SARS-COV-2 nucleocapsid protein hijacks multiple components of the host nuclear transport machinery for distinct functions

Many viruses target the host nuclear transport machinery to traffic their own proteins or restrict trafficking of host cargo, including mediators of antiviral immune signalling. Nucleocapsid (N) proteins from several coronaviruses traffic to the nucleus/nucleolus, with roles in cell cycle regulation. While N-protein from severe acute respiratory syndrome virus 2 (SARS-COV-2), the causative agent of the global COVID-19 pandemic, is widely reported to localise to the cytoplasm, we identify multiple, functionally distinct interactions of N-protein with the hosts nuclear transport machinery. Using quantitative cell imaging, including fluorescence recovery after photobleaching, and protein-protein interaction analysis, we describe a sub-population of SARS-COV-2 N-protein that localises more diffusely between the nucleus and cytoplasm, undergoes active nuclear import, and re-localises nuclear import receptors (karyopherins) KPNB1 and KPNA2 to the nucleus and cytoplasm, respectively. Truncation analyses identify at least two distinct KPNA2/KPNB1 binding sites located in the N-terminal and C-terminal regions of N-protein. Interestingly, while mutation of K/R-rich sites within these domains reduces KPNA2/KPNB1 binding and disables re-localisation of KPNA2, re-localisation of KPNB1 and nuclear import of N-protein remain intact, indicating that these are molecularly and functionally distinct mechanisms. siRNA knockdown confirms a role for KPNB1 in N-protein nuclear trafficking, while KPNA2 binding and mislocalisation may be antagonistic. Thus, SARS-COV-2 N-protein binds to karyopherins via multiple distinct sites to facilitate import and other functions.