[Objectives] Stress granules (SGs) are mRNA-storing intracellular structures that can inhibit translation when a cell is subjected to stress, and were recently revealed to play a role as a scaffold to activate antiviral responses. Previously, we reported that Nishigahara (Ni) strain, which is a lethal fixed rabies virus, inhibited SG formation, but Ni-CE (CE) strain, a non-lethal offshoot of Ni strain, did not. We showed that this phenomenon is related to the rabies virus matrix (M) protein (Masatani et al., 65th JSV meeting): however, the details of the molecular mechanisms by which differences in M proteins lead to differences in SG formation are unknown. Accordingly, we aimed to identify the amino acid of the M protein responsible for SG formation and elucidate the relevant mechanism. [Methods] Since there are only two amino acid differences between the Ni and CE M-proteins, at positions 29 and 95, the following mutants were constructed by swapping amino acids between Ni and CE strains with a single substitution at position 29 or 95: Ni(CEM29), Ni(CEM95), CE(NiM29), and CE(NiM95) strains. Cells were then infected with mutant strains and examined for SG formation by fluorescent microscopy, to determine the amino acid associated with lethality. To elucidate the mechanism of SG formation, eIF2α phosphorylation, a known SG-formation factor, was examined in Ni, CE, Ni(CEM95), and CE(NiM95)-infected cells by western blotting. Furthermore, 293T cells not producing PKR, an eIF2α-phosphorylating intracellular protein kinase, were generated with a CRISPR/Cas9 system. CE-infected PKR-knockout cells were examined for SG formation. [Results and discussion] Ni(CEM29) and CE(NiM95) strains inhibited SG formation, whereas Ni(CEM95) and CE(NiM29) strains did not, indicating that the amino acid at position 95 is a determinant for SG formation. eIF2α phosphorylation was not detected in Ni or CE(NiM95)-infected cells but was detected with CE and Ni(CEM95) strains. PKR-knockout 293T cells were incapable of SG formation and eIF2α phosphorylation. We consider that SG formation induced by CE strain is PKR dependent. In conclusion, we suggest that the amino acid at position 95 in rabies virus M protein is responsible for SG formation and this mechanism is dependent on PKR-activated eIF2α phosphorylation.
