Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

MERS-CoV ORF4b EMPLOYS AN UNUSUAL BINDING MECHANISM TO TARGET IMPα AND BLOCK INNATE IMMUNITY (#161)

Thilini S Munasinghe 1 , Megan R Edwards 2 , Sofiya Tsimbalyuk 1 , Olivia A Vogel 2 , Kate M Smith 3 , Murray Stewart 4 , Justin K Foster 1 , Loretta A Bosence 1 , David Aragão 5 , Justin A Roby 1 , Christopher F Basler 2 6 , Jade K Forwood 1
  1. School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
  2. Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA
  3. Swiss Light Source, Paul Scherrer Institute, Villigen, Aargau, Switzerland
  4. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK
  5. Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxford, UK
  6. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, UK

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) is a highly pathogenic, emerging virus that produces accessory proteins to antagonize the host innate immune response. The MERS-CoV ORF4b protein has been shown to bind preferentially to the nuclear import adapter IMPα3 in infected cells, thereby inhibiting NF-κB-dependent innate immune responses. The structural basis by which ORF4b binding to IMPα can prevent NF-κB function to evade host immunity is not known. Here, we report high-resolution structures of MERS-CoV ORF4b bound to two distinct IMPα family members. Each exhibit highly similar binding mechanisms that, in both cases, lack a prototypical Lys bound at their P2 site. Mutations within the NLS region dramatically alter the mechanism of binding, which reverts to the canonical P2 Lys binding mechanism. Mutational studies, based on the high-resolution structural information, confirm that the novel binding mechanism seen with ORF4b is important for its nuclear import, IMPα interaction, and inhibition of innate immune signaling pathways. In parallel, we determined high-resolution crystal structures of the nuclear binding domain of NF-κB component p50 bound to both IMPα2 and α3. These structures demonstrate that p50 overlaps directly with the MERS-CoV ORF4b binding sites, suggesting a basis for ORF4b-mediated inhibition. Our results provide a detailed structural basis that explains how a virus can target the IMPα nuclear import adapter to impair immunity and also illustrate how small mutations in ORF4b, like those found in closely related coronaviruses such as HKU5, change the IMPα binding mechanism.

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