Title: Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes
Abstract: Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance. Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance. The specter of the HIV pandemic first appeared in the United States Centers for Disease Control Morbidity and Mortality Weekly Report on June 5, 1981. A brief in that report recounted the appearance of Pneumocystis carinii pneumonia cases in the Los Angeles area, a diagnosis typically associated with severely immunocompromised patient populations but unexpected in young men (1Jaffe H.W. The early days of the HIV-AIDS epidemic in the U.S.A.Nat. Immunol. 2008; 9 (18936776): 1201-120310.1038/ni1108-1201Crossref PubMed Scopus (2) Google Scholar). This report unknowingly provided the first description of what would become HIV/AIDS in the medical literature. Today, the AIDS pandemic persists as a global public health crisis, with 38 million people currently living with HIV worldwide. More than 1.7 million new infections and nearly 700,000 HIV-related deaths occurred in 2019, mainly in southern and eastern Africa. Over 30 million people have died from AIDS and related illnesses since the start of the pandemic (UNAIDS 2020 Fact Sheet). The first antiretroviral drug developed for HIV-1 was the nucleoside reverse transcriptase inhibitor (NRTI), zidovudine, which was approved by the United States Food and Drug Administration in 1987 (2Marwick C. AZT (zidovudine) just a step away from FDA approval for AIDS therapy.JAMA. 1987; 257 (3469418): 1281-128210.1001/jama.1987.03390100015002Crossref PubMed Google Scholar). Subsequent development of additional NRTIs facilitated combination therapy that markedly delayed AIDS progression compared with zidovudine alone (3Hammer S.M. Katzenstein D.A. Hughes M.D. Gundacker H. Schooley R.T. Haubrich R.H. 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Because HIV-1 is an integrating lentivirus, identification of curative strategies for HIV-1 infection has proven to be an even more difficult challenge than developing drugs to control infection. Clinical trials of HIV-1 vaccine candidates have been largely unsuccessful, with just one vaccine producing partial efficacy to date (7Rerks-Ngarm S. Pitisuttithum P. Nitayaphan S. Kaewkungwal J. Chiu J. Paris R. Premsri N. Namwat C. de Souza M. Adams E. Benenson M. Gurunathan S. Tartaglia J. McNeil J.G. Francis D.P. et al.Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.N. Engl. J. Med. 2009; 361 (19843557): 2209-222010.1056/NEJMoa0908492Crossref PubMed Scopus (2266) Google Scholar). In addition to vaccines, substantial research effort has addressed therapeutic cures for HIV-1 infection. One well-known cure strategy is based on the concept of “shock and kill,” in which latency reversal agents (LRAs) are used to reactivate the integrated provirus in latent reservoirs in the presence of antiretroviral drugs. Induction of viral protein expression may then promote immune recognition and clearance of latently infected cells (8Kim Y. Anderson J.L. Lewin S.R. Getting the “kill” into “shock and kill”: strategies to eliminate latent HIV.Cell Host Microbe. 2018; 23 (29324227): 14-2610.1016/j.chom.2017.12.004Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Recent examples of LRAs that induce latency reversal in animal models in the presence of antiretroviral drugs include engineered agonists for the interleukin-15 receptor (9McBrien J.B. Mavigner M. Franchitti L. Smith S.A. White E. Tharp G.K. Walum H. Busman-Sahay K. Aguilera-Sandoval C.R. Thayer W.O. Spagnuolo R.A. Kovarova M. Wahl A. Cervasi B. 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The observations that LRAs can induce viral gene expression in the presence of antiretroviral drugs, but fail to prevent viral rebound following antiretroviral drug withdrawal, indicate that additional modalities are required to enhance the host immune response to the reactivated HIV-1 producer cell population. Inhibition of the HIV-1 Nef accessory protein, which plays a key role in immune escape of HIV-infected cells, may provide the additional immune system boost required for reduction of the latent reservoir. HIV-1 Nef is a relatively small (27–34 kDa, depending on the lentivirus), nonenzymatic accessory protein expressed early and throughout the viral life cycle. Nef was initially named on the basis of early evidence that it was a “negative factor” for HIV-1 replication (11Ahmad N. Venkatesan S. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR.Science. 1988; 241 (3262235): 1481-148510.1126/science.3262235Crossref PubMed Google Scholar). On the contrary, Nef plays prominent roles in HIV-1 pathogenesis by promoting viral replication and spread in vivo and by enabling immune escape of HIV-infected cells. Patients infected with HIV-1 harboring mutant or defective nef genes fail to progress to AIDS (12Rhodes D.I. Ashton L. Solomon A. Carr A. Cooper D. Kaldor J. Deacon N. Characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. Australian Long-Term Nonprogressor Study Group.J. Virol. 2000; 74 (11044102): 10581-1058810.1128/jvi.74.22.10581-10588.2000Crossref PubMed Scopus (103) Google Scholar, 13Kirchhoff F. Greenough T.C. Brettler D.B. Sullivan J.L. Desrosiers R.C. Brief report: 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 (859) Google Scholar). 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Nef is comprised of two main parts: the N-terminal anchor region and a folded core (Fig. 1). The N-terminal 60 amino acids that comprise the anchor region are largely unstructured and bear a signal for myristoylation at the N-terminal glycine. NMR studies identified an amphipathic helix within the N-terminal anchor region (20Geyer M. Munte C.E. Schorr J. Kellner R. Kalbitzer H.R. Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.J. Mol. Biol. 1999; 289 (10339411): 123-13810.1006/jmbi.1999.2740Crossref PubMed Scopus (0) Google Scholar), which together with the N-myristoyl group serves to localize Nef to host cell membranes (21Gerlach H. Laumann V. Martens S. Becker C.F. Goody R.S. Geyer M. HIV-1 Nef membrane association depends on charge, curvature, composition and sequence.Nat. Chem. Biol. 2010; 6 (19935658): 46-5310.1038/nchembio.268Crossref PubMed Scopus (69) Google Scholar). Following the anchor region is a folded core domain (∼105 residues) with a large flexible internal loop (∼25 residues). Because the anchor region is flexible, the folded core can move off the membrane (22Akgun B. Satija S. Nanda H. Pirrone G.F. Shi X. Engen J.R. Kent M.S. Conformational transition of membrane-associated terminally acylated HIV-1 Nef.Structure. 2013; 21 (24035710): 1822-183310.1016/j.str.2013.08.008Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23Kent M.S. Murton J.K. Sasaki D.Y. Satija S. Akgun B. Nanda H. Curtis J.E. Majewski J. Morgan C.R. Engen J.R. Neutron reflectometry study of the conformation of HIV Nef bound to lipid membranes.Biophys. J. 2010; 99 (20858440): 1940-194810.1016/j.bpj.2010.07.016Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) to accommodate interactions with diverse host cell proteins, many of which are also associated with the membrane (Fig. 1). Nef does not exhibit any known enzymatic or biochemical activities, functioning instead through a diverse array of protein-protein interactions (24Jäger 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 (443) Google Scholar). Examples of the best-characterized Nef functions and attendant binding partners are summarized below. These include endocytic trafficking molecules followed by protein kinases, which are the primary focus of this review. Nef is remarkably well-adapted to hijack the intracellular trafficking machinery to modulate cell-surface protein expression. Two well-characterized Nef functions in this regard are down-regulation of CD4 and major histocompatibility complex I (MHC-I) proteins, which are orchestrated by Nef via the endocytic adaptor protein complexes, AP-1 and AP-2. Nef also drives down-regulation of the SERINC family of host cell restriction factors via AP-2 to enhance viral infectivity. Structural features and other aspects of these interactions are covered in several recent reviews (25Buffalo C.Z. Iwamoto Y. Hurley J.H. Ren X. How HIV Nef proteins hijack membrane traffic to promote infection.J. Virol. 2019; 93: e0132210.1128/JVI.01322-19Crossref PubMed Scopus (3) Google Scholar, 26Pereira E.A. daSilva L.L. HIV-1 Nef: taking control of protein trafficking.Traffic. 2016; 17 (27161574): 976-99610.1111/tra.12412Crossref PubMed Google Scholar) and are not discussed in detail here. Down-regulation of CD4 by Nef is conserved across virtually all HIV-1 subtypes (27Mariani R. Skowronski J. 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Antigenic peptides derived from proteolytically digested viral proteins are loaded onto MHC-I molecules within the endoplasmic reticulum and presented on the surface of infected cells, triggering recognition and killing of the infected cell by CD8 cytotoxic T cells. In contrast to CD4 down-regulation, Nef-mediated MHC-I down-regulation occurs via AP-1. Two temporally distinct models of Nef-mediated antagonism of MHC-I have been reported. In the first model, also known as the “signaling mode,” Nef is recruited by the phosphofurin acidic cluster 2 (PACS-2) adaptor protein to the trans-Golgi network (TGN), where it drives the activation of Src-family kinases specific to the host cell lineage (Hck in macrophages; Lyn in T cells). Src-family kinase activity initiates a signaling cascade that ultimately increases levels of membrane phosphatidylinositol (3,4,5)-trisphosphate via phosphoinositide 3-kinase, causing activation of the small GTPases Arf1 and Arf6 and endocytosis of cell-surface MHC-I (42Dikeakos J.D. Atkins K.M. Thomas L. Emert-Sedlak L. Byeon I.J. Jung J. Ahn J. Wortman M.D. Kukull B. Saito M. Koizumi H. Williamson D.M. Hiyoshi M. Barklis E. Takiguchi M. et al.Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action.Mol. Biol. Cell. 2010; 21 (20702582): 3279-329210.1091/mbc.E10-05-0470Crossref PubMed Scopus (51) Google Scholar, 43Hung C.H. Thomas L. Ruby C.E. Atkins K.M. Morris N.P. Knight Z.A. Scholz I. Barklis E. Weinberg A.D. Shokat K.M. Thomas G. 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