BASIC AND TRANSLATIONAL SCIENCE Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles Johanna Galaski,*† Fareed Ahmad, PhD,‡§ Nadine Tibroni,*† Francois M. Pujol, PhD,*† Birthe Müller, MA,*† Reinhold E. Schmidt, MD,‡§ and Oliver T. Fackler, PhD*† Objective: HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients. Methods: Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. Results: Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK- T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16- binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef. Conclusions: The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection. Key Words: HIV, Vpu, Nef, NK cell ligands, cell surface down- regulation (J Acquir Immune Defic Syndr 2016;72:1–10) INTRODUCTION The clinical course of HIV infection is determined by the interplay between immune responses of the host, viral evasion strategies thereof and intrinsic replication fitness of the virus. 1–4 Typical immune responses to HIV infection include innate recognition events resulting in the release of antiviral cytokines during acute infection, rapid mounting of cytotoxic T cell responses, and subsequent production of virus-specific antibodies. 5 Despite the potency of these measures, immunological control of virus spread is not achieved in most HIV patients. This reflects the relative resistance of transmitted founder viruses to antiviral cytokines such as interferons, 6,7 the genetic plasticity of HIV that allows evasion from immune recognition by selection of virus variants that lack predominant antigenic epitopes, 2 or shield- ing mechanisms that prevent efficient recognition of target structures by neutralizing antibodies. 8,9 The accessory proteins Vpu and Nef make important contributions to the ability of HIV-1 to evade host immune responses. Dispensable for virus replication in most ex vivo cell culture systems, they optimize virus replication in the infected host and this activity is thought, at least in part, to mirror direct effects of Vpu and Nef on immune recognition of productively infected cells. 10–14 Even though Vpu and Nef do not share any amino acid homology, differ in their membrane topology (Vpu is a transmembrane and Nef a peripheral membrane protein), and are expressed to peak levels early (Nef) or late (Vpu) in the replication cycle, 15 many of their functions are remarkably redundant; although via distinct molecular mechanisms, a major activity of Vpu and Nef is to alter the surface exposure of a large and overlapping set of host cell receptors. This includes the viral entry receptor CD4 for prevention of superinfection, major histocompatibility complex class 1 (MHC-I) molecules for evasion of CD8 + cytotoxic T cells, and many other receptors for which the functional consequences have yet to be established. 16–22 Cell surface levels of the antiviral restriction factor CD317/tetherin are also reduced by HIV-1 Vpu and Nef, however, only Vpu is able to counteract the particle release restriction imposed by CD317/tetherin. 23–25 Cell surface down- regulation of CD4 and CD317/tetherin by Vpu and Nef, also prevent antibody-dependent cell-mediated cytotoxicity. 26–29 In addition to the host immune reactions described above, the importance of natural killer (NK) cells especially at early stages of HIV infection is increasingly recognized as an important mechanism of host defense. 30–32 The NK cell response is determined by the balance of activating and Received for publication September 7, 2015; accepted November 23, 2015. From the *Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Heidelberg, Germany; †German Center for Infection Research, Heidelberg University, Heidelberg, Germany; ‡Department of Clinical Immunology and Rheumatology, Hannover Medical School, Hannover, Germany; and §German Center for Infection Research, Hann- over Medical School, Hannover, Germany. Supported by the German Center for Infection Research (project 8.2 TTU HIV to O.T.F./R.E.S. and an MD student fellowship to J.G.) and by the European Union FP7 consortium HIVERA IRIFCURE grant to O.T.F. The remaining authors have no funding or conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jaids.com). Correspondence to: Oliver T. Fackler, PhD, Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany (e-mail: oliver. fackler@med.uni-heidelberg.de). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. J Acquir Immune Defic Syndr  Volume 72, Number 1, May 1, 2016 www.jaids.com | 1 Copyright © 201 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 5