Identification Of Functional Sites On Rabies Virus M Protein Related With Transcription

Rabies virus(RABV)can cause acute,progressive encephalitis disease with high mortality rates.It's a public health problem worldwide,causing about 70,000 human deaths every year.Now there is no effective method for treatment besides vaccine to prevent rabies virus infection.Rabies virus is an unsegmented negative-stranded RNA virus,encoding five functional proteins N,P,M,G and L.Viral genomic RNA with the N,P and L proteins make up a ribonucleoprotein complex(RNP),that is in charge for replication and transcription.In our previous study,we focus on the M gene of street rabies virus GX01.The full-length cDNA clone of RC-HL strain used as template.When GX01 M gene replaced RC-HL M gene,the recombinant virus can influences replication and transcription.Fortunately,we found that F44L or F44L/S46G mutation was the key for viral inhibition effect.The rRC-HL(F44L/S46G)could significantly decrease the transcription and propagation.In this study,for elucidation of the mechanism of that inhibitory effect,the rRC-HL(GX01M)was used as the template to generate mutant rabies virus.We replaced the 44 and 46 residues of GX01 M gene with the corresponding residues of rRC-HL M gene,and rescued rRC-HLM(L44F),rRC-HLM(G46S)and rRC-HLM(L44F/G46S)mutants.Then the biological characteristics of mutants were detected by using multiple step growth curves,expression of viral proteins and mRNA level of different virus.The results indicated that rRC-HLM(L44F)could facilitate transcription and the expression of viral M proteins,similar to rRC-HL.The rRC-HLM(G46S)had a slight decrease in transcription and the expression of viral M proteins,suggesting that G46S substitution of M protein could inhibit rabies virus activity slightly.However,the replication and transcription capacity of rRC-HLM(L44F/G46S)was slightly higher than rRC-HL(GX01M).These data corresponded with our previous study,proved the results are correct.As described above,rRC-HL(F44L/S46G)could significantly decrease the growth and propagation of rabies virus.We speculated the amino acid serine(Ser)may be a phosphorylation site,so we constructed and rescued three rabies virus mutants rRC-HL(F44L/S46A),rRC-HL(F44L/S46D)and rRC-HL(F44L/S46E).Asp and Glu was used to replace Ser and to simulate phosphorylation site.Meanwhile,Ala as a non-functional residue,we devoted to the biological characteristics of three rabies virus mutants as well.Then,we found both mutants rRC-HL(F44L/S46D)and rRC-HL(F44L/S46E)had a similar activity with rRC-HL.In the early time,rRC-HL(F44L/S46A)could affect transcription,closed to rRC-HL(GX01M).But time flied,rRC-HL(F44L/S46A)had a rapid growth on transcription and propagation.These data indicated that there was no phosphorylation occurred on Ser.All rescued virus infected BSR/T7-9 cells.RIG-I,TBK1 and NF-?B level were detected by Western blot.The results showed that TBK1 and M protein had a negative regulation correlation.When the expression of M protein was high,the expression of TBK1 was inhibited.These implied M protein could inhibit the expression of TBK1,and then escape from the host's immune response.The ELISA assay was used to detect the expression level of TNF-?,IL-1?,IFN-?,IL-6 and IL-12.RABV was infected to BSR/T7-9 cells and collected of cell cultures.We could test less of TNF-? and IL-1?,suggesting that there was few TNF-a and IL-1? expression in the BSR/T7-9 cells infected with rabies virus.However plenty of IFN-y,IL-6 and IL-12 were expressed at 48 hpi,revealed they all had a anti-viral effect in the late of viral infection.Be worth to mention that,the expression of IL-12 was highest and IL-12 played an important role in the anti-viral immune response.