Summary The VP35 protein encoded by the highly pathogenic Ebola virus facilitates immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I like receptors. Here we report the crystal structure of Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which reveals how VP35-dsRNA interactions contribute to immune evasion, and corresponding in vivo studies. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are “end-capped” by a pocket of hydrophobic residues that mimics blunt end dsRNA recognition by RIG-I-like receptors. Residues that are critical for RNA binding are also important for interferon inhibition in vivo, but not for viral polymerase co-factor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

SUMMARY During anti-viral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C-terminus in complex with eVP24. In the complex, eVP24 recognizes a unique non-classical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that maybe required for viral replication unaffected.

SUMMARY During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

Reactive oxygen species (ROS) are generated by virally-infected cells however the physiological significance of ROS generated under these conditions is unclear. Here we show that inflammation and cell death induced by exposure of mice or cells to sources of ROS is not altered in the absence of canonical ROS-sensing pathways or known cell death pathways. ROS-induced cell death signaling involves interaction between the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5−/− mice show exacerbated lung inflammation and proinflammatory cytokines in an ozone exposure model. Similarly, challenge with influenza A virus leads to increased virus infiltration, lymphocytic bronchiolitis and reduced survival of Pgam5−/− mice. This pathway, which we term ‘oxeiptosis’, is a ROS-sensitive, caspase independent, non-inflammatory cell death pathway and is important to protect against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

SUMMARY The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.