Characterising the intrinsically disordered region of ORF6 from SARS-CoV-2

Many viral proteins have flexible and disordered regions that lack a well-defined tertiary structure. These disordered regions are often functionally important in immune evasion and for rapid replication. One such viral protein is the 61-residue protein ORF6, from SARS-CoV-2. ORF6 is a potent interferon antagonist that has been shown to bind to the ribonucleic acid export 1 and GLEBS motif of nucleoporin 98 (Rae1-Nup98) heterodimer via its C-terminal region. The binding of ORF6 to the Rae1-Nup98 heterodimer prevents nuclear export of cellular mRNAs, which suppresses the antiviral immune response.

ORF6 is predicted to be very flexible, and only very distant homologues to ORF6 are available. This makes homology modelling and AlphaFold2 structure predictions essentially impossible. To characterise the C-terminal region of ORF6 in the unbound state, we combined nuclear magnetic resonance spectroscopy (NMR) with advanced all-atom molecular dynamics (MD) simulations. Specifically, we employed enhanced MD sampling techniques with NMR chemical shift restraints to improve the force field accuracy (metadynamic metainference).

Here, I present molecular scale detail on the conformational sampling of the ORF6 C-terminal region and provide a comparison of our MD simulations to experimental results. In agreement with the chemical shifts, the C-terminal region ensemble showed a mainly disordered state. I will also present insights into the mechanisms of ORF6 gained by using this multi-disciplinary approach to characterise the C-terminal region in the unbound state.