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 downregulation 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 HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein-protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3-SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3-SH2 domains of the Src-family kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3-SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3-SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which Src-family kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.

SERINC5 is a multi-pass 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 downregulation from the producer cell membrane through an AP-2-dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 downregulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 downregulation, 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 (L112, Y115, F121) 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, while downregulation 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 downregulation of SERINC5 and subsequent enhancement of viral infectivity requires 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.

HIV‐1 Nef is a virulence factor that is essential for immune escape of HIV‐infected cells, viral pathogenesis and AIDS progression. Nef lacks intrinsic biochemical activity, functioning instead by interacting with host cell effector proteins to hijack multiple signaling and endocytic pathways. Many Nef functions depend on dimerization of the protein, including enhancement of viral infectivity, replication, and immune cell receptor downregulation. Work in our lab, through both X‐ray crystallography and virological assays, has shown that distinct hydrophobic residues (L112, Y115, F121) are key in Nef dimer formation, and disruption of these residues significantly impairs many Nef functions. Pharmacologic disruption of the Nef homodimer may therefore represent a novel approach to antiretroviral therapy. Here we investigated whether Nef dimerization is required for antagonism of the host cell restriction factor SERINC5 (S5). S5 is a potent suppressor of HIV‐1 infectivity that is antagonized by Nef via binding and downregulating S5 from the surface of infected cells, preventing its incorporation into nascent virions. For our studies, HIV‐1 virions were produced in 293T cells transfected with the HIV‐1 provirus and S5, Jurkat WT and S5‐knockout cells, or donor PBMCs (which naturally express high levels of S5). Viral supernatants were harvested from these “producer” cells and normalized for p24 AlphaLISA. Infectivity of producer‐cell viruses was assessed using TZM‐bl reporter cells, which couple the HIV‐1 LTR to expression of luciferase. Mutation of conserved Nef residues essential for homodimerization (L112A/D, Y115D, F121A) reduced the infectivity of HIV‐1 produced from all three producer cell lines in a S5‐dependent manner. S5 incorporation into virions was increased in the presence of Nef dimer interface mutants, suggesting that dimerization is important in its ability to exclude S5 from newly synthesized virions. Recruitment, downregulation, and endocytic trafficking of S5 by wild type and dimerization‐defective Nef mutants were also examined via confocal microscopy. Nef dimer interface mutants also failed to induce colocalization of S5 with Rab7+ late endosomes, suggesting that Nef dimerization is essential for S5 internalization and degradation via the lyso‐endosomal pathway. By contrast, Nef mutants retained their ability to interact with both S5 and subunits of the AP‐2 clathrin adaptor protein essential for S5 endocytosis, as shown via bimolecular fluorescence complementation. Taken together, these results demonstrate that downregulation of S5 and subsequent enhancement of viral infectivity requires Nef homodimerization, providing further support for pharmacological targeting of Nef quaternary structure in HIV‐1 cure strategies.

Nef is an HIV‐1 accessory factor essential for viral pathogenesis and immune escape of HIV‐infected cells. Recently, our group reported the discovery of small molecule Nef antagonists that reverse Nef‐mediated downregulation of MHC‐I and restore the anti‐HIV cytotoxic T cell response. These compounds also inhibit Nef's ability to enhance the inherent infectivity of HIV‐1 virions, although their mechanism of action in this regard is not known. Recent studies have identified the host cell factor SERINC5 (S5) as a suppressor of HIV‐1 infectivity that is countermanded by Nef. Here we investigated whether our Nef inhibitors suppress viral infectivity through a S5‐dependent mechanism. We first confirmed that expression of S5 within viral producer cells results in Nef‐reversible inhibition of HIV‐1 infectivity. For these experiments, we co‐transfected 293T cells with wild‐type and ΔNef HIV‐1 proviral DNA along with various amounts of a S5 expression plasmid. The infectivity of each resulting virus was then quantified using the luciferase‐based HIV‐1 reporter cell line, TZM‐bl. As expected, co‐expression of S5 resulted in suppression of HIV‐1 infectivity. This S5 effect was reduced by Nef in cells producing wild‐type HIV‐1, but not in cells producing the ΔNef virus. Co‐expression of Nef from a separate expression plasmid during the production of the ΔNef virus completely reversed S5 inhibition of viral infectivity, demonstrating Nef dependence in this system. To determine whether our inhibitors block Nef‐enhanced infectivity in a S5‐specific manner, we treated cells producing wild‐type or ΔNef virus in the presence or absence of S5 with either DMSO or the diphenylpyrazolo Nef inhibitor, B9 (3 μM). This compound inhibited the infectivity of wild‐type but not ΔNef HIV‐1 at the producer cell level, consistent with direct action on Nef. However, the effect of the compound was independent of S5 expression, suggesting that mechanisms in addition to S5 may contribute to inhibition of HIV‐1 infectivity by B9 through Nef.Support or Funding InformationT32 AI65380 (R.P.S.)R01 AI057083, R42 GM112516 (T.E.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.