Study On The Mechanisms Of Rabies Virus Inducing Host Shutoff

Host shutoff is a common antiviral response upon viral infection.Many viruses such as influenza virus,herpes virus,and Newcastle disease virus will induce host gene shutoff,while the mechanisms are not the same all the time.It mainly occurs at the level of transcription or translation,which including four measures that inhibiting the nucleation of mature m RNA,promoting m RNA or protein degradation and inhibiting translation initiation.Rabies virus(RABV)is one of the important pathogens that endangers the safety of human and animal life worldwide.It is unknown whether the Evelyn Rokitniki Abelseth(ERA)virus infection induces the host gene shutoff or not.Studying on this question can help us further understand the function of structural proteins RABV coding,replication strategy and pathogenic mechanism of RABV,which provides new theoretical basis for the treatment and prevention of rabies.In this study,the results of the Renilla luciferase reporter gene detection system and ribo-puromycylation assay proved that the ERA virus infection could induce the host shutoff.The matrix protein(M)played an important role in this process.The functional sites of M as arginine at positions 70 and 73 were further identified by the luciferase reporter gene detection system.With the help of reverse genetics technology,M single-point mutant virus ERA-M-R70A-GFP was successfully rescued,and its proliferation ability was greatly attenuated compared with the wild type virus.It convinced us the two amino acids not only impacted the host shutoff,but also played a critical role in virus replication.The level of eIF2? phosphorylation which has a strong regulation of overall translation directly related to the protein expression level in the host cell.Although we proved that ERA virus infection could induce the enhancement of eIF2?phosphorylation,M-induced translational inhibition had nothing to do with eIF2?phosphorylation.When eIF2? phosphorylation was completely reversed,ERA virus infection could still have a negative regulation of translation level,which revealed that there may be another pathway to induce host shutoff.M-induced host gene shutoff mainly occured at the translational level.M specifically interacting with eIF3 A and eIF5 B was verified by the mass spectrum and co-immunoprecipitation experiments,while its mutation M-R70/73 A lost the ability to bind to the translation initiation factors,which indicating that RABV-M protein may interact with the translation initiation factors to inhibit translation in virus-infected cells.ERA virus infection and M overexpression significantly weakened the m TOR signaling pathway which was another pathway that regulated host protein expression.When attenuated m TOR phosphorylation levels were reactivated by the activators of m TOR signaling pathway,ERA virus-induced host shutoff may be partially reversed.Therefore,eIF2?phosphorylation,the interaction between RABV-M and eIFs and the attenuated m TOR signaling pathway all contributed to the host shutoff of RABV infection.