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Viruses frequently carry out replication in specialized compartments within cells. The effect of these structures on virus replication is poorly understood. Recent research supports phase separation as a foundational principle for organization of cellular components with the potential to influence viral replication. In this review, phase separation is described in the context of formation of viral replication centers, with an emphasis on the nonsegmented negative-strand RNA viruses. Consideration is given to the interplay between phase separation and the critical processes of viral transcription and genome replication, and the role of these regions in pathogen-host interactions is discussed. Finally, critical questions that must be addressed to fully understand how phase separation influences viral replication and the viral life cycle are presented, along with information about new approaches that could be used to make important breakthroughs in this emerging field.

[Image: see text] Nipah virus (NiV) is an emerging and deadly zoonotic paramyxovirus that is responsible for periodic epidemics of acute respiratory illness and encephalitis in humans. Previous studies have shown that the NiV V protein antagonizes host antiviral immunity, but the molecular mechanism is incompletely understood. To address this gap, we biochemically characterized NiV V binding to the host pattern recognition receptor MDA5. We find that the C-terminal domain of NiV V (V(CTD)) is sufficient to bind the MDA5(SF2) domain when recombinantly co-expressed in bacteria. Analysis by hydrogen–deuterium exchange mass spectrometry (HDX-MS) studies revealed that NiV V(CTD) is conformationally dynamic, and binding to MDA5 reduces the dynamics of V(CTD). Our results also suggest that the β-sheet region in between the MDA5 Hel1, Hel2, and Hel2i domains exhibits rapid HDX. Upon V(CTD) binding, these β-sheet and adjacent residues show significant protection. Collectively, our findings suggest that NiV V binding disrupts the helicase fold and dynamics of MDA5 to antagonize host antiviral immunity.

Background and objective Few head-to-head evaluations of immune responses to different vaccines have been reported. Methods Surrogate virus neutralization test (sVNT) antibody levels of adults receiving either two doses of BNT162b2 (n = 366) or CoronaVac (n = 360) vaccines in Hong Kong were determined. An age-matched subgroup (BNT162b2 [n = 49] vs. CoronaVac [n = 49]) was tested for plaque reduction neutralization (PRNT) and spike-binding antibody and T-cell reactivity in peripheral blood mononuclear cells. Results One month after the second dose of vaccine, BNT162b2 elicited significantly higher PRNT50 , PRNT90 , sVNT, spike receptor binding, spike N-terminal domain binding, spike S2 domain binding, spike FcR binding and antibody avidity levels than CoronaVac. The geometric mean PRNT50 titres in those vaccinated with BNT162b2 and CoronaVac vaccines were 251.6 and 69.45, while PRNT90 titres were 98.91 and 16.57, respectively. All of those vaccinated with BNT162b2 and 45 (91.8%) of 49 vaccinated with CoronaVac achieved the 50% protection threshold for PRNT90. Allowing for an expected seven-fold waning of antibody titres over 6 months for those receiving CoronaVac, only 16.3% would meet the 50% protection threshold versus 79.6% of BNT162b2 vaccinees. Age was negatively correlated with PRNT90 antibody titres. Both vaccines induced SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at 1 month post-vaccination but CoronaVac elicited significantly higher structural protein-specific CD4+ and CD8+ T-cell responses. Conclusion Vaccination with BNT162b2 induces stronger humoral responses than CoronaVac. CoronaVac induces higher CD4+ and CD8+ T-cell responses to the structural protein than BNT162b2.

People infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes.

Oropouche orthobunyavirus (OROV; Peribunyaviridae ) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein–related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience's Empower microplate reader, and microarray analysis software. Sample volumes of 1 μL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by100-fold (apparent

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids., Graphical Abstract

BackgroundFew head-to-head evaluations of immune responses to difference vaccines have been reported.MethodsSurrogate virus neutralization test (sVNT) antibody levels of adults receiving either 2 doses of BNT162b2 (n=366) or CoronaVac (n=360) vaccines in Hong Kong were determined. An age-matched subgroup (BNT162b2 (n=49) vs CoronaVac (n=49)) were tested for plaque reduction neutralizing (PRNT) and spike binding antibody and T cell reactivity in peripheral blood mononuclear cells (PBMC).FindingsOne month after the second dose of vaccine, BNT162b2 elicited significantly higher PRNT50, PRNT90, sVNT, spike receptor binding, spike N terminal domain binding, spike S2 domain binding, spike FcR binding and antibody avidity levels than CoronaVac. The geometric mean PRNT50 titres in those vaccinated with BNT162b2 and CoronaVac vaccines were 251.6 and 69.45 while PRNT90 titres were 98.91 and 16.57, respectively. All of those vaccinated with BNT162b2 and 45 (91.8%) of 49 vaccinated with CoronaVac achieved the 50% protection threshold for PRNT90. Allowing for an expected seven-fold waning of antibody titres over six months for those receiving CoronaVac, only 16.3% would meet the 50% protection threshold versus 79.6% of BNT162b2 vaccinees. Age was negatively correlated with PRNT90 antibody titres. Both vaccines induced SARS-CoV-2 specific CD4+ and CD8+ T cell responses at 1-month post-vaccination but CoronaVac elicited significantly higher structural protein-specific CD4+ and CD8+ T cell responses.ConclusionVaccination with BNT162b2 induces stronger humoral responses than CoronaVac. CoronaVac induce higher CD4+ and CD8+ T cell responses to the structural protein than BNT162b2.Summary At a GlanceThrough the head-to-head comparison, vaccination with BNT162b2 induces significantly higher levels of SARS-CoV-2 specific binding and neutralizing antibody responses when compared to CoronaVac. CoronaVac induce higher CD4+ and CD8+ T cell responses to the structural protein than BNT162b2.

Rift Valley Fever Virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific clusters Lrp1 and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAP(D3)) and anti-Lrp1 antibodies neutralize RVFV infection in taxonomically diverse cell lines. Mice treated with mRAP(D3) and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Altogether, these data support Lrp1 as a host entry factor for RVFV infection and defines a new target to limit RVFV infections.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

Marburg virus causes sporadic outbreaks of severe hemorrhagic fever with high case fatality rates. Approved, effective, and safe therapeutic or prophylactic countermeasures are lacking. To address this, we used phage display to engineer a synthetic antibody, sFab H3, which binds the Marburg virus VP35 protein (mVP35). mVP35 is a critical cofactor of the viral replication complex and a viral immune antagonist. sFab H3 displayed high specificity for mVP35 and not for the closely related Ebola virus VP35. sFab H3 inhibited viral-RNA synthesis in a minigenome assay, suggesting its potential use as an antiviral. We characterized sFab H3 by a combination of biophysical and biochemical methods, and a crystal structure of the complex solved to 1.7 Å resolution defined the molecular interface between the sFab H3 and mVP35 interferon inhibitory domain. Our study identifies mVP35 as a therapeutic target using an approach that provides a framework for generating engineered Fabs targeting other viral proteins.

Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNβ messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

The trimeric SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and TMPRSS2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. Additionally, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high profile therapeutic target.

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell–cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2–infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell–cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell–cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell–cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell–cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.

SUMMARY Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV., In brief Pei et al. show that NS1 is present in the nucleus in primary airway epithelial, MDDCs, and A549 cells upon RSV infection. This study finds that NS1 functions as an immune antagonist by binding chromatin at the promoters and enhancers of immune response genes and modulating host gene transcription., Graphical Abstract

Nucleocapsid (N) encoded by SARS-CoV-2 plays key roles in the replication cycle and is a critical serological marker. Here we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high affinity RNA binding platform. We also map the RNA binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose-coils, showing how N/RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based upon antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers., Graphical Abstract

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs. In these interactions the largely alpha-helical carboxy-terminal domain of the EBOV VP30 engages with the motif such that the prolines adopt non-canonical orientations, as compared to other proline-rich motifs. Affinity tag-purification mass spectrometry identified additional PPxPxY-containing host proteins, including hnRNP L, hnRNPUL1 and PEG10, as VP30 interactors. Of these, hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV replication, whereas hnRNPUL1 enhances. Further, double knockdown studies support additive effects of RBBP6 and hnRNP L. Binding studies demonstrate variable capacity of PPxPxY motifs to bind VP30 and the extended motif PxPPPPxY is demonstrated to confer optimal binding and to inhibit RNA synthesis, with the fifth proline and the tyrosine being most critical. Competition binding and hydrogen-deuterium exchange studies demonstrate that each protein binds a similar interface on VP30 and impacts VP30 phosphorylation. VP30 therefore represents a novel proline recognition domain that allows multiple host proteins to target a single viral protein-protein interface to modulate viral transcription.

BACKGROUND: Several vaccine platforms have been successfully evaluated for prevention of Ebola virus (EBOV) disease (EVD) in nonhuman primates and humans. Despite remarkable efficacy by multiple vaccines, the immunological correlates of protection against EVD are incompletely understood. METHODS: We systematically evaluated the antibody response to various EBOV proteins in 79 nonhuman primates vaccinated with various EBOV vaccine platforms. We evaluated the serum immunoglobulin (Ig)G titers against EBOV glycoprotein (GP), the ability of the vaccine-induced antibodies to bind GP at acidic pH or to displace ZMapp, and virus neutralization titers. The correlation of these outcomes with survival from EVD was evaluated by appropriate statistical methods. RESULTS: Irrespective of the vaccine platform, protection from EVD strongly correlated with anti-GP IgG titers. The GP-directed antibody levels required for protection in animals vaccinated with virus-like particles (VLPs) lacking nucleoprotein (NP) was significantly higher than animals immunized with NP-containing VLPs or adenovirus-expressed GP, platforms that induce strong T-cell responses. Furthermore, protective immune responses correlated with anti-GP antibody binding strength at acidic pH, neutralization of GP-expressing pseudovirions, and the ability to displace ZMapp components from GP. CONCLUSIONS: These findings suggest key quantitative and qualitative attributes of antibody response to EVD vaccines as potential correlates of protection.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 -/- mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 -/- mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.

The pattern recognition receptors RIG-I-like receptors (RLRs) are critical molecules for cytosolic viral recognition and for subsequent activation of type I interferon production. The interferon signaling pathway plays a key role in viral detection and generating antiviral responses. Among the many pathogens, the non-segmented negative sense RNA viruses target the RLR pathway using a variety of mechanisms. Here, I review the current state of knowledge on the molecular mechanisms that allow non-segmented negative sense RNA virus recognition and antagonism of RLRs.

To develop an antibody (Ab) therapeutic against Staphylococcal Enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “Class T” anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “Class M” Abs. A systematic engineering approach was applied to affinity mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) revealed that the Class T epitope on SEB overlapped with the T-cell receptor (TCR) binding site, whereas other evidence suggested that the Class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ~50-fold more potent toxin-neutralizing efficacy than the best Class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two Class M binding sites and two Class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that Class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bispecific Ab engineering.

Members of Mononegavirales, the order including non-segmented negative sense RNA viruses (NNSV), encode a small number of multifunctional proteins. In members of the Filoviridae family, virus protein 35 (VP35) facilitates immune evasion and functions as an obligatory co-factor for viral RNA-synthesis. VP35 functions in an orthologous manner to phosphoproteins (P proteins) from other NNSVs. Although the critical roles of Ebola viral VP35 (eVP35) in immune evasion and RNA synthesis are well-appreciated, a complete understanding of its organization its role in carrying out its many functions has yet to be fully realized. In particular, we currently lack information on the role of the oligomerization domain within eVP35. To address this limitation, we report here an investigation of the oligomer structure of eVP35 using hybrid methods that include multi-angle light scattering (MALS), small angle X-ray scattering (SAXS), and crosslinking coupled with mass spectrometry (XL-MS) to determine the shape and orientation of the eVP35 oligomer. Our integrative results are consistent with a parallel tetramer, in which the N-terminal regions that are required for RNA synthesis are all oriented in the same direction. Furthermore, these results define a framework to target the symmetric tetramer for structure-based antiviral discovery.

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 biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV viral protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.