The SHFV nsp2 and nucleocapsid proteins recruit G3BP1 to sites of viral replication, but stress granules are not induced by the infection.

Stress granules (SGs) are dynamic, cytoplasmic foci that form in response to environmental stresses, including viral infections, and function to restore cellular homeostasis by regulating mRNA translation, storage, and decay. To inhibit SG formation and subvert their antiviral effects, viruses from diverse families sequester or cleave G3BP1, the key SG nucleating protein. We found that an infection with simian hemorrhagic fever virus (SHFV), a member of the family Arteriviridae, does not induce the formation of bona fide SGs despite inducing phosphorylation of PKR and eIF2α. The SG proteins, G3BP1, G3BP2, TIA-1, Caprin-1, and USP10, but not the translation initiation proteins, eIF3A, eIF4G, and small ribosomal protein S6 (rpS6), were redistributed into foci located in the same intracellular region as the viral dsRNA foci. However, SGs could be induced in infected cells by exogenous inducers. LC-MS/MS analysis of the proteins co-immunoprecipitating with endogenous G3BP1 from SHFV-infected cell lysates detected multiple viral replication/transcription complex proteins. Interaction between G3BP1 and the nsp2 and N proteins of SHFV was observed in reciprocal co-immunoprecipitation assays, and colocalization was detected by IFA. A conserved FGAP motif in nsp2 and a FAEP motif in the N protein were shown to be required for interaction with G3BP1. We also detected G3BP cleavage products in the SHFV-infected cell lysates and hypothesize that cleavage is mediated by a viral protease. These findings suggest that SG formation is not induced by an SHFV infection due to recruitment of G3BP to sites of viral replication and cleavage of G3BP by viral proteins.IMPORTANCEEukaryotic cells shut down translation by assembling stress granules (SGs) in response to environmental stresses, including viral infections. Viruses require cellular translation machinery for protein synthesis and have developed mechanisms to subvert SG assembly. Simian hemorrhagic fever virus (SHFV), a simian arterivirus, causes asymptomatic infections in African cercopithecoid monkeys but fatal hemorrhagic fever disease in Asian macaques. Even though intracellular production of SHFV RNA activates the stress sensor, PKR, SGs are not induced. G3BP1, the main nucleating protein of SGs, is recruited to foci located near viral replication complexes through interaction with the viral proteins nsp2 and N. An FGAP motif in nsp2 and an FAEP motif in the N protein are required for interaction with G3BP1. Cleavage of G3BP1 was identified as an additional mechanism of viral counteraction of SG formation.