An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins. Rift Valley fever virus (RVFV) infection can cause severe illnesses including liver disease, encephalitis, and hemorrhagic fever. Unfortunately, there are no FDA-approved vaccines or therapeutics for human use. The NSs protein is the main virulence factor of RVFV and is a potent inhibitor of the host antiviral immune responses. Autophagy has been previously shown to be antiviral in RVFV infected cells, but RVFV can suppress this antiviral pathway by a previously undetermined mechanism. In this manuscript, we demonstrate that NSs interacts with host LC3-family proteins through an LC3 interacting region (LIR) within its C-terminus. This interaction results in LC3 being retained in the nucleus and autophagy inhibition. Our studies provide mechanistic insights by which NSs subverts antiviral autophagy during RVFV infection.