Joseph Rucker

A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors

Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.

Regions in β-Chemokine Receptors CCR5 and CCR2b That Determine HIV-1 Cofactor Specificity

Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.

ChemR23, a putative chemoattractant receptor, is expressed in monocyte‐derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV‐1 strains

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein‐coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met‐Leu‐Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte‐derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription‐PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2 – 21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV‐1, HIV‐2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV‐2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmac17E‐Fr and SIVsm62A), as well as a primary HIV‐1 strain (92UG024‐2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen‐presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein‐coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1)

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin.

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

ABSTRACT Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epitope conservation, and epitope accessibility—also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAbs specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus. IMPORTANCE Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus E2 protects from disease.

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

Atomic-Level Mapping of Antibody Epitopes on a GPCR

Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

ABSTRACT Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.