Biology subjects

Hyde, J. L.

Publications and source records attributed to Hyde, J. L..

7 recordsLinked to original sources

bioRxiv2026

TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

Innate immune recognition of double-stranded RNA (dsRNA) by germline-encoded receptors initiates antiviral defenses, including type I interferon (IFN) production. The inflammasome-forming sensor NLRP1 binds and is activated by dsRNA in a mitogen-activated protein kinase (MAPK) p38-dependent manner. How dsRNA initiates these events to induce NLRP1 inflammasome activation is unclear. Here we demonstrate that both exogenous and cellular dsRNA triggers NLRP1 inflammasome activation downstream of RIG-I/MDA5-MAVS and/or TLR3-TRIF signaling but is independent of type I IFN. In immortalized and primary human keratinocytes, we find that NLRP1 inflammasome activation by dsRNA, including during viral infection, requires the MAPK kinase kinase TAK1. Mechanistically, TAK1-dependent phosphorylation of the NLRP1 N-terminal disordered region is necessary and sufficient for inflammasome activation. Collectively, we reveal TAK1 as a novel activator of the NLRP1 inflammasome, functioning as a critical signaling hub linking NLRP1 to inflammatory responses in the context of viral infection and autoimmunity.

immunology↗

bioRxiv2025

Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo

BackgroundAlphaviruses, including chikungunya virus (CHIKV), pose a significant global health threat, yet specific antiviral therapies remain unavailable. MethodsWe evaluated combinations of three oral directly acting antiviral drugs (sofosbuvir (SOF), molnupiravir (MPV), and favipiravir (FAV)), which are approved for other indications, against CHIKV, Semliki Forest virus (SFV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) in vitro and in vivo. We assessed antiviral efficacy in human skin fibroblasts and liver cells, as well as in a mouse model of CHIKV-induced arthritis. FindingsIn human skin fibroblasts, synergistic antiviral effects were observed for combinations of MPV + SOF and FAV + SOF against CHIKV, and for FAV + SOF against SFV. In human liver cells, FAV + MPV conferred additive to synergistic activity against VEEV and SINV, while SOF synergized with FAV against SINV. In mice, MPV improved CHIKV-induced foot swelling and reduced systemic infectious virus titres. Combination treatment with MPV and SOF significantly reduced swelling and infectious virus titres compared to monotherapies of each drug. Sequencing of CHIKV RNA from joint tissue revealed that MPV caused dose- dependent increases in mutations in the CHIKV genome. Upon combination therapy of MPV with SOF, the number of mutations was significantly lower compared to monotherapy with several higher doses of MPV. InterpretationCombining these approved oral nucleoside analogues confers potent suppression of multiple alphaviruses in vitro and in vivo with enhanced control of viral genetic evolution in face of antiviral pressure. These drug combinations may ultimately lead to the development of potent combinations of pan-family alphavirus inhibitors. FundingThis work was supported by a PhD fellowship granted to S.V. by the Research Foundation - Flanders (FWO) (11D5923N). L.D.C. was also supported by Research Foundation - Flanders (FWO) PhD fellowship (11L1325N). Dr. Polyak and Schiffer are partially supported by R01AI121129. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSAlphaviruses such as chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) are a major threat for global health. Alphaviruses are responsible for debilitating diseases with major public health implications, yet no antiviral drugs are currently approved for treating these virus infections. Existing treatment options are limited to supportive care and are unlikely to provide protection against future outbreaks of other alphaviruses. Previous studies have shown that oral approved nucleoside analogues such as favipiravir (FAV), molnupiravir (MPV), and sofosbuvir (SOF) have antiviral activity against certain RNA viruses, including alphaviruses. However, systematic in vivo evaluations of these drugs and testing of drug combinations in both in vitro and in vivo settings are limited. Added value of this studyThis study provides a comprehensive evaluation of combinations of FAV, MPV and SOF against multiple alphaviruses in two human cell lines and a CHIKV mouse model. We demonstrate that certain combinations of these drugs confer synergistic antiviral activity, effectively suppressing CHIKV, SFV, SINV, and VEEV replication in vitro. Moreover, in vivo, we show for the first time that MPV treatment results in reduced CHIKV-induced foot swelling and systemic virus replication. Combining MPV with SOF enhances antiviral activity in mice as compared to monotherapy. By sequencing the viral genome, we show that MPV increases the number of mutations in a dose-dependent manner. Combination therapy of MPV and SOF reduces the number of mutations compared to higher doses of MPV. These findings highlight the potential of nucleoside analogue combinations as a promising therapeutic strategy against alphavirus infections. Implications of all the available evidenceThe results of this study suggest that combination therapy with approved nucleoside analogues could provide an effective treatment strategy for alphavirus infections. The observed increased efficacy of drug combinations supports the potential for dose optimization to enhance efficacy while reducing toxicity and development of resistance. Future research should focus on clinical evaluation of these drug combinations, pharmacokinetic studies, and further exploration of their impact on viral evolution. Given the expanding geographical distribution of alphaviruses and the lack of available treatments, these findings provide a foundation for developing pan-alphavirus antiviral therapies that could improve patient outcomes and global outbreak preparedness.

microbiology↗

bioRxiv2023

Regulation of coronavirus nsp15 cleavage specificity by RNA structure

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (NSPS) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established function of nsp15 as a uridine-specific endonuclease, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS 3UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus high probably targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency, suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of recognition of RNA by nsp15.

microbiology↗

bioRxiv2023

Norovirus NS3 protein induces apoptosis through translation repression and dysregulation of BCL-2 pro-survival proteins

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.

microbiology↗

bioRxiv2022

Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.

immunology↗

bioRxiv2021

Sequence diversity in the 3' untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

Alphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3 untranslated region (3UTR) of the virus. We investigated the potential role of variability in the 3UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

microbiology↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗