Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30-eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30-eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.

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 Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

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 (VCTD) is sufficient to bind the MDA5SF2 domain when recombinantly co-expressed in bacteria. Analysis by hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies revealed that NiV VCTD is conformationally dynamic, and binding to MDA5 reduces the dynamics of VCTD. Our results also suggest that the β-sheet region in between the MDA5 Hel1, Hel2, and Hel2i domains exhibits rapid HDX. Upon VCTD 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.

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

Kelch-like ECH-associated protein 1 (Keap1) is a ubiquitin E3 ligase specificity factor that targets transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for ubiquitination and degradation. Disrupting Keap1-Nrf2 interaction stabilizes Nrf2, resulting in Nrf2 nuclear accumulation, binding to antioxidant response elements (AREs), and transcription of cytoprotective genes. Marburg virus (MARV) is a zoonotic pathogen that likely uses bats as reservoir hosts. We demonstrate that MARV protein VP24 (mVP24) binds the Kelch domain of either human or bat Keap1. This binding is of high affinity and 1:1 stoichiometry and activates Nrf2. Modeling based on the Zaire ebolavirus (EBOV) VP24 (eVP24) structure identified in mVP24 an acidic loop (K-loop) critical for Keap1 interaction. Transfer of the K-loop to eVP24, which otherwise does not bind Keap1, confers Keap1 binding and Nrf2 activation, and infection by MARV, but not EBOV, activates ARE gene expression. Therefore, MARV targets Keap1 to activate Nrf2-induced cytoprotective responses during infection.

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 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.

Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (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 on 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.

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.

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. IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

Non-retroviral integrated RNA viral sequences (NIRVs) potentially encoding ∼280 amino acid homologs to filovirus VP35 proteins are present across the Myotis genus of bats. These are estimated to have been maintained for ∼18 million years, indicating their co-option. To address the reasons for co-option, 16 Myotis VP35s were characterized in comparison to VP35s from the extant filoviruses Ebola virus and Marburg virus, in which VP35s play critical roles in immune evasion and RNA synthesis. The Myotis VP35s demonstrated a conserved suppression of innate immune signaling, albeit with reduced potency, in either human or Myotis cells. Their attenuation reflects a lack of dsRNA binding that in the filoviral VP35s correlates with potent suppression of interferon responses. Despite divergent function, evolution has preserved in Myotis the structure of the filoviral VP35s, indicating that this structure is critical for co-opted function, possibly as a regulator of innate immune signaling.

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.

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.

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.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.

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, residues 20-48) 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.

Although interferon-induced proteins with tetratricopeptide repeats (IFIT proteins) inhibit infection of many viruses by recognizing their RNA, the regulatory mechanisms involved remain unclear. Here we report a crystal structure of cap 0 (m7GpppN) RNA bound to human IFIT1 in complex with the C-terminal domain of human IFIT3. Structural, biochemical, and genetic studies suggest that IFIT3 binding to IFIT1 has dual regulatory functions: (1) extending the half-life of IFIT1 and thereby increasing its steady-state amounts in cells; and (2) allosterically regulating the IFIT1 RNA-binding channel, thereby enhancing the specificity of recognition for cap 0 but not cap 1 (m7GpppNm) or 5'-ppp RNA. Mouse Ifit3 lacks this key C-terminal domain and does not bind mouse Ifit1. The IFIT3 interaction with IFIT1 is important for restricting infection of viruses lacking 2'-O methylation in their RNA cap structures. Our experiments establish differences in the regulation of IFIT1 orthologs and define targets for modulation of human IFIT protein activity.

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.IMPORTANCE Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.

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. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).