Construction of rabies virus GX074 P-M^S20F 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 20^th phenylalanine (Phe) of M protein of the strong strain of rabies virus (RABV) strain GX074 to the attenuated strain r RC-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 20^th serine (Ser) of M protein of attenuated strain r RC-HL (GX074P) was re-placed by the 20^th Phe of M protein of strong strain GX074 by reverse genetic technique to construct the infectious c DNA clone of r RC-HL (GX074P-M^S20F) 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 20^th Ser of the M protein of the rescued mutant r RC-HL (GX074P-M^S20F) was success-fully replaced by the 20^th 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 r RC-HL and the control attenuated strain r RC-HL(GX074PM1) at 24,48,72 and 96 h after infection,with 10 times of the parent attenuated r RC-HL.In terms of protein expression,the relative expression levels of N protein and M protein of r RC-HL (GX074P-M^S20F) strain were extremely significantly higher than those of the parental attenuated strain r RC-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 20^th Phe of M protein to the attenuated strain r RC-HL increased the expression of N and M proteins in the mutant.The relative expression levels of N,P and M genes of r RC-HL (GX074P-M^S20F) strain were higher than those of parental attenuated strain r RC-HL and control attenuated strain r RC-HL (GX074PM1) strain at 24 and 48 h after infection.【Conclusion】The replacement of Phe at the 20^th 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 20^th position of M protein may be a key site affecting the replication and transcrip-tion of virus.