Construction And Rescue Of Dicistronic Minigenome Of Human Respiratory Syncytial Virus

Objective:In order to investigate the impact of nucleocapsid proteins, helper proteins, of human respiratory syncytial virus (RSV) on the rescue of recombinant RSV, the cDNA clone of monocistronic or dicistronic RSV minigenomes containing enhanced green fluorescent protein (EGFP) gene or Gaussia luciferase (Glue) gene are constructed and rescued. The optimized encoding genes of helper protein are synthesized and cloned into transcription cassette driven by cytomegalovirus (CMV) promoter to increase the rescue efficiency of recombinant RSV by reverse genetics.Methods:The synthesized transcription cassette including cDNA of mini genome containing EGFP gene and the flanking sequences of T7 promoter and T7 terminator is cloned to the modified vector pBR322B to construct monocistronic minigenome expressing EGFP, the EGFP gene of which is replaced with Glue gene to produce monocistronic minigenome expressing Glue. Then, the resulting two monocistronic minigenome plasmids are modified by inserting an open reading frame (ORF) encoding pseudo-protein in the upstream of the ORF encoding reporter gene of EGPF or Glue to construct dicistronic minigenome containing the ORF1 of pseudo-protein gene and ORF2 of EGFP or Glue gene in the order of 5’to 3’, respectively. The helper plasmids with the optimized encoding genes of the helper proteins and under the control of CMV promoter are constructed and identified by Western blot. Mono-/dicistronic RSV minigenomes containing EGFP gene or Glue gene and the helper plasmids are co-transfected to BHK-T7 cells. The inmpact of helper proteins on the rescue of the above RSV minigenome are analyzed by the expressed EGFP under fluorescent microscope, the expressed Glue by Glue assay kit and the transcribed mRNA of EGFP by Real-time quantitative Polymerase Chain Reaction (RT-qPCR). The rescue efficiency of the helper plasmids under the control of CMV promoter are also compared with the helper plasmids under the control of T7 promoter.Result:The mono-/dicistronic RSV minigenomes containing EGFP or Glue gene based on the T7 transcription cassette, and the helper plasmids encoding the helper proteins with optimized genes and under the control of CMV promoter were constructed successfully. Upon the differential expressions of EGFP and Glue in protein levels and EGFP in mRNA level, from the rescued mono-/dicistronic RSV minigenomes containing EGFP or Gluc gene by helper plasmids, the vital roles played by all the helper proteins, especially for the M2-1 protein capable of elongating the transcription, were analyzed and identified. The rescue efficiency of helper plasmids encoding helper proteins under the control of CMV promoter was observed and compared with the helper plasmids under the control of T7 promoter throuth the expressed EGFP.Conclusion:By exploiting the constructed mono-/dicistronic RSV minigenomes containing EGFP gene or Gluc gene, it has been confirmed that all the helper proteins, including the M2-1 protein, play critical roles in rescue process. Also, the rescue efficiency of helper plasmids encoding codon-optimized helper proteins and using trscription cassette with CMV promoter is higher than with T7 promoter, which could be used in subsequent rescue of the recombinant RSV.