Antiviral RNA Interference in Mammalian Cells

Abstract

In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

“…As mentioned previously, failure to document this process in the context of viral infection is complicated by the possibility that the virus encodes VSRs (Haasnoot et al , 2007; Wu et al , 2010). Therefore, we designed a virus‐free assay to measure long dsRNA‐mediated gene silencing at the single‐cell level and tested it initially in mouse embryonic stem cells (mESCs), in which the presence of dsRNAi and antiviral RNAi has been reported (Billy et al , 2001; Yang et al , 2001; Paddison et al , 2002; Maillard et al , 2013). mESCs stably expressing a GFP reporter gene were transfected with in vitro synthesised Cy5‐labelled long dsRNA corresponding to the first 200 nt of either the GFP (dsRNA‐GFP) or, as a control, the Renilla luciferase (dsRNA‐ RL ) coding sequences; GFP expression in live single Cy5‐positive (transfected) cells was then measured by flow cytometry (Fig 1A and B).…”

“…Consequently, plant and insect viruses have evolved virulence factors called viral suppressors of RNA silencing (VSRs) that block various steps of the antiviral RNAi mechanism (Haasnoot et al , 2007; Wu et al , 2010). Experimental appreciation of the effects of RNAi on virus accumulation therefore requires the use of VSR‐deficient viruses (Deleris et al , 2006; Wang et al , 2006a; Diaz‐Pendon et al , 2007; Maillard et al , 2013). …”

“…Whether RNAi also acts as an antiviral mechanism in mammals is hotly debated (Parameswaran et al , 2010; Cullen et al , 2013; Li et al , 2013; Maillard et al , 2013; Backes et al , 2014; Ding & Voinnet, 2014; Kennedy et al , 2015). Infection of mammalian somatic cells with various viruses results in little accumulation of viRNA (Parameswaran et al , 2010; Girardi et al , 2013; Backes et al , 2014; Bogerd et al , 2014) and, where tested, virus replication is only modestly affected in Dicer‐defective cells (Wang et al , 2006b; Matskevich & Moelling, 2007; Bogerd et al , 2014).…”

“…For example, viral infection of mESCs resulted in RISC loading with viRNAs that could provide antiviral activity towards a VSR‐deficient homologous virus (Maillard et al , 2013). Interestingly, oocytes express an alternative isoform of Dicer that lacks the N‐terminal helicase domain (Flemr et al , 2013) and has enhanced ability to process long dsRNA.…”

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

“…However, Dicer's catalytic function appears mainly confined to precursor microRNA (pre‐miRNA) processing, and its antiviral capacity in mammals remains much debated (Parameswaran et al , 2010; Cullen et al , 2013; Li et al , 2013, 2016; Maillard et al , 2013; Backes et al , 2014; Ding & Voinnet, 2014; Kennedy et al , 2015; Jeffrey et al , 2017; tenOever, 2017). Some studies have reported that RNAi can impact antiviral immunity in mammals during influenza A virus, hepatitis C virus, Nodamura virus and, more recently, human enterovirus 71 infection (Wang et al , 2006; Matskevich & Moelling, 2007; Li et al , 2013, 2016; Maillard et al , 2013; Qiu et al , 2017). In contrast, others have found low abundance of viRNAs in mammalian somatic cells infected with various viruses, and only a modest effect of Dicer deficiency on viral replication, suggesting that RNAi is not an active mechanism of antiviral defence in most mammalian cell types (Parameswaran et al , 2010; Girardi et al , 2013; Backes et al , 2014; Bogerd et al , 2014; Schuster et al , 2017).…”