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.

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.

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.

The COVID-19 pandemic has been accompanied by the largest mobilization of therapeutic convalescent plasma (CCP) in over a century. Initial identification of high titer units was based on dose-response data using the Ortho VITROS IgG assay. The proliferation of SARS-CoV-2 serological assays and non-uniform application has led to uncertainty about their interrelationships. The purpose of this study was to establish correlations and analogous cutoffs between commercially available serological tests (Ortho, Abbott, Roche), a spike ELISA, and a virus neutralization assay using convalescent plasma from a cohort of 79 donors from April 2020. Relationships relative to FDA-approved cutoffs under the CCP EUA were identified by linear regression and receiver operator characteristic curves. Relative to the Ortho VITROS assay, the r 2 of the Abbott, Roche, the anti-Spike ELISA and the neutralizing assay were 0.58, 0.5, 0.82, and 0.44, respectively. The best correlative index for establishing high-titer units was 3.82 S/C for the Abbott, 10.89 COI for the Roche, 1:1,202 for the anti-Spike ELISA, and 1:200 by the neutralization assay. The overall agreement using derived cutoffs compared to the CCP EUA Ortho VITROS cutoff of 9.5 was 92.4% for Abbott, 84.8% for Roche, 87.3% for the anti-S ELISA and 78.5% for the neutralization assay. Assays based on antibodies against the nucleoprotein (Roche, Abbott) and neutralizing antibody tests were positively associated with the Ortho assay, although their ability to distinguish FDA high-titer specimens was imperfect. The resulting relationships help reconcile results from the large body of serological data generated during the COVID-19 pandemic.

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.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.

The experience of individuals with Coronavirus Disease 2019 (COVID-19) ranges from asymptomatic to life threatening multi-organ dysfunction. Specific HLA alleles may affect the predisposition to severe COVID-19 because of their role in presenting viral peptides to launch the adaptive immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this population-based case-control study in the midwestern United States, we performed high-resolution HLA typing of 234 cases hospitalized for COVID-19 in the St. Louis metropolitan area and compared their HLA allele frequencies with those of 22,000 matched controls from the National Marrow Donor Program (NMDP). We identified two predisposing alleles, HLA-DRB1*08:02 in the Hispanic group (OR = 9.0, 95% confidence interval: 2.2-37.9; adjusted p = 0.03) and HLA-A*30:02 in younger African Americans with ages below the median (OR = 2.2, 1.4-3.6; adjusted p = 0.01), and several candidate alleles with potential associations with COVID-19 in African American, White, and Hispanic groups. We also detected risk-associated amino acid residues in the peptide binding grooves of some of these alleles, suggesting the presence of functional associations. These findings support the notion that specific HLA alleles may be protective or predisposing factors to COVID-19. Future consortium analysis of pooled cases and controls is warranted to validate and extend these findings, and correlation with viral peptide binding studies will provide additional evidence for the functional association between HLA alleles and COVID-19.

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.

Reported coronavirus disease 2019 (COVID-19) case counts likely underestimate the true prevalence because mild or asymptomatic cases often go untested. Here, we use a sero-survey to estimate the seroprevalence of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the St. Louis, MO, metropolitan area in a symptom-independent manner. Five hundred three adult and 555 pediatric serum/plasma samples were collected from patients presenting to Barnes-Jewish Hospital or St. Louis Children's Hospital between 14 April 2020 and 12 May 2020. We developed protocols for in-house enzyme-linked immunosorbent assays (ELISAs) using spike and nucleoprotein and used the assays to estimate a seroprevalence rate based on our samples. Overall IgG seropositivity was estimated to be 1.71% (95% credible interval [CI], 0.04% to 3.38%) in pediatric samples and 3.11% (95% CI, 0.92% to 5.32%) in adult samples. Seropositivity was significantly lower in children under 5 years of age than in adults, but rates between adults and children aged 5 or older were similar. Of the 176 samples tested from children under 4 years of age, none were positive.IMPORTANCE This study determined the percentages of both children and adult samples from the greater St. Louis metropolitan area who had antibodies to SARS-CoV-2 in late April to early May 2020. Approximately 1.7 to 3.1% of the tested individuals had antibodies, indicating that they had previously been infected by SARS-CoV-2. These results demonstrate that the extent of infection was about 10 times greater than the number of confirmed cases at that time. Furthermore, it demonstrated that by 5 years of age, children were infected to an extent similar to that of adults.

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.

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.

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.

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.

The COVID-19 pandemic has been accompanied by the largest mobilization of therapeutic convalescent plasma (CCP) in over a century. Initial identification of high titer units was based on dose-response data using the Ortho VITROS IgG assay. The proliferation of severe acute respiratory syndrome coronavirus 2 serological assays and non-uniform application has led to uncertainty about their interrelationships. The purpose of this study was to establish correlations and analogous cutoffs between multiple serological assays.

SARS-CoV-2 triggers a complex systemic immune response in circulating blood mononuclear cells. The relationship between immune cell activation of the peripheral compartment and survival in critical COVID-19 remains to be established. Here we use single-cell RNA sequencing and Cellular Indexing of Transcriptomes and Epitomes by sequence mapping to elucidate cell type specific transcriptional signatures that associate with and predict survival in critical COVID-19. Patients who survive infection display activation of antibody processing, early activation response, and cell cycle regulation pathways most prominent within B-, T-, and NK-cell subsets. We further leverage cell specific differential gene expression and machine learning to predict mortality using single cell transcriptomes. We identify interferon signaling and antigen presentation pathways within cDC2 cells, CD14 monocytes, and CD16 monocytes as predictors of mortality with 90% accuracy. Finally, we validate our findings in an independent transcriptomics dataset and provide a framework to elucidate mechanisms that promote survival in critically ill COVID-19 patients. Identifying prognostic indicators among critical COVID-19 patients holds tremendous value in risk stratification and clinical management.