Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA).
Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-beta and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%).
Kumar
Amit
A
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. amitkothari2006@gmail.com <amitkothari2006@gmail.com>
Panda
Subrat Kumar
SK
Durgapal
Hemlata
H
Acharya
Subrat Kumar
SK
Rehman
Shagufta
S
Kar
Upendra K
UK
D000998:Antiviral Agents / Q000494:pharmacology*; D001688:Biological Products / Q000235:genetics / Q000494:pharmacology*; D002460:Cell Line; D020868:Gene Silencing; D005814:Genes, Viral; D016752:Hepatitis E virus / Q000187:drug effects / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D001688:Biological Products; D034741:RNA, Small Interfering