Abstract
RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.
Highlights
RNA interference (RNAi) is an ancient mechanism of gene silencing for diverse eukaryotes [1]
For the present work a 653 nt long target-sequence for RNAi was selected from the UL29 gene coding for the essential ICP8 protein of herpes simplex virus (HSV)
The selected region covers the target-site of the previously used Small interfering RNA (siRNA) [29], which we here designate as UL29 siRNA molecule (UL29S) (S for small, 21-nt siRNA; Table S1)
Summary
RNA interference (RNAi) is an ancient mechanism of gene silencing for diverse eukaryotes [1]. The siRNAs enter into the RNA Induced Silencing Complex (RISC) with subsequent association of one of the two strands (guide strand) with Argonaute protein, a core component of the RISC. This leads to Argonaute-mediated sequence-specific cleavage of messenger RNA (mRNA) sequence complementary to the bound guide strand [3]. In the course of the enzymatic reaction it is possible to synthesize both siRNA [8,11] and long dsRNA molecules [7,9] The latter can be subsequently cleaved in vitro by Dicer enzyme generating a pool of target-specific siRNAs representing sequences along the entire region of interest [7,9,12,13]. As the concentration of each siRNA species in the pool is low, the pools may be less potent in inducing off-target effects than the single siRNAs [17]
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