Ebolavirus Proteins Suppress the Effects of Small Interfering RNA by Direct Interaction with the Mammalian RNA Interference Pathway

Abstract

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.RNA interference (RNAi) is a sequence-specific gene regulatory pathway widely conserved from plants to mammals (16, 47) that provides a natural cellular response to viral infection. Since mammals have evolved complex protein-based adaptive and innate immunity in response to infection, the existence of a nucleic acid-based innate immunity such as RNAi was initially debated. To date, several studies have provided experimental support to clarify the role of RNAi-based immunity in mammals. The identification of proteins working as suppressors of RNAi in animal virus (38) and of noncoding adenoviral RNAs able to inhibit components of the RNAi machinery (64) as well the presence of virus-derived small RNAs (3,11,48) and the observation that engineered RNAi can successfully restrict viral infection in mammalian cells (4,17,18,28,31) support a role for RNAi as an innate antiviral mechanism (3,11,38,48).The first mechanistic step of the RNAi pathway involves Dicer, an RNase III-type enzyme that processes doublestranded RNAs (dsRNAs) into small interfering RNAs (siRNAs) of 19 to 21 nucleotides (nt) in length, with 2-base 3Ј overhangs (12,16,41,47). The RNA-induced silencing complex (RISC) unwinds duplex siRNAs and selectively incorporates one strand of the pair, known as the guide strand. In the active RISC, the guide strand identifies the target RNA transcript (mRNA) with perfect sequence complementarity. The endonuclease Argonaute2 (Ago2) then cleaves and disrupts the targeted transcript (41). The active RISC includes Dicer, Ago2, TRBP, and PACT. TRBP and PACT are two dsRNAbinding proteins (dsRBPs) that function as partners for Dicer (29), forming a complex with Dicer and Ago2 to achieve siRNA-mediated cleavage of mRNA and facilitate microRNA (miRNA) biogenesis (6,19,20,33). TRBP and PACT also bind the dsRNA-dependent protein k...

Characterization of the RNA Silencing Suppression Activity of the Ebola Virus VP35 Protein in Plants and Mammalian Cells

“…Thus, our results suggest that the microRNA-specific silencing suppression activity of VP35 observed in mammalian cells is not dependent on its ability to bind small RNAs. These results are consistent with a recent report that demonstrated the ability of the RISC-associated proteins PACT and TRBP to bind to EBOV VP35 when overexpressed by transfection (11). These investigators also reported that the VP35 interactions with PACT and TRBP were not dependent upon the presence of siRNA.…”

“…It is noteworthy that both we ( Fig. 2A) and Fabozzi and colleagues (11) have observed that the silencing-independent translational enhancement caused by VP35 is much less than its effect on either microRNA-or siRNA-directed silencing, confirming the utility of the Fluc expression assays to measure microRNA-directed silencing suppression by VP35.…”

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

“…These transcomplementation assays confirm that RRE can act as an RSS in the context of a viral infection, similarly to what has been shown for other RSSs. 22,23,27 Because E1A is synthesized in part from the incoming DNA, 77 the increase of E1A expression from DVA adenovirus but not from WT adenovirus suggests that RRE mainly compensates for the lack of VA when the RNA is transcribed from the incoming DNA. The RRE-mediated increase in DNA replication observed on both DVA and WT adenovirus suggests that RRE further impacts the RNA transcribed from the replicating DNA whether it is mutated in VA or not.…”

“…[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.…”

Host Cell Factors Involved in Filovirus Infection

“…Interestingly, interaction of PACT with VP35 prevents formation of a functional polymerase complex and suppresses viral RNA synthesis [102•]. In addition to antagonism of RIG-I signaling, VP35 was shown to inhibit RNA silencing [103,104] and the PKR-mediated cellular antistress response [105]. Since PACT is involved in both RNA silencing and PKR activation, it might be possible that the VP35-PACT interaction contributes to the suppression of RNA silencing and PKR-mediated antiviral response.…”