Combinatorial Approach to Target Human Metapneumovirus Using shRNA and Ribozyme
We aimed to restrict the propagation of human metapneumovirus (hMPV) in host cells by designing a chimeric construct encoding N gene specific shRNA linked through an intracellular cleavable linker to a ribozyme targeted to the same gene. Constructs expressing shRNA or ribozymes were also designed to evaluate their individual effects. The RNA expressed from the chimeric construct had the capability to cleave the target mRNA corresponding to N gene of hMPV under in vitro condition. Introduction of chimeric construct along with the N gene encoding DNA into A549 cells led to approximately 70% downregulation in the level of target RNA. When the construct expressing only shRNA was used, approximately 35% reduction in the level of target RNA was observed while the construct having only the catalytically active ribozyme caused approximately 45% inhibition in the RNA level. Significant knockdown of viral protein was also observed in the presence of the chimeric construct while the individual shRNA and ribozymes were less effective. Thus, we have demonstrated that the shRNA and ribozyme targeted to the nucleoprotein gene of hMPV acted synergistically to downregulate the gene and this combinatorial approach could thus be effective in reducing the replication of hMPV in infected cells.