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

Abstract

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/ beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs

“…Previous reports have shown that transient overexpression of the P19, NS3, VP35 and E3L proteins can suppress induced RNAi directed against reporter gene constructs in mammalian cells (Dunoyer et al, 2004;Haasnoot et al, 2007;Liu et al, 2012;Schnettler et al, 2008;Zhu et al, 2012). In addition, VP35, E3L and NS3 can complement the RNAi suppressor function of the HIV-1 Tat protein to support HIV-1 replication (Haasnoot et al, 2007;Schnettler et al, 2009).…”

Camouflage and Misdirection: The Full-On Assault of Ebola Virus Disease

“…While subsequent studies found that the C-terminal domain of VP35 bound to siRNA and not to dsRNA, the RNAi silencing suppression activity of VP35 did not correlate with binding to siRNA. It was hypothesized that suppression via RNA binding-independent mechanisms could instead occur through binding to the RISC complex or sequestration of RISC complex proteins prior to their incorporation into the complex (Zhu et al, 2012). This theory is supported by prior experiments that found VP35 interacts with transactivation response RNA-binding protein (TRBP) and PACT (Fabozzi et al, 2011), both components of the RISC RNAi complex and thus proposed to mediate the VP35-dependent RNA silencing suppressor activity.…”

Mutual Antagonism between the Ebola Virus VP35 Protein and the RIG-I Activator PACT Determines Infection Outcome

“…The VP35 proteins are innate immune antagonists that target RIG-I-like receptors (RLRs), PKR, and RNA silencing activity (Bale et al, 2012; Basler et al, 2003; Basler et al, 2000; Cardenas et al, 2006; Fabozzi et al, 2011; Feng et al, 2007; Haasnoot et al, 2007; Ramanan et al, 2012; Schümann et al, 2009; Zhu et al, 2012). RLRs detect viral nucleic acids and signal to induce an interferon (IFN)-α/β response (Leung et al, 2012).…”