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Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and 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...

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Autores principales: Lam, Shirley, Chen, Karen Caiyun, Ng, Mary Mah-Lee, Chu, Justin Jang Hann
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466284/
https://www.ncbi.nlm.nih.gov/pubmed/23056297
http://dx.doi.org/10.1371/journal.pone.0046396
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author Lam, Shirley
Chen, Karen Caiyun
Ng, Mary Mah-Lee
Chu, Justin Jang Hann
author_facet Lam, Shirley
Chen, Karen Caiyun
Ng, Mary Mah-Lee
Chu, Justin Jang Hann
author_sort Lam, Shirley
collection PubMed
description BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and 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. METHODS: Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and 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 and 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. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and 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 and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: 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.
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spelling pubmed-34662842012-10-10 Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication Lam, Shirley Chen, Karen Caiyun Ng, Mary Mah-Lee Chu, Justin Jang Hann PLoS One Research Article BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and 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. METHODS: Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and 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 and 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. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and 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 and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: 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. Public Library of Science 2012-10-08 /pmc/articles/PMC3466284/ /pubmed/23056297 http://dx.doi.org/10.1371/journal.pone.0046396 Text en © 2012 Lam et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Lam, Shirley
Chen, Karen Caiyun
Ng, Mary Mah-Lee
Chu, Justin Jang Hann
Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title_full Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title_fullStr Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title_full_unstemmed Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title_short Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication
title_sort expression of plasmid-based shrna against the e1 and nsp1 genes effectively silenced chikungunya virus replication
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466284/
https://www.ncbi.nlm.nih.gov/pubmed/23056297
http://dx.doi.org/10.1371/journal.pone.0046396
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