Biology subjects

Davey, R. A.

Publications and source records attributed to Davey, R. A..

14 recordsLinked to original sources

bioRxiv2026

Substrate and target selectivity of 4'-fluoroadenosine against viral and host polymerases

Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4'-fluorouridine (4'-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4'-fluoroadenosine (4'-FlA). Like 4'-FlU, 4'-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics show that the triphosphate (4'-FlA-TP) is efficiently incorporated by viral RdRps. A cryo-EM structure of RdRp of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in complex with double-stranded RNA and the incorporated monophosphate (4'-FlA-MP) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4'-FlA-MP inhibit incorporation of complementary UTP across the viral RdRps. However, incorporation of 4'-FIA-TP is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4'-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.

microbiology↗

bioRxiv2026

A Yeast Two-Hybrid Protein Domain Screening Approach for Ebola Virus-Human Protein Interactions Identifies PABPC1 as a Host Factor Required for Replication

Ebola virus (EBOV) causes severe hemorrhagic fever and remains a global health threat despite advances in vaccines and antibody-based therapeutics. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet the full spectrum of these interactions is unknown. To systematically identify EBOV-human protein-protein interactions, we performed more than 200 yeast two-hybrid (Y2H) screens using full-length EBOV proteins and domain fragments as baits and cDNA libraries derived from human liver tissue, macrophages, and interferon-stimulated macrophages. This approach revealed 521 unique interactions supported by confidence scoring based on retest assays and library screen data. The interactome includes proteins involved in RNA metabolism, transcriptional regulation, and post-translational modification, with enrichment for RNA-binding proteins among partners of NP, VP30, and L. By employing multiple constructs for each EBOV gene, interaction interfaces could be localized to specific viral domains. This approach identified a likely MYND domain binding site in the intrinsically ordered region of NP. Similarly, overlapping cDNA fragments from Y2H screens revealed potential binding sites within human proteins, including putative VP30 binding sites on NUFIP2 and PABPC1 that lacked canonical PPPPxY motifs. PABPC1 interacted with VP30 in uninfected human cells and accumulated in inclusion bodies in infected cells that resembled respiratory syncytial virus inclusion body associated granules (IBAGs). Inhibiting PABPC1 expression reduced EBOV replication early in infection. These findings expand the EBOV-host interaction network, identify candidate regulators of viral RNA synthesis, and provide a resource for mechanistic studies and antiviral target discovery. ImportanceEbola virus (EBOV) remains a major global health threat due to its high mortality rate and limited treatment options. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet these interactions are incompletely understood. This study uses a large-scale yeast two-hybrid approach to systematically map EBOV-human protein-protein interactions, revealing hundreds of previously unreported partners and identifying cellular pathways that may be exploited by the virus. By integrating domain mapping, confidence scoring, and functional validation, we provide a resource that advances understanding of EBOV biology and may be used to discover candidates for antiviral development.

microbiology↗

bioRxiv2025

Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling

Ebola virus (EBOV) is a non-segmented, negative-sense virus (NNSV) with a single-stranded RNA genome. EBOV encodes for a limited number of proteins and thus depends on host factors to facilitate viral replication and pathogenesis. Of the virus-encoded proteins, multifunctional EBOV VP35 (eVP35) is necessary for host immune evasion and viral RNA synthesis. Previous proteomics studies identified an interaction between eVP35 and the host E3 ubiquitin ligase Mindbomb 2 (MIB2). Here, we show how a previously uncharacterized NNLNS motif (residues 201-205) within eVP35 serves as a binding site for MIB2. This motif is critical for eVP35-dependent inhibition of MIB2-mediated IFN induction. It is also important for EBOV RNA synthesis as MIB2 binding to eVP35 inhibited EBOV minigenome activity. Altogether, these findings highlight the importance of the eVP35 protein and the role of host factors in EBOV infection. SIGNIFICANCE STATEMENTThe Ebola virus (EBOV) genome encodes for a limited number of proteins and depends on host factors to facilitate viral replication. Identification and characterization of host-viral interactions are needed to define infection, resolution, and to develop new therapeutics. EBOV VP35 (eVP35) is necessary for mediating host immune evasion and a cofactor for viral RNA synthesis. Here we characterized an interaction between eVP35 and MIB2. We show that the 201NNLNS205 motif in eVP35 is necessary and sufficient for MIB2 binding and inhibition of MIB2-mediated IFN production. Our results also reveal how the eVP35-MIB2 interaction impacts virus infection. These results support the importance of the multifunctional eVP35 to EBOV infection and highlight the significance of host proteins, including E3 ligases, during viral infection.

microbiology↗

bioRxiv2025

Ebola's Hidden Target: Virus Transmission to and Accumulation within Skin

Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), remains one of WHOs top ten threats to global health. Infectious EBOV virions can be found on the surface of skin late during systemic infection and passed from the deceased through skin-to-skin contact. Here, we assess viral load and antigen expression in the skin of EBOV-infected non-human primates (NHP) and mouse adapted-EBOV (ma-EBOV) - infected mice and use the low containment viral model, rVSV/EBOV GP, to mechanistically define skin infection in mice. Viral RNA peaked within the skin proximal to the site of injection in EBOV-infected NHPs on day 6. In contrast, mouse skin sites distal to the site of ma-EBOV injection achieved maximal viral loads by day 3. At late times of infection, viral antigen-positive cells co-localized with markers for endothelial, stromal, and immune cells in the dermis. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a potential mechanism of virus trafficking to the epidermal surface. Despite robust viral infection, distal skin sites of ma-EBOV-infected mice had low expression of proinflammatory stimulated genes. A similar cellular tropism was observed in the skin of mice infected with rVSV/EBOV GP, with discrete focal areas of intense infection. When virus was applied to the surface of gently abraded skin to remove the stratum corneum, epidermal keratinocytes were robustly infected, followed by systemic viral dissemination. To define cell surface receptors critical for virus trafficking to and replication within the skin, mice lacking the phosphatidylserine receptors were infected intraperitoneally with rVSV/EBOV GP. At day 3 of infection, skin distal to the site of infection of TIM-1 knock out (KO) mice had significantly lower levels of infectious virus than the control mice, suggesting that TIM-1 is essential for efficient distribution of virus to the skin. Our findings reveal that EBOV targets specific skin cell populations at late times of viral infection and that the host receptor TIM-1 is required for optimal viral dissemination.

microbiology↗

bioRxiv2024

Multiple dermal cell types support productive infection and dynamic translocation of infectious Ebola virus to the apical surface of human skin

Ebola virus (EBOV) within the Filoviridae family causes severe human disease. At late stages of infection, EBOV virions are found on the surface of patients skin; however, the permissive cell types within the skin and how infectious virus translocates to the apical skin surfaces is not known. Here, we describe a human transwell skin explant culture model and show that EBOV infection of human skin tissues via the basal media results in a time- and dose-dependent increase in infectious virus in dermal and epidermal tissue. Infectious virus was detected on the apical epidermal surface within 3 days, indicating that the virus propagates within and traffics through the tissue. In the dermis, EBOV-infected cells were of myeloid, endothelial and fibroblast origins, whereas keratinocytes harbored virus in the epidermis. Complementary studies showed that both purified skin fibroblasts and keratinocytes supported EBOV infection ex vivo and that both cell types required the phosphatidylserine receptor, Axl, and the endosomal protein, NPC1, for virus entry. Our experimental platform identified new susceptible cell types and demonstrated dynamic trafficking of EBOV virions that resulted in infectious virus on the skin surface; findings that may explain person-to-person transmission via skin contact. TeaserUsing a human skin explant model, these studies identify and characterize skin cell populations that support Ebola virus infection.

microbiology↗

bioRxiv2024

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 M. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

microbiology↗

bioRxiv2024

The Role of Ebola Virus VP24 Nuclear Trafficking Signals in Infectious Particle Production

Ebola virus (EBOV) protein VP24 carries out at least two critical functions. It promotes condensation of viral nucleocapsids, which is crucial for infectious virus production, and it suppresses interferon (IFN) signaling, which requires interaction with the NPI-1 subfamily of importin- (IMPA) nuclear transport proteins. Interestingly, over-expressed IMPA leads to VP24 nuclear accumulation and a carboxy-terminus nuclear export signal (NES) has been reported, suggesting that VP24 may undergo nuclear trafficking. For the first time, we demonstrate that NPI-1 IMPA overexpression leads to the nuclear accumulation of VP24 during EBOV infection. To assess the functional impact of nuclear trafficking, we generated tetracistronic minigenomes encoding VP24 nuclear import and/or export signal mutants. The minigenomes, which also encode Renilla luciferase and viral proteins VP40 and GP, were used to generate transcription and replication competent virus-like particles (trVLPs) that can be used to assess EBOV RNA synthesis, gene expression, entry and viral particle production. With this system, we confirmed that NES or IMPA binding site mutations altered VP24 nuclear localization, demonstrating functional trafficking signals. While these mutations minimally affected transcription and replication, the trVLPs exhibited impaired infectivity and formation of shortened nucleocapsids for the IMPA binding mutant. For the NES mutants, infectivity was reduced approximately 1000-fold. The NES mutant could still suppress IFN signaling but failed to promote nucleocapsid formation. To determine whether VP24 nuclear export is required for infectivity, the residues surrounding the wildtype NES were mutated to alanine or the VP24 NES was replaced with the Protein Kinase A Inhibitor NES. While nuclear export remained intact for these mutants, infectivity was severely impaired. These data demonstrate that VP24 undergoes nuclear trafficking and illuminates a separate and critical role for the NES and surrounding sequences in infectivity and nucleocapsid assembly. Author StatementEbola virus (EBOV) viral protein 24 (VP24) interacts with importin- (IMPA) nuclear transport proteins to inhibit interferon signaling. VP24 also contains a nuclear export signal (NES). The capacity to bind IMPA and the presence of a NES suggest that VP24 traffics to the nucleus. However, this had not been demonstrated in the context of EBOV replication. In addition, whether these signals influence virus replication beyond effects on interferon responses has been unclear. Here, we demonstrate that VP24 traffics to the nucleus via IMPA during EBOV infection and in the context of an EBOV transcription and replication competent virus-like particle (trVLP) assay. Using the trVLP system, we also confirmed that VP24 possesses a functional nuclear export signal (NES). Mutations disrupting VP24-IMPA interaction or nuclear export reduced or abrogated trVLP infectivity, respectively. VP24 is known to be necessary for viral nucleocapsid maturation. The VP24 IMPA interface mutants yielded shortened nucleocapsids while VP24 NES mutations led to loss of nucleocapsid formation. Interestingly, VP24 mutants with intact NES function but altered sequences near the NES remain incapable of producing nucleocapsids. Together, these data demonstrate VP24 undergoes nuclear trafficking and reveals additional roles for both the IMPA interaction interface and the NES in nucleocapsid assembly.

microbiology↗

bioRxiv2023

Design of universal Ebola virus vaccine candidates via immunofocusing

Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV) - one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here we describe a universal Ebola virus vaccine approach using structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species. SIGNIFICANCE STATEMENTEbola virus outbreaks cause hemorrhagic fever with high mortality rates. Current viral vaccines require cold-chain storage and are distributed in limited ring vaccination settings. They are only indicated for protection against orthoebolavirus zairense (EBOV), one of three human-pathogenic Ebola virus species. Here we harness hyperglycosylation as an immunofocusing approach to design universal Ebola virus vaccine candidates based on Ebola virus glycoprotein (GP) displayed on ferritin nanoparticles (Fer). Compared with wild-type GP-Fer, immunization with hyperglycosylated GP-Fer elicited potently neutralizing antisera against EBOV, and more importantly, consistent cross-neutralizing activity against the other two orthoebolavirus species. Our work shows that immunofocusing antibody responses toward conserved and neutralizing epitopes of GP represents a promising strategy for vaccine design against antigenically diverse Ebola virus species.

immunology↗

bioRxiv2022

CAPG is required for Ebola virus infection by controlling virus egress from infected cells.

Replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors such as Rac1 and ARP2/3, involved in actin nucleation were shown important in both steps. However, downstream of nucleation, many other cell factors, are needed to control actin dynamics. How these regulate EBOV infection remains largely unknown. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown (KD) of CAPG specifically inhibited viral infectivity and yield of infectious particles. Mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split GFP reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

microbiology↗

bioRxiv2022

Brequinar and Dipyridamole in Combination Exhibits Synergistic Antiviral Activity Against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of currently available vaccines and monoclonal antibody (mAb)-based treatment options for COVID-19. The limited number of authorized small-molecule direct-acting antivirals present challenges with pill burden, the necessity for intravenous administration or potential drug interactions. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

microbiology↗

bioRxiv2021

Multidose evaluation of 6,710 drug repurposing library identifies potent SARS-CoV-2 infection inhibitors In Vitro and In Vivo.

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

microbiology↗

bioRxiv2020

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics

The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1. Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

genetics↗

bioRxiv2020

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection

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.

microbiology↗

bioRxiv2020

Pyronaridine Tetraphosphate Efficacy Against Ebola Virus Infection in Guinea Pig

The recent outbreaks of the Ebola virus (EBOV) in Africa have brought global visibility to the shortage of available therapeutic options to treat patients infected with this or closely related viruses. We have recently computationally identified three molecules which have all demonstrated statistically significant efficacy in the mouse model of infection with mouse adapted Ebola virus (ma-EBOV). One of these molecules is the antimalarial pyronaridine tetraphosphate (IC50 range of 0.82-1.30 {micro}M against three strains of EBOV and IC50 range of 1.01-2.72 {micro}M against two strains of Marburg virus (MARV)) which is an approved drug in the European Union and used in combination with artesunate. To date, no small molecule drugs have shown statistically significant efficacy in the guinea pig model of EBOV infection. Pharmacokinetics and range-finding studies in guinea pigs directed us to a single 300mg/kg or 600mg/kg oral dose of pyronaridine 1hr after infection. Pyronaridine resulted in statistically significant survival of 40% at 300mg/kg and protected from a lethal challenge with EBOV. In comparison, oral favipiravir (300 mg/kg dosed once a day) had 43.5 % survival. The in vitro metabolism and metabolite identification of pyronaridine and another of our EBOV active molecules, tilorone, which suggests significant species differences which may account for the efficacy or lack thereof, respectively in guinea pig. In summary, our studies with pyronaridine demonstrates its utility for repurposing as an antiviral against EBOV and MARV, providing justification for future testing in non-human primates. ImportanceThere is currently no antiviral small molecule drug approved for treating Ebola Virus infection. We have previously used machine learning models to identify new uses for approved drugs and demonstrated their activity against the Ebola virus in vitro and in vivo. We now describe the pharmacokinetic properties of the antimalarial pyronaridine in the guinea pig. In addition, we show that this drug is effective against multiple strains of EBOV and MARV in vitro and in the guinea pig model of Ebola virus infection. These combined efforts indicate the need to further test this molecule in larger animal efficacy studies prior to clinical use in humans. These findings also may be useful for repurposing this drug for use against other viruses in future.

pharmacology and toxicology↗