Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

Abstract

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells. R NA silencing is a unique RNA-guided gene regulatory mechanism that operates in a wide range of eukaryotic organisms from plants to mammals (1). A feature common to all RNA silencing processes is the production of 21-to 26-nt small RNAs from structured or double-stranded RNA (dsRNA) by the endoribonuclease Dicer (2-6). These small interfering RNAs (siRNAs) control the specificity of RNA silencing in a homology-dependent manner by means of an RNA-induced silencing complex (RISC), of which Argonaute-2 (AGO2) is an essential protein component (1,7,8). RNA silencing in fungi, plants, and worms involves a cellular RNA-dependent RNA polymerase (RdRP); however, the multiple-turnover RISC may mediate RNA silencing in absence of a cellular RdRP in Drosophila and mammalian cells (1, 9-11).We reported recently that infection of cultured Drosophila cells with the plus-strand RNA Nodavirus flock house virus (FHV), triggers specific silencing of FHV RNAs that is associated with accumulation of 22-nt siRNAs (12). Silencing of the replicating viral RNAs is RISC-dependent and sensitive to inhibition by the FHV B2 protein, as shown by the observation that B2 is essential for FHV infection of WT Drosophila cells but dispensable in cells depleted for AGO2 (12). These findings provided an example indicating an antiviral role for RNA silencing in the animal kingdom (12, 13), as has been established in higher plants (14)(15)(16)(17)(18).In this article, we report that specific RNA silencing was induced in mosquito cells in response to viral RNA replication and show that this mosquito antiviral immunity is RISCdependent and sensitive to suppression by the B2 protein encoded by either FHV or nodamura virus (NoV). We demonstrate th...

Viral Virulence Protein Suppresses RNA Silencing–Mediated Defense but Upregulates the Role of MicroRNA in Host Gene Expression[W]

“…Recent work has demonstrated a similar antiviral role for RNA silencing in the animal kingdom (Li et al, , 2004. The antiviral response may be triggered by virusspecific dsRNA intermediates produced during genome replication, by transcription from converging promoters, or through recognition of viral RNAs by host RdRP, as proposed in plants (Voinnet et al, 2000;Szittya et al, 2002).…”

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…[17][18][19] The apparent absence of RNAi restriction of some viruses could be explained by the counteraction of encoded viral RNA silencing suppressors (RSSs). Several mammalian viruses encode RSSs, 17,[20][21][22][23][24][25][26][27][28][29] although the efficiency of a few of them has been debated. 16,[30][31][32] Proposed RSSs encoded by HIV-1 are the transactivator Tat 20,27 and the Trans Activation Response (TAR) RNA.…”

Inhibition of host innate immune responses and pathogenicity of recombinant Newcastle disease viruses expressing NS1 genes of influenza A viruses

“…In particular, mutational analysis of H5N1 NS1 protein indicated that both the RNA-binding and effector domains affect virus pathogenicity synergistically. Our study also demonstrated that expression of H1N1/09 NS1 resulted in enhanced replication of rNDV in human cells, indicating that function of the NS1 proteins can be host-species-specific.Influenza A viruses are associated with significant morbidity and mortality, causing worldwide epidemics yearly and pandemics sporadically (Li et al, 2004). The genome of influenza A virus consists of eight RNA segments that encode nine structural proteins and two non-structural proteins, NS1 and PB1-F2 (Palese & Shaw, 2007).…”

“…Influenza A viruses are associated with significant morbidity and mortality, causing worldwide epidemics yearly and pandemics sporadically (Li et al, 2004). The genome of influenza A virus consists of eight RNA segments that encode nine structural proteins and two non-structural proteins, NS1 and PB1-F2 (Palese & Shaw, 2007).…”

“…In addition, point (E92A) and deletion mutations in the effector domain of H5N1 NS1 resulted in enhanced levels of IFN-b mRNA synthesis in virus-infected cells, indicating that the effector domain also plays an important role in antagonizing IFN-b synthesis. Indeed, elimination of the effector domain has been shown to alter the dsRNA-binding affinity of NS1 significantly (Li et al, 2004). The effector domain is also known to bind to the cellular proteins that are essential for the processing of cellular pre-mRNA, including IFN-b mRNA (Noah et al, 2003).…”