[Objectives] Stress granules (SGs) are intracellular structures that store mRNA and inhibit transcription when cells undergo stress, and reportedly function as a scaffold for antiviral factor activation. One factor reportedly localized in SGs and activated after detection of viral RNA is the IFN-b-transcription initiator retinoic acid-inducible gene I (RIG-I), and RIG-I accumulation may thus be useful in evaluation of SG scaffold functionality. We previously reported that SG formation was inhibited by Nishigahara (Ni) strain, a lethal fixed rabies virus, but not by its non-lethal offshoot, Ni-CE (CE) strain, in a phenomenon related to the amino acid position at 95 in the rabies virus matrix protein (M95; Kojima et al., 68th JSV meeting); however, the role of SG formation in the response to the rabies virus is not fully understood. Accordingly, we aimed to further elucidate this SG-related phenomenon by comparing RIG-I accumulation and IFN-b transcription between cells infected with Ni, CE, and mutant strains (which differ in the amino acid at M95).
[Methods] HEK293T cells were infected with the Ni strain, CE strain, or mutant strains replacing M95 between the Ni and CE strain [Ni(CEM95) and CE(NiM95), respectively]. At 24 hours post-infection, cells were examined for RIG-I accumulation and SG formation by fluorescent microscopy. To elucidate the antiviral effects of RIG-I accumulation and SG formation, IFN-b mRNA was quantified by real-time PCR for extracts from 293T cells infected with each strain.
[Results and discussion] Ni and CE(NiM95) strains, which possess the same amino acid at M95 as the Ni strain, inhibited SG formation and RIG-I accumulation, whereas CE and Ni(CEM95) strains, which possess the same amino acid at M95 as the CE strain, did not, suggesting that SGs induced by the CE strain act as a scaffold for RIG-I accumulation. CE and Ni(CEM95)-infected cells showed higher quantities of IFN-b mRNA than CE(NiM95) and Ni-infected cells, respectively. In conclusion, we suggest that M95-related induction of SGs facilitates activation of RIG-I, which initiates IFN-b mRNA transcription.
