Viruses have evolved several strategies to replicate successfully within an infected host while avoiding antiviral responses such as apoptosis, IFN, RNAi, and autophagy (2
). Regardless of the host species or the genomic nucleic acid composition (RNA versus DNA), many viruses resist RNAi by encoding SRSs (37
). In this study, we showed that EBOV encodes three proteins that suppress host RNAi-based immunity. We designed sequence-specific siRNAs that silenced viral genes expressed as RNA polymerase II-driven transcripts. When cells treated with the same siRNAs were infected with the virus, however, siRNA-mediated RNAi was unable to silence the same viral genes, suggesting that the virus possesses a mechanism to counter RNAi. Using a reporter-based RNAi assay, we identified VP30, VP35, and VP40 as SRSs.
We focused on identifying the molecular mechanisms by which VP30 and VP35 interact with the RNAi machinery and observed direct associations with components of RNAi. There are several features of the three proteins identified that support their role as SRSs in the context of the EBOV life cycle. (i) All SRSs identified to date are ssRNA- or dsRNA-binding proteins (36
). All three EBOV protein candidates fit this paradigm. (ii) It is important to viral replication that any SRS should act early in the postentry life cycle to efficiently suppress the host RNAi apparatus. It is therefore of note that two of the three proteins identified, VP30 and VP35, are components of the EBOV RNP transcriptional replication complex. (iii) The exclusively cytosolic intracellular life cycle of EBOV leaves the viral genome accessible to the cytosolic RNAi machinery; the panhandle-like dsRNA may trigger host RNAi. Encoding multiple independent mechanisms to suppress RNAi might be necessary for a virus displaying such susceptibility.
Many known mammalian SRSs are dsRBPs and potent IFN antagonists. The SRS activity of EBOV VP35 has been postulated (21
) based on the viral protein function as a dsRBP and IFN antagonist like other mammalian SRSs (38
). By use of VP35 point mutants defective in dsRNA binding/IFN antagonism (R312A and K309A mutants) that abolish shRNA-RNAi suppression, it has been proposed that the dsRNA binding ability of VP35 mediates the SRS function (21
). In this study, we observed that VP35 interacts with RNAi in an siRNA-independent fashion. We also found that transfected siRNAs do not induce IFNs in HEK293 cells (Fig. ). Thus, we propose that VP35 contains SRS functions independent of two other known properties of VP35, the dsRNA binding activity and IFN antagonism. Finally, our observations provide evidence that the dsRBP/IFN antagonist criterion (38
) is not universally applicable to all viruses.
Here, we report for the first time the molecular interactions between VP35 and RNAi machinery. Our data show that VP35 interacts with RNAi components through protein-protein interactions. VP35 is in a complex with Dicer and its partners TRBP and PACT and directly interacts only with Dicer partners. TRBP and PACT heterodimerize through domains A and B in an RNA-independent fashion and contact Dicer or PKR through domain C (Fig. ) (29
). VP35 simultaneously contacts TRBP-PACT, implying that this interaction occurs through domains A and B, which are not directly involved with Dicer or PKR binding. As a consequence, one of the Dicer partners (TRBP or PACT) can still interact with PKR, and this might explain previous results that failed to detect a physical interaction between VP35 and PKR (15
), despite a reduction in PKR level, as well as residual IFN inhibition (5
). Also, either TRBP or PACT can still bind to Dicer, present at any step of the RNAi pathway, e.g., at the dsRNA cleavage step. This might help to interpret results showing that VP35 suppresses shRNA-mediated RNAi (21
). shRNAs, indeed, require Dicer cleavage or Dicer partners TRBP and PACT, which assist Dicer in this step (29
). The use of VP35 mutants showed that point mutations K309A and R312A present at the C terminus of VP35 in the dsRNA binding/IFN domain are not required for the interaction with Dicer partners TRBP and PACT. Recently, structural data of the VP35 carboxyl-terminal IFN domain have suggested that additional basic residues may play a role in the dsRNA binding activity/IFN functions (34
Upon dsRNA-mediated IFN induction or viral infection, PKR is strongly enhanced, whereas PKR remains latent in unstimulated cells until activation is mediated by the cellular factor PACT (39
). VP35 recruits TRBP and PACT, positive and negative regulators of PKR and partners of Dicer, which are shared between RNAi and the dsRNA/PKR recognition pathway. Though coimmunoprecipitation of VP35 with TRBP and PACT is an indirect demonstration of the suppressive role of VP35, we propose a model in which the viral protein mediates the cross talk between these two pathways by sequestering key components of host cellular RNA-mediated antiviral immunity (Fig. ). Despite the presence of viral dsRNA, VP35 employs a molecular mechanism that might result in more advantages in antagonizing host RNA defenses, not only affecting RNAi steps which require both TRBP and PACT (6
) but also regulating the PKR-mediated pathway. PKR, indeed, is activated by PACT (39
). PACT-mediated PKR activation is regulated by TRBP concentration or by stress-induced dissociation of the TRBP-PACT complex (10
The transcriptional factor VP30 also reveals a novel molecular mechanism of RNAi suppression. Though the association seen between VP30 and RNAi components cannot prove directly that the viral protein mediates the inhibition of RNAi, we have considered the interactions seen and the behavior of mutant proteins to propose a model. The important features of the model are the presence of an siRNA bridge in the interaction with TRBP and the absence of PACT association when VP30 is in a complex with RNAi components. Dicer interacts with VP30 regardless of the presence of siRNA. This interaction with Dicer and TRBP, but not PACT, suggests that VP30 prevents PACT from entering the RNAi machinery (Fig. ).
Our data favor a possible mode of action for VP30 in which the viral protein acts at the level of RISC loading and prevents any further RISC activity that requires PACT. By directly interacting with Dicer or with TRBP while TRBP passes the duplex siRNA to the RISC, VP30 limits the interactions between TRBP and PACT or between Dicer and PACT (Fig. , inset). We hypothesize that VP30 may interfere with the TRBP-Dicer-bound siRNA complex at the level of RISC loading and that it intervenes at the step in which Dicer and TRBP contact the different ends of siRNA. By using mutants with mutations in the N terminus region of VP30, we have excluded the possibility that the RNA binding activity of the protein participates in RNAi suppression (Fig. ).
We tested the hypothesis that VP30 interacts with the RNase IIIb domain by using mutant proteins with mutations in the C-terminal region of VP30, carrying alanine changes in the residues contacting the Dicer RNA-binding groove at or near the Mg ion-binding sites of Dicer. VP30 mutant proteins corresponding to the three predicted docking configurations of the VP30 RNase IIIb domain behave like the wild-type VP30 protein in reverting siRNA RNAi (Fig. ), suggesting either that these residues of VP30 do not mediate the effect of VP30 to block RNAi or that RNAi suppression by VP30 does not require a direct interaction of the viral protein with Dicer. Our result excludes the possibility that the interaction of VP30 with the Dicer RNase IIIb domain limits further interactions of Dicer with the Piwi domain of Ago2 (58
) or with other RNAi components (Ago2-PACT). It might be possible that the viral protein interacts with other Dicer domains (e.g., PAZ). Alternatively, VP30 can directly interact with the TRBP-siRNA complex with which Dicer is in contact.
EBOV has developed redundant mechanisms to counterattack host RNAi-based immunity. We found a third SRS, the EBOV matrix protein VP40. Preliminary experiments show that VP40 was not seen in association with siRNA protein complexes, suggesting that it may function by a different mechanism. It has been shown that plant-infecting viruses encoding multiple SRSs may disable host RNAi in a temporal and cellular location-related manner during infection (43
). In mammalian cells, two Dicer-Ago2 complexes have been found to localize mainly in the cytosol and with a small but a significant fraction with the membrane (25
). We speculate a mode of action in which VP40 may recruit components of RNAi that are associated with the membranes but at levels too low for detection by immunoprecipitation. This is in accordance with the role played by VP40 during the late stages of the virus life cycle, when it interacts with the RNP complex and binds to the membrane during budding and virion release. Further studies will help to dissect the molecular mechanism by which VP40 blocks RNAi.
So far, mammalian RNAi suppressors have been studied by using overexpressed systems. Here, we demonstrate that overexpressed EBOV proteins suppress an experimentally initiated RNAi. One limitation of our study is that overexpressed proteins cannot address the physiological relevance of EBOV suppressors during infection. In addition, an overexpressed system limits the possibility to study in a temporal manner the role of a protein, such as the matrix protein VP40, known to be expressed later during infection, after the less abundant VP35 (13
Here, we show that EBOV has evolved a mechanism to subvert RNAi, similar to what has been reported for influenza A virus and HIV-1 (3
). It should be noted that EBOV replication and subsequent lethal effects in primates can be reduced by treatment with siRNA (18
). However, siRNA treatment is required over time to maintain the efficacy, demonstrating a balance between viral replication and innate immune response by the host (18
) and a potential role for EBOV SRSs.
EBOV is the first mammalian virus for which more than one SRS has been identified. So far, Citrus tristeza virus
(CTV) is the only known plant-infecting virus to possess a sophisticated mechanism of RNA silencing suppression by encoding three SRSs (43
). Regardless of genome polarity and the targeted host, EBOV- and CTV-encoded functions are either present early in the life cycle or abundantly expressed. SRSs from unrelated viruses have no sequence or structural similarity, even though they may have similar biochemical functions, suggesting that they have evolved independently (36
). Consistent with this hypothesis, we observed that CTV p23 and EBOV VP30 possess an RNA binding activity within a region containing basic residues and a zinc finger domain (27
), suggesting that this is unlikely to be a mere coincidence; rather, it might be an example of an evolutionary convergence by which SRSs evolved independently such that they share a common function in distinct protein folds.
Why does EBOV have multiple SRSs? The simplest explanation may be grounded in the fact that EBOV contains multiple, distinct RNA-binding proteins that offer several opportunities to evolve antagonistic binding against host RNAi machinery. All three EBOV suppressors display potential unique binding specificities. We have confirmed this hypothesis by asking whether secondary structural elements related to the αβββα fold common to dsRBPs are present in EBOV SRSs. None of the three EBOV SRSs contain a dsRBD with the αβββα architecture. Thus, the absence of the canonical dsRBD fold suggests that RNAi suppressors undergo an independent evolution of protein folds to be able to bind RNAi activators.
Virus replication results in the release of many RNAs into the host cell cytosol. To prevent activation of RNA silencing, it may be more efficient for viruses to block RNAi protein effectors than to block the RNA stimuli, the latter of which could adversely affect virus propagation in the host. The identification of three viral SRSs demonstrates the importance of RNA-based immunity in the evolution of RNA viruses and provides important insight into viral pathogenesis and host defenses.