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Funder: NIHR Cambridge BRC, Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.Author summaryTechniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses.

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ–inducible, and binds to MHC class I coupled with β2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

RNA-binding E3 ubiquitin ligases were recently identified, though their function remains unclear. While studying the regulation of the MHC class I (MHC-I) pathway, we here characterize a novel role for ubiquitin in mRNA degradation. MHC-I molecules provide ligands for both cytotoxic T-lymphocytes as well as natural killer (NK) cells, and play a central role in innate and adaptive immunity. MHC-I cell-surface expression is closely monitored by NK cells, whose killer immunoglobulin-like receptors encode MHC-I-specific activatory and inhibitory receptors, implying that MHC-I expression needs to be tightly regulated. In a functional siRNA ubiquitome screen we identified MEX-3C, a novel RNA-binding ubiquitin E3 ligase, as responsible for the post-transcriptional, allotype-specific regulation of MHC-I. MEX-3C binds the 3′UTR of HLA-A2 mRNA, inducing its RING-dependent degradation. The RING domain of MEX-3C is not required for HLA-A2 cell-surface downregulation, but regulates the degradation of HLA-A2 mRNA. We have therefore uncovered a novel post-transcriptional pathway for regulation of HLA-A allotypes and provide a link between ubiquitination and mRNA degradation.

The Kaposi’s sarcoma-associated herpesvirus-encoded ubiquitin E3 ligase K3 ubiquitinates cell-surface MHC class I molecules (MHC I), causing the internalization and degradation of MHC I via the endolysosomal pathway. K3 recruits the cellular E2 ubiquitin-conjugating enzyme Ubc13 to generate lysine-63–linked polyubiquitin chains on MHC I, leading to the clathrin-mediated endocytosis and lysosomal degradation of MHC I. In this study, we identify a ubiquitin isoleucine-44-alanine mutant (I44A) that inhibits K3-mediated downregulation of MHC I by preventing MHC I polyubiqitination. This E3-specific inhibition by I44A prevents dissociation of the MHC I–K3–Ubc13–ubiquitin complex, allows the in vivo visualization of a transient substrate–E3–E2–ubiquitin complex interaction, and highlights a potential substrate hierarchy between the different MHC I alleles downregulated by K3. The I44A mutant also increases cell-surface MHC I expression in control cells in the absence of K3, predicting the presence of an endogenous E3 ubiquitin ligase required for cell-surface MHC I regulation.

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.

The Kaposi's sarcoma-associated herpes virus gene product K3 (KK3) subverts the MHC class I antigen presentation pathway by downregulating MHC class I from the plasma membrane. We now show that KK3 associates with MHC class I molecules and promotes ubiquitylation of class I after export from the endoplasmic reticulum. Ubiquitylation requires the KK3 N-terminal plant homeodomain and provides the signal for class I internalization at the plasma membrane. Once internalized, ubiquitylated MHC class I is targeted to the late endocytic pathway, where it is degraded. Depletion by small interfering RNA of TSG101, a ubiquitin enzyme 2 variant protein involved in late endosomal sorting, prevents class I degradation and preserves cell surface class I expression in KK3-expressing cells. These results suggest a mechanism by which the KK3-induced class I ubiquitylation provides a signal for both internalization and sorting to the late endosomal pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin-dependent endosomal sorting machinery.

The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.

Cell-cell contacts are essential for morphogenesis and tissue function and play a vital role in mediating endothelial cohesion within the vascular system during vessel growth and organization. We identified a novel junctional adhesion molecule, named JAM-2, by a selective RNA display method, which allowed identification of transcripts encoding immunoglobulin superfamily molecules regulated during coculture of endothelial cells with tumor cells. The JAM-2 transcript is highly expressed during embryogenesis and is detected in lymph node and Peyer's patches RNA of adult mice. Accordingly, antibodies specific for JAM-2 stain high endothelial venules and lymphatic vessels in lymphoid organs, and vascular structures in the kidney. Using real time video microscopy, we show that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is further suggested by the reduced paracellular permeability of cell monolayer transfected with JAM-2 cDNA, and by the localization of JAM-2 to tight junctional complexes of polarized cells. Taken together, our results suggest that JAM-2 is a novel vascular molecule, which participates in interendothelial junctional complexes.

The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins.

The mK3, K3, and K5 gene products from the gamma2 group of gamma-herpesviruses are the founding members of a family of membrane-associated ubiquitin E3 ligases. As part of the viral immunoevasion strategy, expression of these proteins results in a decrease in cell-surface major histocompatibility complex class I molecules and other immunoreceptors including intercellular adhesion molecule-1, CD86, and CD1d. These viral gene products all possess a characteristic cytosolic N-terminal RING-CH domain, responsible for ubiquitination of the target protein, and two membrane-spanning segments required for substrate specificity. For the majority of substrates, ubiquitination at the cell surface leads to rapid internalization and endolysosomal degradation, while mK3 ubiquitinates class I molecules associated with the peptide-loading complex resulting in proteasome-mediated degradation. Related viral genes with similar functions have been found in poxviruses, suggesting appropriation of these genes from the eukaryotic host. Ten membrane-associated RING-CH (MARCH) human genes with a similar organization have now been identified, and their overexpression leads to ubiquitination and downregulation of a variety of cell-surface immunoreceptors. While all the MARCH proteins are predicted to act as ubiquitin E3 ligases, their physiological role and substrates remain to be defined.

Throughout embryonic and early postnatal development, endothelial cells proliferate and differentiate to form new blood vessels via vasculogenesis and angiogenesis (1, 2). In adult organisms the endothelium defines the blood-tissue barrier and consists of non-cycling quiescent cells. These polarized cells are linked to each other by tight junctions and adherens junctions to form a continuous layer of cells. JAM (hereafter referred as JAM-1) and VE-cadherin were characterized as endothelial adhesion molecules participating in tight and adherens intercellular junctions respectively (3, 4). These molecules were shown to regulate vascular functions such as monocyte transmigration or paracellular permeability, probably as a consequence of their structural contribution to the vessel wall. It is well established that the regulated and coordinated expression of adhesion molecules is necessary to maintain normal vascular functions such as tissue homeostasis, vascular permeability, leukocyte emigration, fibrinolysis, coagulation and vasotonus. Temporary changes in the adhesion properties of vascular endothelium have been observed during inflammation, tumor growth, wounding or angiogenesis (5-7). The presence of a growing tumor increases the local concentration of angiogenic factors leading to a switch from non-cycling quiescent endothelial cells to proliferating endothelium. As a result, endothelial cells of existing vessels degrade the extracellular matrix (ECM) and invade the surrounding tissue, which leads to vascularization of tumors. During the angiogenic switch, the pattern of endothelial gene expression is modified, and the level of transcripts encoding protein s involved in cell migration or cell division is affected. For example, the balance between proteases/antiproteases such as PA/PAI1 is changed, leading to increased endothelial cell motility (8). Moreover, the treatment of endothelial cells with angiogenic factors results in a fourfold increase in αVβ3 integrin expression, an adhesion molecule implicated in cell migration (9). In addition, angiogenesis was shown to modify the endothelial inflammatory response leading to abnormal expression of inflammatory adhesion molecules (10, 11). These examples do not constitute an exhaustive list, but support the hypothesis that during the angiogenic switch, the global adhesion behavior of endothelial cells is changed.

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.