) were repeated three times independently. (D) Working model of how UAP56 facilitates NP-mediated influenza genome packaging. Our results suggest that UAP56 initially acts on RNA-free NP through interactions with both NTE and the core of UAP56. Such configurations may prime NP for RNA interaction. Upon NP deposition on viral RNA, UAP56 is displaced due to the mutually exclusive binding of UAP56-NTE and RNA to NP.
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