title: Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.
abstract: Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.
authors.affiliation: Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110007, India.
authors.forename: Binod
authors.initials: B
authors.lastname: Kumar
authors.forename: Roopali
authors.initials: R
authors.lastname: Rajput
authors.forename: Dibya Ranjan
authors.initials: DR
authors.lastname: Pati
authors.forename: Madhu
authors.initials: M
authors.lastname: Khanna
mesh_terms: D002460:Cell Line; D021881:DNA, Catalytic / Q000235:genetics / Q000502:physiology; D004277:DNA, Single-Stranded / Q000235:genetics / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000472:pathogenicity*; D012367:RNA, Viral / Q000235:genetics; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
chemical_list: D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012367:RNA, Viral; C402582:RNA-cleaving DNA 10-23