5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

There are five families of vertebrate Cys loop receptors as follows: the nicotinic acetylcholine receptor (nAChR), 3 the 5-hydroxytryptamine type 3 receptor (5-HT 3 R), the zinc-activated ion channel, the ␥-aminobutyric acid type A receptor, and the strychnine-sensitive glycine receptor (1, 2). Structural analysis by cryo-EM of tubular crystals prepared from the Torpedo marmorata electric organ revealed that five subunits combine in nAChRs, forming a rosette around the central ion channel (3). The second transmembrane (M2) domain of each subunit participates in lining the channel pore, and collectively they present a hydrophobic constriction adjacent to what is traditionally believed to be the rate-limiting portion of the ion conduction pathway that controls single channel conductance (␥) and ionic selectivity (2, 4).The homomeric 5-HT 3A receptor is unique among Cys loop receptors, having a ␥ below the resolution of single channel recording, estimated by variance analysis to be in the femtosiemen range. The incorporation of the 5-HT 3B subunit into human heteromeric 5-HT 3A/B receptors increases ␥ to 16 pS, enabling direct observation of events by single channel recording from outside-out patches (5). The use of chimeric 5-HT 3A -5-HT 3B constructs and site-directed mutagenesis revealed a critical role of three arginine residues within the MA helix of the M3-M4 cytoplasmic loop in determining ␥ (6, 7). This has prompted speculation that the MA helix may participate in the control of ␥ in other Cys loop receptors (4,7,8). In support of this interpretation, cryo-EM analysis revealed portals within the Torpedo nAChR formed, in part, by the MA helices of adjacent subunits that may participate in the ion conduction pathway (9).In this study we investigated the influence of the 5-HT 3A subunit's MA helix Arg-432 (Ϫ4Ј), Arg-436 (0Ј), and Arg-440 (4Ј) residues in the control of ␥. We investigated the effect of introducing arginine into the equivalent locations within the nAChR ␣ 4 and ␤ 2 subunits. Our data confirm the critical role of MA Ϫ4Ј, 0Ј, and 4Ј residues in controlling ␥ of 5-HT 3A receptors and support the hypothesis that the MA helix also forms part of the ion conduction pathway of nAChRs. Our functional data provide support for the existence of cytoplasmic portals depicted in the 4 Å structural model of the nAChR (10). EXPERIMENTAL PROCEDURES DNA Constructs and Transient Transfection of Subunit cDNAs-cDNAs encoding rat wild-type (WT) nAChR ␣ 4 and ␤ 2 subunits (Dr. J. M. Boulter, Department of Psychiatry and Biobehavioral Sciences, UCLA), human WT 5-HT 3A subunits, and mutant nAChR and 5-HT 3A subunits were cloned into pGW1 (11). Point mutations were introduced using standard molecular biological techniques (7). All cDNAs were sequenced to confirm fidelity. Transfection of tsA-201, or HEK-293 cells, with subunit cDNAs, at equimolar ratios when appropriate, was performed by either the calcium phosphate precipitation method or electroporation (400 V, infinite resistance, 125 microfarads) using a Bio-Rad gene ...

Human IgA is both a major serum immunoglobulin and the most abundant antibody class in seromucous secretions (1). The mucosal surfaces bathed by these secretions, such as those of the respiratory, gastrointestinal, and genitourinary tracts, are major potential sites of invasion due to their vast surface area. IgA therefore serves as a key first line of defense against many invading pathogens. Like all antibodies, IgA is capable of both recognizing the foreign invader and triggering its elimination. The latter process is frequently mediated by the interaction of the Fc region of IgA with Fc␣ receptors (Fc␣R) 1 present on the surface of neutrophils, macrophages, monocytes, and eosinophils (2, 3). The human myeloid Fc␣R, CD89, possesses two extracellular Ig-like domains and displays homology to the three classes of human IgG Fc receptors (Fc␥RI, Fc␥RII and Fc␥RIII) and the high affinity IgE receptor Fc⑀RI, albeit at a lower level than these receptors do to each other (4). Interaction of CD89 with IgA, aggregated either by binding to antigen or artificially, acts as a potent trigger for an array of myeloid cell functions including phagocytosis, antibody-dependent cellmediated cytotoxicity, superoxide generation, enzyme and inflammatory mediator release, and clearance of immune complexes (3). A detailed understanding of the molecular basis of the IgA-Fc␣R interaction is clearly important if the increasingly appreciated potential of recombinant IgA in numerous therapeutic applications (5-7) is to be fully realized.Although others have described expression of human IgA in insect (8) and plant cells (5), we have expressed hapten-specific recombinant human IgA of both subclasses, IgA1 and IgA2, in mammalian cell hosts (9 -11). Here, we have used an extensive panel of chimeric and site-directed mutant IgAs expressed in CHO K1 cells to identify residues critical for Fc␣R binding. We have constructed domain swap antibodies through exchange of the homologous C-terminal CH3 domains between human IgA1 and IgG1 in order to ascertain the contribution of each Fc domain to the interaction with Fc␣R. An IgA1 lacking the C-terminal 18 amino acid tailpiece has also been assayed for ability to bind the receptor. To allow more precise localization of the interaction site, a number of IgA1 mutants with single substitutions in loop regions lying at the interface of the CH2 and CH3 domains have been generated. The effects of such mutations on the ability to bind Fc␣R are consistent with the interdomain region of the Fc playing a critical role in binding to the receptor. Further support for this proposal is lent through correlation of the binding ability of IgAs derived from other species with sequence differences in these loops.To more readily assess receptor interaction, we have developed stable CHO cell transfectants expressing high levels of CD89, which have allowed, for the first time, comparison of the relative binding affinities of the different IgA molecules. As an additional, more physiologically relevant test for function, we hav...

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To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fc␣ receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e., epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate surfaces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To the best of our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity toward their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key binding residues by simply changing the amino acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key binding residue for binding monoclonal antibody V3.2.

Objective To determine the prevalence of the hepatitis C virus among pregnant women, to gauge the noninjecting, particularly sexual, risk of them being hepatitis C virus infected and to assess the potential impact of selective antenatal screening. Population Antenatal clinic attenders and women undergoing termination of pregnancy in 1997.Setting Ninewells Hospital, Dundee. Design Unlinked anonymous hepatitis C virus antibody testing of residual sera from specimens sent to the virus laboratory for routine serological testing. The results were linked to non-identifying risk information. Results Overall anti-hepatitis C virus prevalence was 0.6% (23/3548). Prevalences among injecting drug users, non-injectors who had a sexual partner who injected, and those with neither risk respectively were 41% (7/17), 15% (5/33) and 0.3% (11/3498). Relative risks for being an injector and a sexual partner of an injector respectively were 131 (95% CI 58-297) and 48 (95% CI 5-32). It is estimated that one of the 18 antenatal clinic attenders gave birth to an infected child. Conclusion Findings suggest that non-injecting partners of injectors may be at considerable risk of acquiring hepatitis C virus sexually. Efforts to promote the use of condoms among injectors and their sexual partners should be increased. Selective anti-hepatitis C virus screening of women who reported high risk behaviour would have failed to detect half the cases. Research to gauge the views of women of childbearing age on antihepatitis C virus testing is required.