TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.

ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp61413-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.

hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing.

The Polycomb-repressive complexes PRC1 and PRC2 play a key role in chromosome silencing induced by the non-coding RNA Xist. Polycomb recruitment is initiated by the PCGF3/5-PRC1 complex, which catalyzes chromosome-wide H2A lysine 119 ubiquitylation, signaling recruitment of other PRC1 complexes, and PRC2. However, the molecular mechanism for PCGF3/5-PRC1 recruitment by Xist RNA is not understood. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing and reversing Xist-induced chromatin inaccessibility. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment. Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA.

The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2.

The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Severe Acute Respiratory Syndrome Coronavirus 2 Serosurveillance in a Patient Population Reveals Differences in Virus Exposure and Antibody-Mediated Immunity According to Host Demography and Healthcare Setting.

Identifying drivers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and quantifying population immunity is crucial to prepare for future epidemics. We performed a serial cross-sectional serosurvey throughout the first pandemic wave among patients from the largest health board in Scotland. Screening of 7480 patient serum samples showed a weekly seroprevalence ranging from 0.10% to 8.23% in primary and 0.21% to 17.44% in secondary care, respectively. Neutralization assays showed that highly neutralizing antibodies developed in about half of individuals who tested positive with enzyme-linked immunosorbent assay, mainly among secondary care patients. We estimated the individual probability of SARS-CoV-2 exposure and quantified associated risk factors. We show that secondary care patients, male patients, and 45-64-year-olds exhibit a higher probability of being seropositive. The identification of risk factors and the differences in virus neutralization activity between patient populations provided insights into the patterns of virus exposure during the first pandemic wave and shed light on what to expect in future waves.

The phosphorylation of ARPP19 by Greatwall renders the auto-amplification of MPF independently of PKA in Xenopus oocytes.

Entry into mitosis or meiosis relies on the coordinated action of kinases and phosphatases that ultimately leads to the activation of Cyclin-B-Cdk1, also known as MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest that is tightly controlled by high PKA activity. Re-entry into meiosis depends on activation of Cdk1, which obeys a two-step mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein-synthesis-dependent manner; then a regulatory network known as the MPF auto-amplification loop is initiated. This second step is independent of PKA and protein synthesis. However, none of the molecular components of the auto-amplification loop identified so far act independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiologically intact cell system, the Xenopus oocyte, we show that the phosphorylation of ARPP19 at S67 by the Greatwall kinase promotes its binding to the PP2A-B55δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and has an essential role within the Cdk1 auto-amplification loop for entry into the first meiotic division. Moreover, once phosphorylated by Greatwall, ARPP19 escapes the negative regulation exerted by PKA. It also promotes activation of MPF independently of protein synthesis, provided that a small amount of Mos is present. Taken together, these findings reveal that PP2A-B55δ, Greatwall and ARPP19 are not only required for entry into meiotic divisions, but are also pivotal effectors within the Cdk1 auto-regulatory loop responsible for its independence with respect to the PKA-negative control.

Preservation of memory B cell homeostasis in an individual producing broadly neutralising antibodies against HIV-1.

Immunological determinants favouring emergence of broadly neutralising antibodies are crucial to the development of HIV-1 vaccination strategies. Here, we combined RNAseq and B cell cloning approaches to isolate a broadly neutralising antibody (bnAb) ELC07 from an individual living with untreated HIV-1. Using single particle cryogenic electron microscopy (cryo-EM), we show that the antibody recognises a conformational epitope at the gp120-gp41 interface. ELC07 binds the closed state of the viral glycoprotein causing considerable perturbations to the gp41 trimer core structure. Phenotypic analysis of memory B cell subsets from the ELC07 bnAb donor revealed a lack of expected HIV-1-associated dysfunction, specifically no increase in CD21-/CD27- cells was observed whilst the resting memory (CD21+/CD27+) population appeared preserved despite uncontrolled HIV-1 viraemia. Moreover, single cell transcriptomes of memory B cells from this bnAb donor showed a resting memory phenotype irrespective of the epitope they targeted or their ability to neutralise diverse strains of HIV-1. Strikingly, single memory B cells from the ELC07 bnAb donor were transcriptionally similar to memory B cells from HIV-negative individuals. Our results demonstrate that potent bnAbs can arise without the HIV-1-induced dysregulation of the memory B cell compartment and suggest that sufficient levels of antigenic stimulation with a strategically designed immunogen could be effective in HIV-negative vaccine recipients.

Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1, Cioce et al. (2021)2, and Cioce et al. (2022)3.

Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern.

The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.

Evolution of the SARS-CoV-2 spike protein in the human host.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Simple, sensitive, specific self-sampling assay secures SARS-CoV-2 antibody signals in sero-prevalence and post-vaccine studies.

At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.

Cell-specific bioorthogonal tagging of glycoproteins.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility.

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100 % on 825 pre COVID-19 samples and a potential sensitivity of 99.6 % on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralization and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.