SUMOylation of SARS-CoV-2 Nucleocapsid protein enhances its interaction affinity and plays a critical role for its nuclear translocation

Abstract

AbstractViruses, such as SARS-CoV-2, infect hosts and take advantages of host cellular machinery for their genome replication and new virion production. Identification and elucidation of host pathways for viral infection are critical for understanding the viral life cycle and novel therapeutics development. SARS-CoV-2 N protein is critical for viral RNA(vRNA) genome packaging in new virion formation, Here, we report that identification of SUMOylation sites of SARS-CoV-2 N protein and role of SUMO modification in N protein interaction affinity with itself using our qFRET/MS coupled method. We found, for the first time, that the SUMO modification of N protein can significantly increase its interaction affinity with itself and may support its oligomer formation. One of the identified Lys residues, K65 was critical for N protein translocation to nucleus, where the vRNA replication and packaging take place. The in vitro assessment of the affinity of N protein to N protein with SUMO mutants provides insight of the oligomerized N protein formation after SUMO modification. These results suggest that the host human SUMOylation pathway may be very critical for N protein functions in viral replication. The host SUMOylation pathway may be a critical host factor for the SARS-CoV-2 virus life cycle. Identification and inhibition of critical host SUMOyaltion could provide a novel strategy for future anti-viral therapeutics development, such as SARS-CoV-2 and other viruses.ImportanceThe SARS-CoV-2 virus N protein plays a critical role critical for viral RNA(vRNA) genome packaging in host cell nucleus for new virion formation. Therefore, deciphering the molecular mechanisms modulating N activity could be a strategy to identify potential targets amenable to therapeutics. Here, we identify a comprehensive SUMOylation sites of N proteins using an in vitro reconstitute SUMOyaltion assay containing SUMO E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. We find that SUMOylation modification of N protein can significantly enhance it interaction affinity with itself, indicating an increased oligomerization capability, which is critical for N protein activity for vRNA genome packaging. In addition, we find one of SUMOylation sites of N protein is critical for its nucleus translocation, which is a critical for viral genome packaging. The SUMOylation modification may represent novel potential approach to design new antivirals with the ability to modulate SARS-CoV-2 virus replication.