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.
Highlights
RNA interference (RNAi) is a conserved evolutionary mechanism designed to impart an antiviral defence system across organisms ranging from plants, fungi, arthropods and nematodes[1,2,3,4]
While it was previously thought that flaviviruses do not possess any RNAi suppressors, it was recently proved that these viruses do possess viral suppressors of RNAi (VSRs) that are active in both insect and mammalian cells[26,27]
We undertook the present study to systematically evaluate chikungunya virus (CHIKV) proteins (Fig. 1a) for possible RNAi suppressor activity using a Sf21 RNAi sensor cell line developed in our lab[16]
Summary
RNA interference (RNAi) is a conserved evolutionary mechanism designed to impart an antiviral defence system across organisms ranging from plants, fungi, arthropods and nematodes[1,2,3,4]. In response to RNAi in plants and insects, viruses infecting these systems utilise some of their own proteins/sequence elements that counteract the antiviral RNAi pathway[12]. These viral suppressors of RNAi (VSRs) act through different mechanisms, either by binding to long dsRNA and protecting them from Dicer[7,13] or short RNAs14 or through multi-mode mechanisms[15,16]. Systematic analysis of all CHIKV proteins using a Sf21 RNAi sensor cell line based assay revealed that non-structural proteins nsP2 and nsP3 exhibited RNAi suppressor activity We further validated this finding in its natural hosts, namely, an Aedes and mammalian cell line. Aligning known sequences of nsP2 and nsP3 across all alphavirus species, we observed that these RNA binding motifs are conserved across species thereby confirming the importance of these motifs in alphavirus-host interactions vis-à-vis RNAi pathway
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