Construction of rabies virus GX074 P-MS20F mutant and identification of its growth characteristics
[Objective]To clarify the growth characteristics of the recombinant mutant with the replacement of P pro-tein and the 20th phenylalanine(Phe)of M protein of the strong strain of rabies virus(RABV)strain GX074 to the attenua-ted strain rRC-HL in host cells,and to provide theoretical basis for the study of the transcription and replication mecha-nism of the P and M proteins.[Method]The 20th serine(Ser)of M protein of attenuated strain rRC-HL(GX074P)was re-placed by the 20th Phe of M protein of strong strain GX074 by reverse genetic technique to construct the infectious cDNA clone of rRC-HL(GX074P-MS20F)mutant and rescue the virus.BSR/T7-9 cell was infected by recombinant mutant,and multi-step growth curve determination was performed to compare the growth ability of the recombinant mutant and that of the parent strain.The relative expression levels of N,P and M proteins were detected by Western blotting,and the rela-tive expression levels of N,P and M genes were detected by real-time fluorescence quantitative PCR.[Result]RT-PCR and sequencing confirmed that the 20th Ser of the M protein of the rescued mutant rRC-HL(GX074P-MS20F)was success-fully replaced by the 20th Phe.The results of the multiple-step growth curve demonstrated that the viral titer of the recombi-nant mutant was higher than that of both the parental attenuated strain rRC-HL and the control attenuated strain rRC-HL(GX074PM1)at24,48,72 and 96 h after infection,with 10times of the parent attenuated rRC-HL.In terms of protein expression,the relative expression levels of N protein and M protein of rRC-HL(GX074P-MS20F)strain were extremely significantly higher than those of the parental attenuated strain rRC-HL at 48 h after infection(P＜0.01).These results indi-cated that the substitution of the P protein of strong strain GX074 with the 20th Phe of M protein to the attenuated strain rRC-HL increased the expression of N and M proteins in the mutant.The relative expression levels of N,P and M genes of rRC-HL(GX074P-MS20F)strain were higher than those of parental attenuated strain rRC-HL and control attenuated strain rRC-HL(GX074PM1)strain at 24 and 48 h after infection.[Conclusion]The replacement of Phe at the 20th posi-tion of M protein of strong strain GX074 can improve the proliferation ability of virus,and enhance the replication and transcription ability of virus.Phe at the 20th position of M protein may be a key site affecting the replication and transcrip-tion of virus.
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