Biology subjects

Ramage, H.

Publications and source records attributed to Ramage, H..

5 recordsLinked to original sources

bioRxiv2026

Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

Powassan virus (POWV) is an emerging neurotropic tick-borne flavivirus, yet the mechanisms by which POWV evades host antiviral immunity remain poorly defined. Here, we identify multiple mechanisms of POWV innate immune antagonism with the viral polymerase NS5 protein as a central inhibitor of cytokine signaling. Both POWV lineages potently inhibited type I interferon (IFN) signaling, and NS5 expression suppressed signaling and downstream interferon-stimulated gene expression. Affinity purification-mass spectrometry identified the host kinase TYK2 as a conserved NS5 interactor. POWV NS5 binds the TYK2 kinase domain through a discrete interface within the RNA-dependent RNA polymerase (RdRp) region between catalytic motifs B and C and inhibits TYK2 phosphorylation. Disruption of this interface abrogated TYK2 binding and reduced NS5-mediated IFN antagonism, while revealing additional TYK2-independent mechanisms of immune suppression. POWV NS5 also inhibited TYK2-dependent IFN-{lambda} and IL-12 signaling, demonstrating that its immune antagonism extends beyond type I IFN. Together, these findings identify TYK2 as a central target of POWV immune evasion and implicate the variable RdRp B-C region as an interface for flavivirus-host interactions. More broadly, our results reveal how POWV can coordinately suppress multiple antiviral cytokine pathways and provide insight into mechanisms that may shape tick-borne flavivirus host adaptation and pathogenesis.

microbiology↗

bioRxiv2026

Interferon-α and -β subtypes have temporally distinct roles in containing viral spread and protecting vital organs.

Type I interferons (IFN-Is), including a single IFN-{beta} and over a dozen IFN-s, induce the anti-viral state. In vitro, IFN-{beta} transcription requires the assembly of the "enhanceosome" composed of the constitutive transcription factors NF-{kappa}B and AP-1, and either constitutive IRF3 or IFN-I-inducible IRF7. IRF3 and IRF7 transcribe mouse IFN-4 and human IFN-1/13. Only IRF7 transcribes other IFN-s. How IFN-I subtype multiplicity and their differential constitutive/IFN-I-inducible versus only IFN-I-inducible transcription help control viruses in vivo remains unknown. Using novel genetically modified mice, we demonstrate that most or all IFN-I subtypes, regardless of their transcriptional control, are necessary to curb the systemic dissemination of lymph-borne ectromelia virus (ECTV) but do not necessarily suppress ECTV or West Nile Virus (WNV) replication in the liver or the brain, or promote survival to their infection. Individually, the most critical IFN-I subtype to survive ECTV and WNV infections is IFN-{beta}. IFN-4 potentiates IFN-{beta} but is not essential. PRDII, the IFN-{beta} promoters NF-{kappa}B binding site, which is required for enhanceosome assembly, is dispensable for in vivo IFN-{beta} production but complements IRF7-IFN-{beta} transcription to restrain ECTV and WNV. IFN-{beta} alone protects IFN--deficient mice from ECTV but not from WNV lethality. Control of WNV replication in the brain successively requires IFN-, IFN-{beta}, and then PRDII-dependent IFN-{beta}, suggesting that brain protection requires IFN-I production by various cell types and pathways. Thus, contrary to the prevailing view, in vivo, IFN-{beta} does not function earlier than IFN-, and IFN-I subtypes non-redundantly cooperate to restrain viruses in the periphery and in vital organs.

immunology↗

bioRxiv2025

Nuclear activities and interactome of the NS5 protein of Tick-Borne Encephalitis Virus

Orthoflaviviruses are RNA viruses responsible for significant diseases in humans, domesticated animals and wildlife. Their NS5 protein is central in viral replication, functioning both as an RNA-dependent RNA polymerase and a methyltransferase, while also modulating cellular processes, including the interferon response. Although viral replication is cytoplasmic, the NS5 protein of several mosquito-borne orthoflaviviruses cycles between the cytoplasm and the nucleus of infected human cells. However, the nuclear localization and function of NS5 of tick-borne orthoflaviviruses, such as tick-borne encephalitis virus (TBEV), remained poorly understood. Microscopy analysis and cell fractionation revealed that the NS5 protein of TBEV localized to both the cytoplasm and nucleoplasm of infected cells. Mutagenesis studies identified critical residues required for its nuclear targeting. Mutating these residues in a TBEV replicon abolished viral replication. Immunoprecipitation-mass spectrometry analyses performed in two human cell lines infected with TBEV recovered 352 NS5 partners. Among them, 187 were nuclear or partially nuclear. By integrating our interactome data with that of Powassan virus (POWV), another tick-borne orthoflavivirus, we refined a list of 20 high-confidence NS5 partners, including splicing factors and chromatin modulators. Functional analysis revealed that seven of these nuclear partners significantly modulated viral replication, further underscoring the importance of nuclear NS5 in the viral life cycle. Our work advances our understanding of the nuclear function of the NS5 proteins of tick-borne orthoflaviviruses. ImportanceTick-borne orthoflaviviruses are emerging globally, spreading across Europe, Asia, and North America, where they infect humans, domesticated animals, and wildlife. These viruses produce a protein called NS5, which drives viral replication and helps evade the innate immune response. We observed that the NS5 protein of tick-borne encephalitis virus (TBEV) localized both in the cytoplasm and nucleoplasm of infected human cells. We identified the specific residues responsible for its nuclear addressing and showed that it interacts with numerous nuclear proteins, including some involved in regulating gene expression. Seven of these nuclear partners significantly influenced viral replication, highlighting the importance of NS5s nuclear activity. This work sheds light on how tick-borne orthoflaviviruses manipulate host cells, deepening our understanding of their replication strategies.

microbiology↗

bioRxiv2023

Motif-VI Loop Acts as a Nucleotide Valve in the West Nile Virus NS3 Helicase

The flavivirus NS3 helicase (NS3h), a highly conserved protein, plays a pivotal role in virus replication and thus represents a potential drug target for flavivirus pathogenesis. NS3h utilizes nucleotide triphosphate, such as ATP, for hydrolysis energy (ATPase) to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. The intermediate states along the ATP binding and hydrolysis cycle, as well as the conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. We use extensive molecular dynamics simulations of apo, ATP, ADP+Pi, and ADP bound to WNV NS3h+ssRNA to model the conformational ensembles along this cycle. Energetic and structural clustering analyses on these trajectories depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). These findings were experimentally corroborated using viral replicons encoding three mutations at the D471 position. Replication assays using these mutants demonstrated a substantial reduction in viral replication compared to the wild-type. Molecular simulations of the D471 mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open valve conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a valve for the ATP-pocket, presenting a promising target for antiviral development.

biochemistry↗

bioRxiv2020

Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2

There are an urgent need for antivirals to treat the newly emerged SARS-CoV-2. To identify new candidates we screened a repurposing library of ~3,000 drugs. Screening in Vero cells found few antivirals, while screening in human Huh7.5 cells validated 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we found that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH-independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we found 9 drugs are antiviral in lung cells, 7 of which have been tested in humans, and 3 are FDA approved including Cyclosporine which we found is targeting Cyclophilin rather than Calcineurin for its antiviral activity. These antivirals reveal essential host targets and have the potential for rapid clinical implementation.

microbiology↗