Function Analysis Of Minireplicon Of Human Respiratory Syncytial Virus

Objective: Human Respiratory Syncytial Virus (RSV) is the most important pathogen of pediatric respiratory tract disease worldwide. Till now, no effective preventive way is available. The reverse genetics technology is an important way to develop genetically engineered attenuated living RSV vaccine candidates. In order to develop this technology, as the initial step, we plan to construct minireplicon and investigate its biological activity. The minireplicon of RSV contains Leader or genomic promoter (Le), gene start (GS), multiple cloning site (MCS), gene end (GE), and Trailer or antigenomic promoter (Tr)。By further positioned under the control of T7 RNA promoter and, cloned into px8δT vector, we can obtain the plasmid of RSV minireplicon. After this plasmid is transfected into the cells expressing T7 RNA polymerase amd all the necessary proteins for RSV replication, the function of RSV minireplicon can be investigated. In this study, we construct two RSV proteis of Large protein (L) and transcription elongation/antitermination factor (M2 ORF 1 protein, M2-1) important for RSV replication. Additionally, we finish the function analysis of RSV minireplicon based on some previous work.Methods: During the cDNA backbone of RSV minireplicon, including Le, GS, MCS, GE and Tr from 5′to 3′end, was previously synthesized and nominated as GSGE. GSGE1 and GSGE2 were obtained after adding T7 RNA promoter to 5′and 3′end of GSGE, respectively, by PCR, and then cloned into px8δT to produce the replicon plasmid of RSV, px8δT/GSGE1 and px8δT/GSGE2. After the open reading frame (ORF) of enhanced green fluorescent protein (EGFP) was cloned into px8δT/GSGE1 and px8δT/GSGE2, the RSV recombinant minireplicon plasmids of px8δT/GSGE1/EGFP and px8δT/GSGE2/EGFP were obtained. In this study, two ORFs of nucleocapsid proteins of L protein and M2-1 protein were cloned and constructed to produce pcDNA3.1/L and pcDNA3.1/M2-1. Finally, a RSV minireplicon plasmid and four nucleocapsid protein pasmids, including two previously constructed plasmids of pcDNA3.1/P and pcDNA3.1/N encoding RSV phosphoprotein (P) and nucleoprotein (N), respectively, were co-transfected into BSR T7/5 cell lines expressing T7 RNA polymerase by lipofectamine 2000, and the expression of EGFP was analyzed by inverted fluorescent microscopy and FACS, respectively.Results: The analyses of restriction endonuclease digestion and nucleic acid sequencing showed that pcDNA3.1/L and pcDNA3.1/M2-1 were successfully constructed. The expression of L and M2-1 was confirmed by RT-PCR and western blot analyses. The result of co-transfection displayed that EGFP can be observed in the transfected BSR T7/5 cells under fluorescent microscopy and by FACS.Conclusion: The constructed RSV minireplicon is able to replicate and transcript, which provides a solid foundation for further RSV vaccine study by RSV reverse genetics.