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      Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses

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          ABSTRACT

          HIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV + sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., the Renilla luciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4 + T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4 + T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

          IMPORTANCE In-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

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          Up on the tightrope: natural killer cell activation and inhibition.

          Natural killer (NK) cells circulate through the blood, lymphatics and tissues, on patrol for the presence of transformed or pathogen-infected cells. As almost all NK cell receptors bind to host-encoded ligands, signals are constantly being transmitted into NK cells, whether they interact with normal or abnormal cells. The sophisticated repertoire of activating and inhibitory receptors that has evolved to regulate NK cell activity ensures that NK cells protect hosts against pathogens, yet prevents deleterious NK cell-driven autoimmune responses. Here I highlight recent advances in our understanding of the structural properties and signaling pathways of the inhibitory and activating NK cell receptors, with a particular focus on the ITAM-dependent activating receptors, the NKG2D-DAP10 receptor complexes and the CD244 receptor system.
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            Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control

            Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune–recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.
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              Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

              Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Virol
                J. Virol
                jvi
                jvi
                JVI
                Journal of Virology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0022-538X
                1098-5514
                18 April 2018
                13 June 2018
                1 July 2018
                13 June 2018
                : 92
                : 13
                : e00484-18
                Affiliations
                [a ]Centre de Recherche du CHUM, Montreal, QC, Canada
                [b ]Department of Microbiology, Infectious Diseases and Immunology, Université de Montréal, Montreal, QC, Canada
                [c ]Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
                [d ]Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada
                Icahn School of Medicine at Mount Sinai
                Author notes
                Address correspondence to Christina Ochsenbauer, ochsenba@ 123456uab.edu , or Andrés Finzi, andres.finzi@ 123456umontreal.ca .

                Citation Prévost J, Richard J, Medjahed H, Alexander A, Jones J, Kappes JC, Ochsenbauer C, Finzi A. 2018. Incomplete downregulation of CD4 expression affects HIV-1 Env conformation and antibody-dependent cellular cytotoxicity responses. J Virol 92:e00484-18. https://doi.org/10.1128/JVI.00484-18.

                Article
                00484-18
                10.1128/JVI.00484-18
                6002730
                29669829
                4515d11b-e89f-4ef3-8bdd-8b8ee719f766
                Copyright © 2018 Prévost et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 21 March 2018
                : 11 April 2018
                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 84, Pages: 16, Words: 10946
                Funding
                Funded by: CAVD;
                Award ID: 1032144
                Award Recipient :
                Funded by: HHS | National Institutes of Health (NIH), https://doi.org/10.13039/100000002;
                Award ID: AI100645
                Funded by: HHS | National Institutes of Health (NIH), https://doi.org/10.13039/100000002;
                Award ID: U01-AI067854
                Award Recipient :
                Funded by: HHS | National Institutes of Health (NIH), https://doi.org/10.13039/100000002;
                Award ID: AI129769
                Award Recipient :
                Funded by: Canada Research Chairs (Chaires de recherche du Canada), https://doi.org/10.13039/501100001804;
                Award ID: RCHS0235
                Award Recipient :
                Funded by: Gouvernement du Canada | Canadian Institutes of Health Research (CIHR), https://doi.org/10.13039/501100000024;
                Award ID: 352417
                Award Recipient :
                Categories
                Virus-Cell Interactions
                Custom metadata
                July 2018

                Microbiology & Virology
                hiv-1,nef,cd4,env,gp120,adcc,nkg2d,luciferase,imc,a32,cd4i
                Microbiology & Virology
                hiv-1, nef, cd4, env, gp120, adcc, nkg2d, luciferase, imc, a32, cd4i

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