Title: Nef homodimers down-regulate SERINC5 by AP-2–mediated endocytosis to promote HIV-1 infectivity
Abstract: SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2–dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5. SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2–dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5. The HIV-1 Nef accessory protein is expressed early in the HIV-1 life cycle where it promotes viral replication and AIDS progression (1Laguette N. Brégnard C. Benichou S. Basmaciogullari S. Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins.Mol. Aspects Med. 2010; 31 (20594957): 418-43310.1016/j.mam.2010.05.003Crossref PubMed Scopus (44) Google Scholar). Sole expression of Nef within the CD4-positive cell compartment induces an AIDS-like syndrome in transgenic mice (2Hanna Z. Kay D.G. Rebai N. Guimond A. Jothy S. Jolicoeur P. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95 (9790524): 163-17510.1016/S0092-8674(00)81748-1Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Conversely, patients harboring Nef-defective strains of HIV-1 exhibit reduced viral loads and delayed progression to AIDS (3Deacon N.J. Tsykin A. Solomon A. Smith K. Ludford-Menting M. Hooker D.J. McPhee D.A. Greenway A.L. Ellett A. Chatfield C. Lawson V.A. Crowe S. Maerz A. Sonza S. Learmont J. et al.Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.Science. 1995; 270 (7481804): 988-99110.1126/science.270.5238.988Crossref PubMed Scopus (1007) Google Scholar, 4Kirchhoff F. Greenough T.C. Brettler D.B. Sullivan J.L. Desrosiers R.C. Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection.N. Engl. J. Med. 1995; 332 (7808489): 228-23210.1056/NEJM199501263320405Crossref PubMed Scopus (872) Google Scholar). Enhancement of lentiviral pathogenicity by Nef is conserved across species. Disruption of the nef ORF of simian immunodeficiency virus (SIV) significantly reduces viral replication and prevents the development of AIDS-like disease in SIV-infected macaques (5Kestier 3rd, H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Importance of the nef gene for maintenance of high viral loads and for development of AIDS.Cell. 1991; 65 (2032289): 651-66210.1016/0092-8674(91)90097-IAbstract Full Text PDF PubMed Scopus (1408) Google Scholar). These are just a few examples of the evidence supporting a central role for Nef in HIV-1 and SIV pathogenicity. Nef is a small protein (molecular mass range of 27–34 kDa, depending on the viral subtype) that is associated with host cell membranes by virtue of N-terminal myristylation (6Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain.Cell. 1994; 76 (8124721): 853-86410.1016/0092-8674(94)90360-3Abstract Full Text PDF PubMed Scopus (597) Google Scholar). Nef lacks inherent enzymatic activity, functioning instead through multiple protein–protein interactions that alter host cell signaling and protein trafficking networks involving as many as 70 host cell proteins (7Jäger S. Cimermancic P. Gulbahce N. Johnson J.R. McGovern K.E. Clarke S.C. Shales M. Mercenne G. Pache L. Li K. Hernandez H. Jang G.M. Roth S.L. Akiva E. Marlett J. et al.Global landscape of HIV–human protein complexes.Nature. 2011; 481 (22190034): 365-37010.1038/nature10719Crossref PubMed Scopus (472) Google Scholar). Nef selectively binds and activates members of the Src and Tec protein-tyrosine kinase families (8Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.J. Biol. Chem. 1997; 272 (9218412): 17899-1790210.1074/jbc.272.29.17899Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 9Trible R.P. Emert-Sedlak L. Smithgall T.E. HIV-1 Nef selectively activates SRC family kinases HCK, LYN, and c-SRC through direct SH3 domain interaction.J. Biol. Chem. 2006; 281 (16849330): 27029-2703810.1074/jbc.M601128200Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 10Tarafdar S. Poe J.A. Smithgall T.E. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain–dependent manner.J. Biol. Chem. 2014; 289 (24722985): 15718-1572810.1074/jbc.M114.572099Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 11Li W.F. Aryal M. Shu S.T. Smithgall T.E. HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane.J. Biol. Chem. 2020; 295 (32144207): 5163-517410.1074/jbc.RA120.012536Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Disruption of Nef-dependent kinase activation through genetic or pharmacological means impairs Nef-mediated enhancement of HIV-1 infectivity and replication (10Tarafdar S. Poe J.A. Smithgall T.E. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain–dependent manner.J. Biol. Chem. 2014; 289 (24722985): 15718-1572810.1074/jbc.M114.572099Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 11Li W.F. Aryal M. Shu S.T. Smithgall T.E. HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane.J. Biol. Chem. 2020; 295 (32144207): 5163-517410.1074/jbc.RA120.012536Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 12Emert-Sedlak L.A. Narute P. Shu S.T. Poe J.A. Shi H. Yanamala N. Alvarado J.J. Lazo J.S. Yeh J.I. Johnston P.A. Smithgall T.E. Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.Chem. Biol. 2013; 20 (23352142): 82-9110.1016/j.chembiol.2012.11.005Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Emert-Sedlak L. Kodama T. Lerner E.C. Dai W. Foster C. Day B.W. Lazo J.S. Smithgall T.E. Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds.ACS Chem. Biol. 2009; 4 (19807124): 939-94710.1021/cb900195cCrossref PubMed Scopus (56) Google Scholar, 14Readinger J.A. Schiralli G.M. Jiang J.K. Thomas C.J. August A. Henderson A.J. Schwartzberg P.L. Selective targeting of ITK blocks multiple steps of HIV replication.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18443296): 6684-668910.1073/pnas.0709659105Crossref PubMed Scopus (56) Google Scholar). In terms of protein trafficking, Nef drives the down-regulation of cell-surface receptors essential for immune recognition and viral entry, including MHC-I, CXCR4, CCR5, and CD4 (6Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain.Cell. 1994; 76 (8124721): 853-86410.1016/0092-8674(94)90360-3Abstract Full Text PDF PubMed Scopus (597) Google Scholar, 15Schwartz O. Maréchal V. Le G.S. Lemonnier F. Heard J.M. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.Nat. Med. 1996; 2 (8612235): 338-34210.1038/nm0396-338Crossref PubMed Scopus (853) Google Scholar, 16Sloan R.D. Donahue D.A. Kuhl B.D. Bar-Magen T. Wainberg M.A. Expression of Nef from unintegrated HIV-1 DNA downregulates cell surface CXCR4 and CCR5 on T-lymphocytes.Retrovirology. 2010; 7 (20465832): 4410.1186/1742-4690-7-44Crossref PubMed Scopus (29) Google Scholar). Receptor down-regulation involves Nef interactions with endosomal trafficking proteins including the adaptor protein complexes 1 and 2 (AP-1 and AP-2) (17Pereira E.A. daSilva L.L. HIV-1 Nef: taking control of protein trafficking.Traffic. 2016; 17 (27161574): 976-99610.1111/tra.12412Crossref PubMed Scopus (67) Google Scholar, 18Janvier K. Craig H. Hitchin D. Madrid R. Sol-Foulon N. Renault L. Cherfils J. Cassel D. Benichou S. Guatelli J. HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes.J. Biol. Chem. 2003; 278 (12486136): 8725-873210.1074/jbc.M210115200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Down-regulation of CD4 requires simultaneous engagement of Nef with both the cytoplasmic tail of CD4 and a hemicomplex formed by the α and σ2 subunits of AP-2 (19Ren X. Park S.Y. Bonifacino J.S. Hurley J.H. How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4.eLife. 2014; 3 (24473078): e0175410.7554/eLife.01754Crossref PubMed Google Scholar). Nef–CD4–AP-2 complexes form clathrin-coated pits at the cell surface, leading to endocytosis and lysosomal degradation of internalized CD4 (20Gondim M.V. Wiltzer-Bach L. Maurer B. Banning C. Arganaraz E. Schindler M. AP-2 is the crucial clathrin adaptor protein for CD4 downmodulation by HIV-1 Nef in infected primary CD4+ T cells.J. Virol. 2015; 89 (26423947): 12518-1252410.1128/JVI.01838-15Crossref PubMed Scopus (12) Google Scholar). Nef-mediated endocytosis of the SERINC5 restriction factor, a process essential for enhancement of HIV-1 infectivity, is also mediated by the AP-2 trafficking pathway. In the absence of Nef, SERINC5 is present on the surface of HIV-1–infected cells and incorporated into the membrane of newly synthesized virions (21Rosa A. Chande A. Ziglio S. De S.V. Bertorelli R. Goh S.L. McCauley S.M. Nowosielska A. Antonarakis S.E. Luban J. Santoni F.A. Pizzato M. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.Nature. 2015; 526 (26416734): 212-21710.1038/nature15399Crossref PubMed Scopus (250) Google Scholar, 22Usami Y. Wu Y. Göttlinger H.G. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.Nature. 2015; 526 (26416733): 218-22310.1038/nature15400Crossref PubMed Scopus (249) Google Scholar, 23Sood C. Marin M. Chande A. Pizzato M. Melikyan G.B. SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins.J. Biol. Chem. 2017; 292 (28179429): 6014-602610.1074/jbc.M117.777714Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Incorporation of SERINC5 disrupts viral fusion with host cells and delivery of the viral core through a cryptic, Env-dependent mechanism. Nef antagonizes SERINC5 in part by promoting AP-2–dependent down-regulation of SERINC5 from the plasma membrane of infected cells, thereby preventing incorporation into budding virions (22Usami Y. Wu Y. Göttlinger H.G. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.Nature. 2015; 526 (26416733): 218-22310.1038/nature15400Crossref PubMed Scopus (249) Google Scholar). Following down-regulation by Nef, internalized SERINC5 is targeted for degradation via the endolysosomal pathway (24Shi J. Xiong R. Zhou T. Su P. Zhang X. Qiu X. Li H. Li S. Yu C. Wang B. Ding C. Smithgall T.E. Zheng Y.H. HIV-1 Nef antagonizes SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome system.J. Virol. 2018; 92 (29514909): e00118-e0019610.1128/JVI.00196-18Crossref Scopus (46) Google Scholar). Although Nef uses distinct structural motifs to recognize diverse host cell partners, many Nef functions also require homodimer formation. Mutants of Nef that are defective for homodimerization are unable to induce host-cell tyrosine-kinase activation, even though they retain interaction with the kinase proteins (11Li W.F. Aryal M. Shu S.T. Smithgall T.E. HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane.J. Biol. Chem. 2020; 295 (32144207): 5163-517410.1074/jbc.RA120.012536Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). This observation is consistent with the formation of Nef–kinase dimer complexes necessary for kinase activation by transautophosphorylation. Dimerization-defective Nef mutants are also unable to down-regulate CD4 from the host-cell surface, and these mutations reduce HIV-1 replication efficiency in cell lines (25Shu S.T. Emert-Sedlak L.A. Smithgall T.E. Cell-based fluorescence complementation reveals a role for HIV-1 Nef protein dimerization in AP-2 adaptor recruitment and CD4 co-receptor down-regulation.J. Biol. Chem. 2017; 292 (28031466): 2670-267810.1074/jbc.M116.770016Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 26Poe J.A. Smithgall T.E. HIV-1 Nef dimerization is required for Nef-mediated receptor downregulation and viral replication.J. Mol. Biol. 2009; 394 (19781555): 329-34210.1016/j.jmb.2009.09.047Crossref PubMed Scopus (57) Google Scholar). In this study, we investigated the role of Nef homodimerization in HIV-1 infectivity and restriction by SERINC5. Using a series of Nef mutants defective for homodimer formation in cells, we demonstrated that Nef dimers enhance infectivity of HIV-1 produced from T-cell lines and donor PBMCs, as well as 293T cells co-transfected with SERINC5. These same Nef mutants were attenuated in their ability to exclude SERINC5 from newly synthesized virions. Impairment of Nef dimerization reduced down-regulation of SERINC5 from the cell surface and prevented intracellular trafficking to Rab7+ late endosomes. Critically, cell-based fluorescence complementation assays revealed that these Nef mutants retained interaction with both SERINC5 and the AP-2 α and σ2 subunits, indicating that the reduced antagonism of SERINC5 was not due to the loss of Nef association with either partner protein. Taken together, our data demonstrate that Nef antagonizes the SERINC5 restriction factor as a homodimer, which may serve as a bridge linking SERINC5 with AP-2 for endocytosis and degradation. In this study, we explored the role of Nef homodimerization in the down-regulation of SERINC5 and promotion of HIV-1 infectivity using a genetic approach involving Nef mutants defective for homodimer formation. X-ray crystallography of Nef in complex with the regulatory SH3 and SH2 domains of Src-family kinases has shown that Nef forms homodimers through a hydrophobic interface involving side chains of residues in the Nef αB helices and adjacent loops (27Lee C.-H. Saksela K. Mirza U.A. Chait B.T. Kuriyan J. Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain.Cell. 1996; 85 (8681387): 931-94210.1016/S0092-8674(00)81276-3Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 28Alvarado J.J. Tarafdar S. Yeh J.I. Smithgall T.E. Interaction with the Src homology (SH3–SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.J. Biol. Chem. 2014; 289 (25122770): 28539-2855310.1074/jbc.M114.600031Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). An example is provided by the structure of HIV-1 Nef in complex with the SH3–SH2 regulatory region of the Src-family kinase, Hck (PDB code 4U5W; a model of the Nef homodimer is shown in Fig. 1A) (28Alvarado J.J. Tarafdar S. Yeh J.I. Smithgall T.E. Interaction with the Src homology (SH3–SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.J. Biol. Chem. 2014; 289 (25122770): 28539-2855310.1074/jbc.M114.600031Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In this structure, three conserved residues from one Nef subunit (Leu112, Tyr115, and Phe121) converge to create a “hydrophobic cup” that clasps the side chain of Val70 from the opposing Nef monomer in a reciprocal fashion (Fig. 1B). A recent study has shown that recombinant Nef proteins in which Leu112 or Tyr115 is replaced with aspartate or in which Phe121 is substituted with alanine fail to form dimers in solution, providing direct evidence that these residues are essential for dimer stabilization (11Li W.F. Aryal M. Shu S.T. Smithgall T.E. HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane.J. Biol. Chem. 2020; 295 (32144207): 5163-517410.1074/jbc.RA120.012536Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Mutations at these positions attenuate multiple Nef functions, including host-cell kinase activation, enhancement of HIV-1 replication, and receptor down-regulation including CD4 (see the introduction). Although the role of Nef homodimers in SERINC5 antagonism has not been explored, the mechanistic similarity between Nef-mediated down-regulation of CD4 and SERINC5 suggests a key role. Both processes involve clathrin-mediated endocytosis via the tetrameric trafficking adaptor protein AP-2, which interacts directly with Nef through a hemicomplex of its α and σ2 subunits (19Ren X. Park S.Y. Bonifacino J.S. Hurley J.H. How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4.eLife. 2014; 3 (24473078): e0175410.7554/eLife.01754Crossref PubMed Google Scholar, 29Ramirez P.W. Sharma S. Singh R. Stoneham C.A. Vollbrecht T. Guatelli J. Plasma membrane–associated restriction factors and their counteraction by HIV-1 accessory proteins.Cells. 2019; 8 (31480747): 102010.3390/cells8091020Crossref Scopus (8) Google Scholar). Alignment of the Nef 4U5W homodimer with the crystal structure of Nef bound to the AP-2 α/σ2 hemicomplex shows that the Nef homodimer interface residues do not contact AP-2 (Fig. 1C), suggesting that the dimer interface mutations described above will not affect AP-2 recruitment. These mutants were therefore chosen to explore the requirement for Nef dimers in SERINC5 regulation. Although Nef is present in HIV-1 virions and expressed early within the viral life cycle (29Ramirez P.W. Sharma S. Singh R. Stoneham C.A. Vollbrecht T. Guatelli J. Plasma membrane–associated restriction factors and their counteraction by HIV-1 accessory proteins.Cells. 2019; 8 (31480747): 102010.3390/cells8091020Crossref Scopus (8) Google Scholar), previous analyses have examined the impact of mutations on Nef self-association 40–48 h post-transfection (25Shu S.T. Emert-Sedlak L.A. Smithgall T.E. Cell-based fluorescence complementation reveals a role for HIV-1 Nef protein dimerization in AP-2 adaptor recruitment and CD4 co-receptor down-regulation.J. Biol. Chem. 2017; 292 (28031466): 2670-267810.1074/jbc.M116.770016Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 26Poe J.A. Smithgall T.E. HIV-1 Nef dimerization is required for Nef-mediated receptor downregulation and viral replication.J. Mol. Biol. 2009; 394 (19781555): 329-34210.1016/j.jmb.2009.09.047Crossref PubMed Scopus (57) Google Scholar), which corresponds to the end of the HIV-1 life cycle (30Holmes M. Zhang F. Bieniasz P.D. Single-cell and single-cycle analysis of HIV-1 replication.PLoS Pathog. 2015; 11 (26086614): e100496110.1371/journal.ppat.1004961Crossref PubMed Scopus (43) Google Scholar). We therefore explored the kinetics of WT Nef homodimer formation and membrane association at 12-h intervals using a cell-based bimolecular fluorescence complementation (BiFC) assay (31Kerppola T.K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.Annu. Rev. Biophys. 2008; 37 (18573091): 465-48710.1146/annurev.biophys.37.032807.125842Crossref PubMed Scopus (447) Google Scholar). In this approach, WT and mutant Nef coding sequences are fused to an N-terminal fragment of the YFP variant, Venus (Nef-VN), whereas complementary Nef constructs are fused to a C-terminal Venus fragment (Nef-VC). Co-expression of the Nef-VN and Nef-VC fusion proteins in the same cell results in protein–protein interaction between Nef molecules, leading to complementation of the Venus fluorophore and a bright fluorescent signal. Cultures of 293T cells were transfected with Nef-VN and Nef-VC expression vectors (WT or the dimerization interface mutants L112A, L112D, Y115D, and F121A) and fixed with paraformaldehyde 12, 24, 36, and 48 h later. The cells were then immunostained to confirm Nef protein expression, allowing normalization of the resulting BiFC signal to Nef expression levels within each culture. Fig. 2A shows representative confocal images of Nef BiFC (homodimer formation) and immunofluorescence (Nef protein expression) at the 48-h end point of the experiment, whereas Nef BiFC to immunofluorescence intensity ratios across all four time points are plotted in Fig. 2B. Little fluorescence complementation was observed with any of the Nef BiFC pairs at 12 h, which likely reflects the low levels of Nef expression at this time point. After 24 h, WT Nef self-association was readily observed and increased in a linear fashion over 48 h. In contrast to WT Nef, the L112D, Y115D, and F121A mutants demonstrated statistically significant defects in Nef homodimerization across all time points (Fig. S1). The Y115D mutant showed the lowest ratios at each time point, with the L112D and F121A mutants exhibiting an intermediate phenotype. By contrast, the L112A mutation did not decrease the BiFC signal, suggesting that substitution of leucine with alanine does not disrupt homodimer formation. Inspection of the BiFC and immunofluorescence images suggests that all four Nef mutants remained associated with the plasma membrane, although the Y115D mutant showed increased cytoplasmic localization. To test the requirement for Nef homodimers in SERINC5 antagonism, we first validated a 293T producer cell system to assess virion incorporation of SERINC5 and impact on viral infectivity. For these studies, 293T cells were transfected with proviral plasmids encoding WT HIV-1 or a mutant that fails to express Nef (ΔNef) in the presence and absence of a SERINC5 expression vector. Newly synthesized virions were concentrated from the culture supernatant 2 days later, and levels of virion-incorporated SERINC5 were analyzed via immunoblot. The infectivity of each viral supernatant was assessed in parallel using the TZM-bl reporter cell line (32Gervaix A. West D. Leoni L.M. Richman D.D. Wong-Staal F. Corbeil J. A new reporter cell line to monitor HIV infection and drug susceptibility in vitro.Proc. Natl. Acad. Sci. U.S.A. 1997; 94 (9114046): 4653-465810.1073/pnas.94.9.4653Crossref PubMed Scopus (203) Google Scholar). Expression of SERINC5 in 293T producer cells inhibited HIV-1 ΔNef infectivity but had little impact on the infectivity of the WT virus, consistent with previous results (Fig. 3A) (21Rosa A. Chande A. Ziglio S. De S.V. Bertorelli R. Goh S.L. McCauley S.M. Nowosielska A. Antonarakis S.E. Luban J. Santoni F.A. Pizzato M. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.Nature. 2015; 526 (26416734): 212-21710.1038/nature15399Crossref PubMed Scopus (250) Google Scholar, 22Usami Y. Wu Y. Göttlinger H.G. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.Nature. 2015; 526 (26416733): 218-22310.1038/nature15400Crossref PubMed Scopus (249) Google Scholar). Nef antagonism of SERINC5 within this system is consistent with levels of virion-incorporated SERINC5 observed by immunoblot, with WT virions exhibiting decreased levels of SERINC5 compared with the ΔNef virions (Fig. 3B). Co-transfection of an expression plasmid encoding Nef along with SERINC5 and the ΔNef proviral plasmid rescued infectivity (Fig. 3C) and promoted exclusion of SERINC5 from newly synthesized virions (Fig. 3D). These observations validate the use of the 293T producer cell system to explore the role of Nef homodimerization in SERINC5-mediated restriction of HIV-1 infectivity. We next transfected 293T cells with HIV-1 proviral DNA bearing the Nef dimerization interface mutations L112A, L112D, Y115D, and F121A in the presence and absence of the SERINC5 expression vector. The infectivity of HIV-1 expressing the L112D, Y115D, and F121A Nef mutants was reduced in the presence of SERINC5 to the levels observed with the ΔNef virus, whereas the L112A mutant exhibited a partial loss of function (Fig. 4A). This finding is consistent with BiFC results showing that L112A has little impact on Nef homodimer formation in this system. Immunoblot analysis confirmed that each of the Nef mutants was expressed in transfected 293T cells (Fig. 4B). In a complementary set of experiments, we explored the role of Nef dimerization in SERINC5 antagonism using the Jurkat JTAg producer cell system, which expresses a relatively high level of endogenous SERINC5 like that observed in normal CD4 T cells. An isogenic Jurkat JTag cell population in which both SERINC5 alleles are inactivated by CRISPR/Cas9 was studied in parallel (21Rosa A. Chande A. Ziglio S. De S.V. Bertorelli R. Goh S.L. McCauley S.M. Nowosielska A. Antonarakis S.E. Luban J. Santoni F.A. Pizzato M. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.Nature. 2015; 526 (26416734): 212-21710.1038/nature15399Crossref PubMed Scopus (250) Google Scholar). Jurkat JTAg WT and SERINC5-knockout cells were transfected with HIV-1 proviral constructs bearing the same Nef dimerization interface mutations, along with WT and ΔNef HIV-1. The infectivity of the viruses expressing the L112D, Y115D, and F121A Nef mutants produced in WT Jurkat JTAg cells was reduced to that of the ΔNef virus, consistent with the results in 293T producer cells co-expressing SERINC5. The infectivity of these Nef mutant viruses was restored when produced in the SERINC5-knockout cells, providing evidence of a SERINC5-dependent mechanism in this T-cell line (Fig. 4C). WT HIV-1 produced from either of the Jurkat producer cell lines displayed similar infectivity, consistent with the ability of WT Nef to antagonize SERINC5. Infectivity of the Nef-L112A mutant virus was also less affected by the presence or absence of SERINC5, consistent with the lack of an effect of this mutation on Nef self-association in the BiFC assay. Expression of WT and mutant forms of Nef in Jurkat producer cells was verified by immunoblot analysis (Fig. 4D). We also addressed whether Nef dimerization is important for infectivity of HIV-1 produced during a spreading infection in primary host-cell culture. SERINC5-mediated restriction has been previously observed in donor PBMCs, which also express high levels of endogenous SERINC5 (21Rosa A. Chande A. Ziglio S. De S.V. Bertorelli R. Goh S.L. McCauley S.M. Nowosielska A. Antonarakis S.E. Luban J. Santoni F.A. Pizzato M. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.Nature. 2015; 526 (26416734): 212-21710.1038/nature15399Crossref PubMed Scopus (250) Google Scholar