Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

Activation of alpha/beta interferon (IFN-α/β) production is a key step in the innate immune response to viral infection. Double-stranded RNA (dsRNA) has long been used as an experimental inducer of IFN-α/β and is potentially synthesized during the replication of many viruses. Thus, viral dsRNA has been hypothesized to be a trigger of cellular antiviral responses (29). A number of cellular dsRNA recognition proteins have been implicated in the IFN-induced antiviral response to infection. These include the dsRNA-dependent protein kinase PKR, the 2′,5′-oligoadenylate synthase, and ADAR1 (20, 44, 59, 63). More recently, two IFN-induced, caspase recruiting domain (CARD)-containing, DExD/H family helicases, the retinoic acid-inducible gene I (RIG-I) protein and the melanoma differentiation-associated gene 5 (MDA-5) protein, have been implicated as key sensors of viral infection (1, 30, 56, 66, 67). These proteins are activated by viral infection, possibly through recognition of dsRNA or of ribonucleoprotein complexes produced during infection, and transduce downstream signaling to activate the IFN-α/β responses (34). The transcription factor interferon regulatory factor 3 (IRF-3) plays a critical role in the activation of the IFN-α/β gene. A cytoplasmic protein in its inactive state, IRF-3 becomes hyperphosphorylated on serine and threonine residues, dimerizes, and accumulates in the nucleus, where it participates in initial IFN-α/β gene expression (37, 68, 69). RIG-I and MDA-5 activate IRF-3 in response to dsRNA or to viral infection upstream of the IRF-3 kinases TBK-1 and IKKɛ (18, 30, 54, 66). This signaling appears to involve the homotypic interaction of the CARDs of the helicases with another CARD-containing protein termed alternatively IPS-1, MAVS, VISA, or Cardif (31, 41, 53, 65). Viruses have evolved a variety of mechanisms to avoid recognition or to block the antiviral responses mediated by IFNs (2, 19). Several such proteins also exhibit dsRNA-binding activity. Examples of dsRNA-binding proteins that counteract cellular antiviral responses include the NS1 proteins of influenza A (NS1A) and B (NS1B) viruses, the E3L protein of vaccinia virus, the σ3 protein of reovirus, and the pTRS1 protein of human cytomegalovirus (5, 11, 12, 14, 16, 17, 22, 27, 57). Although dsRNA binding appears to contribute to IFN antagonist function, NS1A, NS1B, and E3L possess additional dsRNA-binding-independent mechanisms to inhibit the cellular antiviral response. The Ebola virus (EBOV) VP35 protein has been found to inhibit IFN-α/β production and the activation of IRF-3 (3, 4, 9, 23, 48). Recently, it was suggested that VP35 may contain a dsRNA-binding motif similar to that found in the NS1 protein of influenza A virus (23). It was further suggested that this dsRNA-binding activity may be required for the ability of VP35 to inhibit IFN production (23). Here, we provide evidence that VP35 has dsRNA-binding activity that may contribute to IFN antagonism. It is likely that VP35 also possesses a dsRNA-binding-independent mechanism(s) that targets a point at or downstream of the IRF-3 kinases TBK-1 and IKKɛ.