The Effects And Underlying Mechanisms Of Interferon Stimulated Iigp1 On Rabies Virus

Rabies is a zoonotic and fulminating infectious disease induced by rabies virus(RABV).When rabies occurs in human or animal,the mortality rate is almost 100%.According to the statistical analysis of the world health organization(WHO),rabies causes more than 59000 human deaths every year and gives a serious threat to more than three hundred million people’s lives in more than 150 countries and regions.Developing countries are the high-risk areas of rabies outbreak,the number of rabies deaths in China ranked second in the world,only behind India.At present,the pathogenic mechanism of RABV is remains unclear,and the study of the pathogenesis of RABV will benefit the controling or even elimination of human rabies.In this study,we established a mouse model of RABV infection with moderately toxic B2C,and then detected the differential expressed proteins by the isobaric tags for relative and absolute quantitation(iTRAQ)technology.We selected an upregulated protein-immune related GTPasea6 subtype(Immunity-related GTPase,Irga6 or IIGP1)and then inserted its gene into the RABV genome of B2C using reverse genetics technology,so as to construct the recombinant RABV expressing IIGP1(B2C-IIGP1).Indirect immunofluorescence(IFA)and Western blotting results showed that IIGP1 was successfully expressed by B2C-IIGP1.The multi-step growth curves of B2C-IIGP1 were determined on NA,U251 and BV2 cells which had successively enhanced immune competence.The results showed that IIGP1 could inhibit the proliferation of RABVIn order to investigate whether the inhibition of RABV is related to the GTPase activity of IIGP1,the key sites of IIGP1 related to GTPase activity were individually mutated to construct IIGP1 eukaryotic expression plasmids expressing GTPase deletion.These plasmids were transfected into 293T cells,and then the cells were inoculated with B2C.RABV titers in the culture medium supernatant and the expression of RABV proteins were detected.The result showed the level of RABV titers and the expression level of RABV proteins were recovered after mutating 106 amino acid of IIGP1.Activity of GTPase was detected by ELISA.The results showed that the GTPase activity of IIGP1 significantly decreased after mutating 106 amino acid of IIGP1.The results showed that the GTPase activity of IIGP1 affects the proliferation of RABVIn order to explore the other mechanism of IIGP1 inhibiting the proliferation of RABV,we expressed IIGP1 in 293T cells in a gradient increasing form,after inoculation with B2C,RABV titers and the expression of RABV N and P protein were decreased with the increasing expression of IIGP1,but the expression of G protein was not changed.RABV N protein,G protein and IIGP1 eukaryotic expression plasmids were co-transfected into 293T cells,Western blotting and laser confocal results showed that the expression level of N protein was not changed with the increasing expression of IIGP1,whereas the expression of G protein was increased with the increasing expression of IIGP1,mainly located in the cytoplasm.The results showed that IIGP1 affects the localization of G protein.Next,we tested the inhibitory effect of IIGP1 on RABV in vivo.Mice were individually inoculated through the ears of the subcutaneous injection(6×103 FFU),intranasal injection(100 FFU)and hind leg muscle injection(6×104 FFU).The results showed that overexpression of IIGP1 reduced the mortality rate of mice compared with the parent virus B2C in the three attacking mode,especially the ears of the subcutaneous injection and intranasal injection.In the intranasal injection,the viral loads were detected in different regions of mice brain respectively in 6,9,12d after challenged,the results indicated that B2C-IIGP1 virus loads were significantly decreased compared with the parental virus in the olfactory bulb,hippocampus,cerebral cortex and brainstem.In conclusion,this study confirmed that IIGP1 inhibits the proliferation of RABV through its GTPase activity and its ability to limit the localization of G protein,which is a novel mechanism of the body against RABV invasion.This study provides a potential target for the treatment of rabies.