Pseudorabies virus IE180 protein hijacks G3BPs into the nucleus to inhibit stress granule formation.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that can infect different animal species and cause pruritus and lethal encephalitis. Stress granules (SGs) are membrane-free cytoplasmic structures formed by liquid-liquid phase separation of G3BP proteins during cell translation inhibition, which generally plays an antiviral role in various viral infections. In this study, we found that infection with different PRV strains inhibits the formation of SGs in host cells. We found that IE180, the only immediate early protein of PRV, has a distinct inhibitory effect on SG formation and colocalizes with SG-nucleating G3BP proteins (G3BP1/2) in the nucleus during PRV infection. Co-immunoprecipitation assays demonstrated an interaction between IE180 and G3BP1/G3BP2, and this interaction appears to depend on the Herpesvirus ICP4-like protein N-terminal (ICP4L-N) domain of IE180 and both NTF2L and RBD domains of G3BP1. Since G3BPs mainly function in the cytoplasm to induce SG formation, we constructed several IE180 protein truncations lacking a nuclear localization sequence to alter the subcellular localization of IE180 to the cytoplasm. Mutant IE180 protein was mainly expressed in the cytoplasm and still suppressed SG formation induced by arsenite or poly(I:C), but its ability to inhibit SG formation was weakened. Importantly, knockout of G3BPs facilitated PRV replication in H1299 cells, while exogenous expression of G3BPs and formation of SGs in wild-type H1299 cells suppressed PRV replication. In summary, our study indicates that PRV IE180 suppresses SG formation and hijacks G3BPs into the nucleus to benefit virus replication.IMPORTANCEHerpesviruses, including pseudorabies virus (PRV), have evolved different strategies to compromise host immune responses. Stress granules (SGs) are one of the targets that viruses can overcome in order to increase replication. The related herpes simplex virus 1 (HSV-1) inhibits SG formation to promote virus replication, but the underlying mechanisms remain unknown. In this study, we confirmed that infection with different PRV strains inhibits SG formation. Interestingly, we found that the PRV immediate early protein IE180 interacts with G3BP proteins and hijacks these proteins into the nucleus to prevent SG formation. In line with the antiviral effect of SGs on PRV replication, exogenous expression of G3BPs and formation of SGs in G3BP1/2 knockout H1299 cells significantly compromised PRV replication. The reported mechanism appears to be also utilized by HSV-1 to prevent SG formation. Therefore, our study elucidates a novel mechanism by which alphaherpesviruses inhibit SG formation, which provides a new perspective to inquire into the immune escape of PRV and other alphaherpesviruses.