title: Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.
abstract: RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Kalika
authors.initials: K
authors.lastname: Mathur
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Anand
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Dubey
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Neeti
authors.initials: N
authors.lastname: Sanan-Mishra
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Raj K
authors.initials: RK
authors.lastname: Bhatnagar
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Sunil
mesh_terms: D000330:Aedes / Q000378:metabolism / Q000821:virology; D000818:Animals; D002646:Chikungunya virus / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D034622:RNA Interference; D061987:Sf9 Cells; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus