Biology subjects

Winstone, H.

Publications and source records attributed to Winstone, H..

10 recordsLinked to original sources

bioRxiv2025

MERS-CoV antagonizes PKR activation by inhibiting its condensation at viral replication complexes

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that antagonizes innate immune responses, including the protein kinase R (PKR) pathway. Here, we examine the process of PKR activation in response to an immunostimulatory MERS-CoV mutant encoding an inactive endoribonuclease U and a deletion of accessory protein NS4a. We show that PKR condenses and activates on viral dsRNA proximal to viral double-membrane vesicles (DMVs). Condensates composed of activated PKR disassociate from dsRNA and dissolve, releasing activated PKR molecules into the cytosol where they phosphorylate eIF2 to initiate the integrated stress response. MERS-CoV NS4a protein prevents PKR activation by condensing on dsRNA and occluding PKR binding. Lastly, PKR condensation coincided with its activation in response to Zika virus. These findings establish a comprehensive model for PKR activation in response to positive-strand RNA viruses that replicate within membrane-associated complexes and elucidate how MERS-CoV antagonizes this crucial antiviral pathway. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/670656v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@130c4b1org.highwire.dtl.DTLVardef@c16a1borg.highwire.dtl.DTLVardef@1d4023org.highwire.dtl.DTLVardef@5dbaea_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPKR condenses on viral dsRNA exposed at membrane-associated replication complexes C_LIO_LIPKR condensation initiates autophosphorylation of PKR C_LIO_LIActivated p-PKR molecules disassociate from dsRNA and re-localize to the cytosol C_LIO_LIMERS-CoV NS4a inhibits PKR activation by occluding PKR condensation on dsRNA. C_LI

immunology↗

bioRxiv2025

Antibody escape drives emergence of diverse spike haplotypes resembling variants of concern in persistent SARS-CoV-2 infections

Evolution of SARS-CoV-2 in long-term persistent infections is hypothesised to be a major source of variants of concern (VOC). However, determination of viral subpopulations by linkage of intra-host variants into haplotypes has been limited by commonly used genomic sequencing techniques. We developed a sequencing and analysis methods for identifying full-length intra-host spike haplotypes and analysed their diversification over the course of persistent infections in individuals with inherited or acquired immunodeficiencies. Mutations frequently emerged in positions found in VOCs that confer escape from neutralising antibodies, while selection analyses showed strong evidence of positive selection and identified specific amino acid positions undergoing selection. In a single chronic infection lasting more than 500 days from the first wave of the pandemic, we detailed the evolution of spike as it gradually acquired mechanisms to evade both autologous and heterologous neutralising antibodies, redolent of Omicron variants. This provides one of the strongest pieces of evidence for persistent infections being the source of immune-evasive variants with accelerated evolution, underscoring their impact on the evolutionary trajectory of SARS-CoV-2.

microbiology↗

bioRxiv2022

The P681H mutation in the Spike glycoprotein escapes IFITM restriction and is necessary for type I interferon resistance in the SARS-CoV-2 alpha variant

The appearance of new dominant variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the COVID-19 pandemic. Of these, the alpha variant (also known as B.1.1.7) that appeared initially in the UK became the dominant variant in much of Europe and North America in the first half of 2021. The Spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation in the polybasic cleavage site that has been suggested to enhance S cleavage. Here, we show that the alpha S protein confers a level of resistance to the effects of interferon-{beta} (IFN{beta}) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN{beta} and context-dependent resistance to IFITMs in the alpha S. However, while this appears to confer changes in sensitivity to endosomal protease inhibition consistent with enhanced cell-surface entry, its reversion does not reduce cleaved S incorporation into particles, indicating a role downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOCs may well also confer replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCEThe emergence of Variants of Concern of SARS-CoV-2 has been a key challenge in the global response to the COVID-19 pandemic. Accumulating evidence suggests VOCs are being selected to evade the human immune response, with much interest focussed on mutations in the Spike protein that escape from neutralizing antibody responses. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type-1 interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2, and enhancement by its paralogue IFITM3, that block virus entry into target cells. The key determinant of this is a proline to histidine change at position 681 in S adjacent to the furin-cleavage site that we have shown previously modulates IFITM2 sensitivity. Unlike other VOCs, in the context of the alpha spike, P681H modulates cell entry pathways of SARS-CoV-2, further reducing its dependence one endosomal proteases. Reversion of position 681 to a proline in viruses bearing the alpha spike is sufficient to restore interferon and IFITM2 sensitivity without reducing furin-mediated spike cleavage, suggesting post cleavage conformational changes in S are changing the viral entry pathway and therefore sensitivity to interferon. These data highlight the dynamic nature of the SARS CoV-2 S as it adapts to both innate and adaptive immunity in the human population.

microbiology↗

bioRxiv2022

A nuclear export signal in KHNYN required for its antiviral activity evolved as ZAP emerged in tetrapods

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin- mediated degradation may control its homeostasis and regulation of protein turnover is separatable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final two positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCEThe interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon- stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell to interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.

microbiology↗

bioRxiv2021

Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

The zinc finger antiviral protein (ZAP) restricts a broad range of viruses by binding CpG dinucleotides in viral RNA to target it for degradation and inhibit its translation. KHNYN was recently identified as an antiviral protein required for ZAP to inhibit retroviral replication, though little is known about its functional determinants. KHNYN contains an N-terminal extended di-KH-like domain, a PIN endoribonuclease domain and a C-terminal CUBAN domain that binds NEDD8 and ubiquitin. We show that deletion of the extended di-KH domain reduces its antiviral activity. However, despite its similarity to RNA binding KH domains, the extended di-KH domain in KHNYN does not appear to bind RNA. Mutation of residues in the CUBAN domain that bind NEDD8 increase KHNYN abundance but do not alter its antiviral activity, suggesting that this interaction regulates KHNYN homeostatic turnover. In contrast, a CRM1-dependent nuclear export signal (NES) at the C-terminus of the CUBAN domain is required for antiviral activity. Deletion of this signal retains KHNYN in the nucleus and inhibits its interaction with ZAP. Interestingly, this NES appeared in the KHNYN lineage at a similar time as when ZAP evolved in tetrapods, indicating that these proteins may have co-evolved to restrict viral replication. AUTHOR SUMMARYAntiviral proteins restrict viral replication in many different ways, including inhibiting viral gene expression. ZAP is an antiviral RNA binding protein that must interact with other cellular proteins to inhibit viral protein synthesis. KHNYN is a ZAP cofactor that is required for it to inhibit retroviral replication. Because little is known about how KHNYN functions in this role, we have analyzed how two of its domains regulate its antiviral activity. We first show that the extended di-KH domain in KHNYN is required for its antiviral activity. While it is related to di-KH domains in RNA binding proteins, it appears to have lost its ability to bind RNA and KHNYN likely acts in the restriction pathway after ZAP binds a target viral RNA. Second, we show that the KHNYN CUBAN domain regulates both its protein abundance and trafficking within the cell. The CUBAN domain contains a nuclear export signal and, when this signal is mutated, KHNYN is sequestered in the nucleus, has substantially reduced antiviral activity and does not interact with ZAP. Overall, we show that the extended di-KH and CUBAN domains in KHNYN are required for it to act as a cofactor for ZAP to restrict viral replication.

microbiology↗

bioRxiv2021

The P681H mutation in the Spike glycoprotein confers Type I interferon resistance in the SARS-CoV-2 alpha (B.1.1.7) variant

Variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) threaten the global response to the COVID-19 pandemic. The alpha (B.1.1.7) variant appeared in the UK became dominant in Europe and North America in early 2021. The Spike glycoprotein of alpha has acquired a number mutations including the P681H mutation in the polybasic cleavage site that has been suggested to enhance Spike cleavage. Here, we show that the alpha Spike protein confers a level of resistance to the effects of interferon-{beta} (IFN{beta}) in lung epithelial cells. This correlates with resistance to restriction mediated by interferon-induced transmembrane protein-2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is necessary for comparative resistance to IFN{beta} in a molecularly cloned SARS-CoV-2 encoding alpha Spike. Overall, we suggest that in addition to adaptive immune escape, mutations associated with VOCs also confer replication advantage through adaptation to resist innate immunity.

microbiology↗

bioRxiv2021

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spikes polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

microbiology↗

bioRxiv2021

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike

The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ~45% showed neutralizing activity of which ~20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

immunology↗

bioRxiv2020

The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein (S). The presence of a polybasic cleavage site in SARS-CoV-2 S at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the S precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons, and more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2 and the degree of this restriction is governed by route of viral entry. Removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH- and cathepsin-dependent in late endosomes where, like SARS-CoV-1 S, it is more sensitive to IFITM2 restriction. Furthermore, we find that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 S may reduce viral replication through the activity of type I IFNs. IMPORTANCEThe furin cleavage site in the S protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2, and that the sensitivity is exacerbated by deletion of the furin cleavage site which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus may represent a potential therapeutic target for COVID-19 treatment development.

immunology↗

bioRxiv2019

IFN-independent cell cycle arrest and SAMHD1 activation following TLR4 activation in macrophages

Monocyte-derived macrophages mostly reside in a resting, G0 state, expressing high levels of dephosphorylated, active SAMHD1. We have previously shown that macrophages can re-enter the cell cycle without division, into a G1-like state. This cell cycle re-entry is accompanied by phosphorylation of the dNTP hydrolase/ antiviral restriction factor SAMHD1 at T592 by the cyclin-dependent kinase CDK1. HIV-1 successfully infects macrophages in G1 through exploiting this naturally occurring window of opportunity where SAMHD1 antiviral activity is de-activated.\n\nHere we demonstrate for the first time that LPS activation of the pathogen associated molecular pattern (PAMP) receptor TLR4 induces G0 arrest in human macrophages. We show this G0 arrest is MyD88-independent and therefore NFkB independent. Furthermore, the effect of TLR4 activation on cell cycle is regulated by (a) the canonical IFN-dependent pathway following TBK1 activation and IRF3 translocation and (b) an IFN-independent pathway that occurs prior to TBK1 activation, and that is accompanied by CDK1 downregulation, p21 upregulation and SAMHD1 dephosphorylation at T592. Furthermore, we show by siRNA knockdown of SAMHD1 that the interferon independent pathway activated by TLR4 is able to potently block HIV-1 infection in macrophages specifically via SAMHD1. Finally, ingestion of whole E. Coli and TLR4 activation by macrophages also activates SAMHD1 via the interferon independent pathway.\n\nTogether, these data demonstrate that macrophages can rapidly activate an intrinsic cell arrest and anti-viral state by activation of TLR4 prior to IFN secretion, thereby highlighting the importance of cell cycle regulation as a response to danger signals in human macrophages. Interferon independent activation of SAMHD1 by TLR4 represents a novel mechanism for limiting the HIV-1 reservoir size and should be considered for host-directed therapeutic approaches that may contribute to curative interventions.

microbiology↗