Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient’s leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient’s dermal fibroblasts and induced pluripotent stem cell (iPSC)–derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

During the last few decades, the global incidence of dengue virus (DENV) has increased dramatically, and it is now endemic in more than 100 countries. To establish a productive infection in humans, DENV uses different strategies to inhibit or avoid the host innate immune system. Several DENV proteins have been shown to strategically target crucial components of the type I interferon system. Here, we report that the DENV NS2B protease cofactor targets the DNA sensor cyclic GMP-AMP synthase (cGAS) for lysosomal degradation to avoid the detection of mitochondrial DNA during infection. Such degradation subsequently results in the inhibition of type I interferon production in the infected cell. Our data demonstrate a mechanism by which cGAS senses cellular damage upon DENV infection.

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.

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.

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, encodes a small hydrophobic (SH) protein of unknown function. Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, also encodes an SH protein, which inhibits tumor necrosis factor alpha (TNF-␣) signaling. In this study, recombinant PIV5 viruses without their own SH but containing RSV SH (from RSV strain A2 or B1) in its place (PIV5⌬SH-RSV SH) and RSV lacking its own SH (RSV⌬SH) were generated and analyzed. The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-␣ signaling.Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children (17). RSV, along with the prototype paramyxovirus parainfluenza virus 5 (PIV5; formerly known as simian virus 5), is a member of the Paramyxoviridae family, which includes important human and animal pathogens. Both RSV and PIV5 encode small hydrophobic (SH) proteins, which are type II transmembrane proteins. The SH protein of RSV contains 64 (RSV subgroup A) or 65 (RSV subgroup B) amino acid residues (Fig. 1A) (3)(4)(5)14). Some studies have suggested that the RSV SH protein may have a role in viral fusion (9, 19) or in changing membrane permeability (15). However, RSV lacking the SH gene (RSV⌬SH) is viable, causes syncytium formation, and grows as well as the wild-type virus (1, 10, 11), indicating that the SH protein is not necessary for virus entry into host cells or syncytium formation (19). RSV⌬SH is attenuated in animals, indicating that RSV plays an important role in viral pathogenesis (1). Interestingly, recombinant PIV5 lacking the SH gene (rPIV5⌬SH) has a similar phenotype: it has normal growth in vitro, but it is attenuated in vivo (7). Studies of rPIV5⌬SH have shown that the SH protein is necessary for the inhibition of tumor necrosis factor alpha (TNF-␣)-induced apoptosis in L929 cells (12). Recent work suggests that the SH protein of mumps virus is a functional counterpart of the PIV5 SH protein (22), even though the PIV5 and mumps SH proteins have no sequence homology. We hypothesized that the SH protein of RSV may be functionally similar to other SH proteins from members of the Paramyxoviridae family. To test this hypothesis, recombinant viruses that contained the RSV SH gene of strain A2 or B1 in place of the PIV5 SH gene were produced and confirmed by reverse transcription (RT)-PCR (Fig. 1B). The rPIV5 and rPIV5⌬SH viruses grow to similar titers, although rPIV5⌬SH virus grows slightly faster in the first stages of infection (Fig. 1C) (6, 22). Growth of the rPIV5⌬SH-RSV SH recombinant viruses was comparable to that of rPIV5 and rPIV5⌬SH up to 2 days postinfection (dpi). Occasionally, a delay in the growth of one or both of the recombinant viruses was observed, but by 24 or 36 h the viruses had always reached titers comparable to that of the wild-type virus (Fig. 1C). The plaques formed by the rPIV5, rPIV5⌬SH, and rPIV5⌬SH-RSV SH viruses in BHK cells were of a...

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-β and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients’ iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN–mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.

SUMMARY Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologies that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.KEYWORDS TRIM6, tripartite motif (TRIM) protein, VP35, viral RNA polymerase, Ebola virus, innate immunity, ubiquitination, unanchored ubiquitin, virus-host interactions