Biology subjects

Turville, S. G.

Publications and source records attributed to Turville, S. G..

13 recordsLinked to original sources

bioRxiv2025

High-stability, clamp-free soluble Sarbecovirus spike trimers and their potential for pan-Sarbecovirus vaccine development.

Broadly effective vaccines are needed to protect against future pandemics caused by severe acute respiratory syndrome coronavirus (SARS CoV)-like coronaviruses (sarbecoviruses). The development of simple trimeric subunit vaccines based on the Sarbecovirus spike (S) has proven problematic due to the unstable nature of the S trimer. Here we developed clamp-free, highly stable soluble S trimers by truncating the stem helix to maximize yield and covalently linking the 3 monomers via engineered disulfides to increase thermostability. In K18hACE2 mice, covalently linked SARS CoV-2 S trimers elicited >10-fold higher neutralizing antibody (NAb) titres than parental unlinked trimers and protected the mice against viral challenge. A trivalent vaccine formulation comprised of covalently stabilized spikes derived from 3 divergent ACE2-using Sarbecovirus clades elicited broad and potent neutralizing activity in mice. The covalently linked S trimers were stable at 37{degrees}C for 112 days and remained intact following lyophilization and storage at ambient temperature for 6 months. This study establishes a framework for producing simple and stable highly immunogenic pan-Sarbecovirus S subunit vaccines that can be stored and distributed in the absence of a cold chain.

microbiology↗

bioRxiv2025

Replication of SARS-CoV-2 Omicron lineages is defined by TMPRSS2 use in environments where ACE2 is complexed with solute carriers SLC6A19 and SLC6A20.

The Omicron variant of SARS-CoV-2 emerged in late 2021 and since then Omicron subvariants have continued to evolve and dominate globally. The viral S protein evolved towards highly efficient antibody evasion and replicative capacity in the upper respiratory tract resulting in high transmissibility. At the same time, the mutations acquired in the S protein diminish infection of the lung epithelium and pathogenic potential. The changing entry requirements for Omicron sub-lineages that lead to this shift in tropism remain poorly understood. We resolve the changing replication requirements of SARS-CoV-2 to be related to two distinct pools of ACE2. The first pool relates to ACE2s role in the renin angiotensin system (RAS) and this pool can complex with TMPRSS2 (RAS-ACE2). The second pool relates to ACE2s role as a protein solute carrier chaperone than cannot complex with TMPRSS2 (Chaperone ACE2). Here, we demonstrate that pre-Omicron lineages replicate in a TMPRSS2 dependent manner across both ACE2 pools, whilst Omicron lineages can only spread and replicate using chaperone ACE2. This provides a mechanistic basis for the evolving infectivity requirements of SARS-CoV-2 and furthermore provides approaches to track and monitor ACE2 utilizing coronaviruses. Graphical AbstractMechanistic basis for shift in SARS-CoV-2 tropism with the arrival of Omicron. A. Chaperone ACE2 is defined structurally as a heterodimer of dimers with a solute carrier protein-SLC6A19 or SLC6A20. Here this ACE2 structure can exist uncomplexed from TMPRSS2 and enables TMPRSS2 use by both pre-Omicron and Omicron lineages. B. Renin Angiotensin ACE2 is defined by ACE2 with an exposed collectrin-like domain (CLD), which enables binding of TMPRSS2 or ADAM-17. Here ACE2 can form a complex with TMPRSS2 in a manner that allows pre-Omicron but not Omicron lineages to utilize TMPRSS2 to facilitate infection. Here Omicron lineages are heavily attenuated as they cannot use TMPRSS2 to spread. C. to E. Based on single cell profiles, ACE2 can exist as a chaperone with SLC6A20 in C. the Nasal Cavity or D. primarily as RAS-ACE2 in the lung to respond to acute lung injury. E. The largest pool of ACE2 in our body resides within the small intestine on enterocytes and this further facilitates replication in this tissue by pre-Omicron and Omicron lineages. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/663433v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@172aa47org.highwire.dtl.DTLVardef@109285eorg.highwire.dtl.DTLVardef@394f8corg.highwire.dtl.DTLVardef@958e31_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution

Understanding viral evolutionary dynamics is crucial to pandemic responses, prediction of virus adaptation over time, and virus surveillance for public health strategies. Whole-genome sequencing (WGS) of SARS-CoV-2 has enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a complementary controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. In this study, nine virus lineages, including four "variants of concern" and three former "variants under investigation", were sampled over [≥]33 serial passages (range 33-100) in Vero E6 cells. WGS was used to examine virus evolutionary dynamics and identify key mutations with implications for fitness and/or transmissibility. Viruses accumulated mutations regularly during serial passaging. Many low-frequency variants were lost but others became fixed, suggesting either in vitro benefits, or at least a lack of deleterious effect. Mutations arose convergently both across passage lines, and when compared with contemporaneous SARS-CoV-2 clinical sequences. These mutations included some hypothesised to drive lineage success through host immune evasion (e.g. S:A67V, S:H655Y). The appearance of these mutations in vitro suggested key mutations can arise convergently even in the absence of a multicellular host immune response through mechanisms other than immune-driven mutation. Such mutations may provide other benefits to the viruses in vitro, or arise stochastically. Our quantitative investigation into SARS-CoV-2 evolutionary dynamics spans the greatest number of serial passages to date, and will inform measures to reduce the effects of SARS-CoV-2 infection on the human population. ImportanceThe ongoing evolution of SARS-CoV-2 remains a challenge for long term public health efforts to minimise the effects of COVID-19. Whole-genome sequencing of outbreak cases has enabled global contact tracing efforts and the identification of mutations of concern within the virus genome. However, complementary approaches are necessary to inform our understanding of virus evolution and clinical outcomes. Here we charted evolution of the virus within a controlled cell culture environment, focusing on nine different virus lineages. Our approach demonstrates how SARS-CoV-2 continues to evolve readily in vitro, with changes mirroring those seen in outbreak cases globally. Findings of the study are important for i) investigating the mechanisms of how mutations arise, ii) predicting the future evolutionary trajectory of SARS-CoV-2, and iii) informing treatment and prevention design.

evolutionary biology↗

bioRxiv2023

TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2

Continued high-level spread of SARS-CoV-2 has enabled an accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) dependent entry by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on TMPRS22 association with the ACE2 Collectrin-Like Domain (CLD). In engineered cells overexpressing ACE2 and TMPRSS2, ACE2/TMPRSS2 complexes led to either augmentation or attenuation of viral infectivity of pre-Omicron and Omicron lineages, respectively. Mutagenesis of the ACE2-CLD TMPRSS2 cleavage site in ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 activation of viral fusion. Our data supports the evolution of Omicron lineages towards the use of ACE2 unable to form complexes with TMPRSS2 and consistent with ACE2 structure and function as a chaperone for many tissue specific amino acid transport proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/558930v4_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1c86cf0org.highwire.dtl.DTLVardef@16815feorg.highwire.dtl.DTLVardef@7be9e1org.highwire.dtl.DTLVardef@137de1c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO ACE2-TMPRSS2 pool model and evolution of SARS-CoV-2 tropism.A. &-B. ACE2-TMPRSS2 pool model to reconcile the evolving entry requirements of SARS-CoV-2 and changes in viral tropism in vivo. A. Both SARS-CoV-1 and early SARS-CoV-2 (pre-Omicron) lineages have molecular dual tropism, with efficient entry when ACE2 (blue protein) can form complexes with TMPRSS2 (green protein) and in settings where TMPRSS2 is excluded from ACE2 (C4-ACE2 CLD). In both settings ACE2 initially engages SARS-CoV-2 spike and fusion is then triggered through TMPRSS2 cleavage of the Spike S2 domain B. Over time, the dual tropism for two distinct pools of ACE2 (with and without TMPRSS2) has been lost, with consolidation towards ACE2 where TMPRSS2 is no longer in a complex. With the arrival of Omicron lineages, ACE2-TMPRSS2 complexes could no longer enable efficient Spike S2 cleavage and fusogenic activation (TMPRSS2 "off" confirmation"). Rather, only TMPRSS2 uncoupled from ACE2 could facilitate the latter cleavage of S2. C. Over time this has further consolidated over generations of omicron lineages from 2022 lineages (BA.1, BA.2 and BA.5) through to 2023 lineages (XBB.1.5) and now in 2024 JN.1 lineages such as KP.3. Overall, this supports the initial molecular tropism of early SARS-CoV-2 clades to be similar to that observed for SARS-CoV-1, with dual tropism across both ACE2 pools and replication proceeding in tissues where ACE2-TMPRSS2 complexes would be prevalent (e.g. Lung). The evolution away from ACE2-TMPRSS2 complexes towards ACE2 where TMPRSS2 is structurally uncoupled (e.g. ACE2 as a chaperone for solute carriers SLCA619 or SLCA620) is consistent selection of this ACE2 pool in a manner that has sustained transmission fitness within the human population. C_FIG

microbiology↗

bioRxiv2022

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. Significance statementThis study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.

biochemistry↗

bioRxiv2022

Enhanced stability of the SARS CoV-2 spike glycoprotein trimer following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity formed by Ala1016 and Ala1020 that form part of the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. The trimer stabilizing effects of filling the Ala1016/Ala1020 cavity was linked to improved S glycoprotein membrane fusion function. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited very high titers of neutralizing antibodies to ancestral and Delta-derived viruses (1/2,700-1/5,110), while neutralization titer was somewhat reduced with Omicron BA.1 (1/210-1,1744). The antigens elicited antibody specificities that could compete with ACE2-Fc for binding to the receptor-binding motif (RBM) and NAbs directed to key neutralization epitopes within the receptor-binding domain (RBD), N-terminal domain (NTD) and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S ectodomain trimers in the absence of an external trimerization motif (T4 foldon). The VI mutation represents a method for producing an intrinsically stable trimeric S ectodomain glycoprotein vaccine in the absence of a foreign trimerization tag. AUTHOR SUMMARYFirst-generation SARS CoV-2 vaccines that generate immune responses to ancestral Spike glycoprotein sequences have averted at least 14.4 million deaths, but their effectiveness against the recently emerged Omicron lineages is reduced. The updating of booster vaccines with variant Spike sequences are therefore likely required to maintain immunity as the pandemic continues to evolve. The Spike is a trimeric integral membrane protein with a membrane spanning sequence at its C-terminus. The Spike protein-based vaccine that is currently licensed for human use is produced by a complex process that reconstitutes the Spike in an artificial membrane. Alternatively, production of the Spike trimer as a soluble protein generally requires replacement of the membrane spanning sequence with a foreign often highly immunogenic trimerization motif that can complicate clinical advancement. We used systematic structure-directed mutagenesis coupled with functional studies to identify an alternative stabilization approach that negates the requirement for an external trimerization motif or membrane-spanning sequence. The replacement of 2 alanine residues that form a cavity in the core of the Spike trimer with bulkier hydrophobic residues resulted in increased Spike thermal stability. Thermostable Spike mutants retained major conserved neutralizing antibody epitopes and the ability to elicit broad and potent neutralizing antibody responses. One such mutation, referred to as VI, enabled the production of intrinsically stable Omicron variant Spike ectodomain trimers in the absence of an external trimerization motif. The VI mutation potentially enables a simplified method for producing a stable trimeric S ectodomain glycoprotein vaccine.

microbiology↗

bioRxiv2022

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

immunology↗

bioRxiv2022

Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure

The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here we investigate the effect of Lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that Lenacapavirs potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.

biophysics↗

bioRxiv2021

Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

biochemistry↗

bioRxiv2020

Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

biochemistry↗

bioRxiv2020

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.

biochemistry↗

bioRxiv2020

Rapid HIV-1 capsid interaction screening using fluorescence fluctuationspectroscopy

The HIV capsid is a multifunctional protein capsule for delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labelled capsid-binding analytes ( prey molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using fluorescent capsid as bait further allows quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for discovery and characterization of molecules used by HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

biophysics↗

bioRxiv2019

HIV corrupts the Cdc42-IQGAP1 axis of actin regulation to promote cell-cell viral spread

F-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, provided Cdc42 and IQGAP1 were present. From these observations we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

microbiology↗