Study On The Role Of Interferon-stimulated Gene Ifit Family In Rabies Virus Infection

Rabies caused by rabies virus(RABV)is a fatal zoonotic infectious disease.Currently,rabies is still circulating in more than 150 countries and regions around the world,which seriously threatens the health of human and animals.Once the clinical symptoms of rabies appear,the mortality rate is near 100%,and there are still no effective clinical therapeutic treatments for rabies.Further strengthening of clarifying the immune response induced by RABV and its pathogenesis will provide a theoretical basis for clinical therapy of RABV.Interferon(IFN)signalling pathway plays an important role in the innate immune response during RABV infection.The activated interferon signaling pathway can express a variety of effector molecules,namely IFN-stimulated genes(ISGs),which perform their own particular functions to restrict and eliminate the invaded pathogens.Among the numerous ISGs,Ifit protein family(IFN-induced protein with tetratricopeptide repeats,Ifit)is a prominent representative,which play an essential role in the immune response against virus infection,but its role in the infection of RABV has not been clarified.Therefore,we conducted a systematic study on the role and mechanism of Ifit protein family in RABV infection,as follows:We demonstrated that the mRNA and protein expression levels of numerous ISGs were increased in mouse afterRABV infection by RNA-seq and iTRAQ,particularly the Ifit protein family.And it was confirmed that the expression of Ifit protein family was up-regulated in RABV infected mouse by RT-qPCR and WB.In addition,the expression of Ifit1,Ifit2 and Ifit3 was also highly up-regulated in murine N2 a cells and BV2 cells afterRABV infection.By constructing and the rescuing the recombinant rabies virus rRABV-Ifit1,rRABV-Ifit2 and rRABV-Ifit3 that over-expressing individual Ifit family protein,it was found that over-expression of individual Ifit protein family member alone had no significancent effect on the replication of RABV in vitro.The effect of individual Ifit family protein over-expressed by rRABV-Ifit1,rRABV-Ifit2 and rRABV-Ifit3 on the pathogenicity of RABV in mice was further explored with comparison of the i.n.,s.c.and i.m.routes of administration,and the results showed that it was varied from the routes of administration.In detail,over-expression of Ifit1 by rRABV-Ifit1 had no significancent effect on the pathogenicity of RABV in mice following i.n.,s.c.and i.m.administration;over-expression of Ifit2 by rRABV-Ifit2 could significantly reduce the pathogenicity of RABV in mice following i.n.administration;over-expression of Ifit3 by rRABV-Ifit3 could significantly reduce the pathogenicity of RABV in mice following s.c.and i.m.administration.These results above indicated that Ifit family protein played an essential role in the pathogenicity of RABV in mice and the impact was related to the route of administration.Strikingly,the over-expression of Ifit3 had the most prominent effect on the pathogenicity of RABV in mice,so the next action in the present study focused on the role of Ifit3 during RABV infection.The test results of viral loads of RABV in different tissues and brain regions of mice after i.m.administration showed that over-expression of Ifit3 by rRABV-Ifit3 could significantly reduce the viral loads of RABV in muscle of mice at day4 post infection and in cerebrum at day 6,day 8 post infection.Deletion of the gene Ifit3 could significantly increase the replication and pathogenicity of RABV in mice.Over-expressed Ifit3 by rRABV-Ifit3 could compensate for the effect of Ifit3 gene deficiency on the replication and pathogenicity of RABV in mice.Finally,in this study,the correlation between members of the Ifit protein family was explored.Over-expression of Ifit1,Ifit2 and Ifit3 individually,in pairwise combinations or all three together by transfection,it was found that simultaneous over-expression of Ifit2 and Ifit3 could synergistically inhibit the replication of RABV in vitro.Co-immunoprecipitation experiments found that there was an interaction between Ifit2 and Ifit3,and the 1-93 aa and 170-403 aa segments of Ifit3 alone could interact with Ifit2.Regarding the recognition of viral RNA by Ifit2 and Ifit3,it was found that over-expression of Ifit2 individual or Ifit3 individual or simultaneous over-expression of Ifit2 and Ifit3 all could not affect the replication of RABV mutants rRABV-K1685 A and rRABV-K1829 A with impaired methyltransferase activity in N2 a cells.In terms of inducing apoptosis,it was found that over-expression of Ifit2 by rRABV-Ifit2 could significantly increase cell apoptosis induced by RABV in N2a cells,but rRABV-Ifit1 and rRABV-Ifit3 couldn't.Interestingly,Ifit2 and Ifit3 could also synergistically inhibit the replication of negative-strand RNA virus such as RABV,VSV and DNA virus HSV-1,indicating that the synergistic antiviral effect between Ifit2 and Ifit3 had a certain broad spectrum.In conclusion,in this study,we revealed the role and mechanisms of Ifit protein family,especially Ifit3 in RABV infection,which laid a foundation for further elucidating the immune response and pathogenesis mechanism during RABV infection.