Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication
Autor(es): Lam Shirley, Chen Karen Caiyun, Ng Mary Mah-Lee, Chu Justin Jang Hann
Resumo: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever - persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 - nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting - transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. Cell clones expressing shRNAs against CHIKV E1 - nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones - non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression - transmission electron microscopy of shRNA E1 - nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects - broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus - Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong - sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.
Imprenta: PloS One, v. 7, n. 10, p. e46396, 2012
Identificador do objeto digital: 10.1371/journal.pone.0046396
Descritores: Chikungunya virus - Cell ; Chikungunya virus - Molecular structure ; Chikungunya virus - Pathogenesis ; Chikungunya virus - RNA ; Chikungunya virus - Molecular methods ; Chikungunya Virus - Virus ; Chikungunya virus - Transmission ; Chikungunya virus - Vaccine ; Chikungunya virus - Chikungunya fever ; Chikungunya virus - Dengue
Data de publicação: 2012
