A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation.

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery-it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of dimethylsulfoxide and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

NTB-A and 2B4 Natural Killer Cell Receptors Modulate the Capacity of a Cocktail of Non-Neutralizing Antibodies and a Small CD4-Mimetic to Eliminate HIV-1-Infected Cells by Antibody-Dependent Cellular Cytotoxicity.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

HIV-1 Nef is carried on the surface of extracellular vesicles.

Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle-producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV-related co-morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef-containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV-associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV-associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef-containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV-associated co-morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV-associated co-morbidities.

Recognition of HIV-1-infected fibrocytes lacking Nef-mediated HLA-B downregulation by HIV-1-specific T cells.

Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE: Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.

Extracellular vesicles produced by HIV-1 Nef-expressing cells induce myelin impairment and oligodendrocyte damage in the mouse central nervous system.

HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.

Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Beyond Impairment of Virion Infectivity: New Activities of the Anti-HIV Host Cell Factor SERINC5.

Members of the serine incorporator (SERINC) protein family exert broad antiviral activity, and many viruses encode SERINC antagonists to circumvent these restrictions. Significant new insight was recently gained into the mechanisms that mediate restriction and antagonism. In this review, we summarize our current understanding of the mode of action and relevance of SERINC proteins in HIV-1 infection. Particular focus will be placed on recent findings that provided important new mechanistic insights into the restriction of HIV-1 virion infectivity, including the discovery of SERINC's lipid scramblase activity and its antagonism by the HIV-1 pathogenesis factor Nef. We also discuss the identification and implications of several additional antiviral activities by which SERINC proteins enhance pro-inflammatory signaling and reduce viral gene expression in myeloid cells. SERINC proteins emerge as versatile and multifunctional regulators of cell-intrinsic immunity against HIV-1 infection.

Kinetic investigation reveals an HIV-1 Nef-dependent increase in AP-2 recruitment and productivity at endocytic sites.

The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.

Neuro-HIV-New insights into pathogenesis and emerging therapeutic targets.

HIV-associated neurocognitive disorders (HAND) is a term describing a complex set of cognitive impairments accompanying HIV infection. Successful antiretroviral therapy (ART) reduces the most severe forms of HAND, but milder forms affect over 50% of people living with HIV (PLWH). Pathogenesis of HAND in the ART era remains unknown. A variety of pathogenic factors, such as persistent HIV replication in the brain reservoir, HIV proteins released from infected brain cells, HIV-induced neuroinflammation, and some components of ART, have been implicated in driving HAND pathogenesis in ART-treated individuals. Here, we propose another factor-impairment of cholesterol homeostasis and lipid rafts by HIV-1 protein Nef-as a possible contributor to HAND pathogenesis. These effects of Nef on cholesterol may also underlie the effects of other pathogenic factors that constitute the multifactorial nature of HAND pathogenesis. The proposed Nef- and cholesterol-focused mechanism may provide a long-sought unified explanation of HAND pathogenesis that takes into account all contributing factors. Evidence for the impairment by Nef of cellular cholesterol balance, potential effects of this impairment on brain cells, and opportunities to therapeutically target this element of HAND pathogenesis are discussed.

Relative resistance of patient-derived envelope sequences to SERINC5-mediated restriction of HIV-1 infectivity.

IMPORTANCE: Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions.

Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

Association of Viral and Host Genetic Architecture with the Status of Neurocognitive Disorder in HIV-Infected Individuals.

The polymorphisms in host genes such as CCR5, CCR2, stromal derived factor (SDF), and MBL (mannose-binding lectin) as well as the viral nef gene have been shown to influence human immunodeficiency virus (HIV) infection, followed by the development of HIV-associated neurocognitive disorder (HAND). In this preliminary study with a limited number of samples, we have tried to associate the genetic polymorphism from the host and viral genetic factors with the neurocognitive status along with immuno-virological parameters. The total RNA was isolated from 10 unlinked plasma samples containing 5 samples from each group with and without HAND based on the International HIV Dementia Scale (IHDS) score <9.5 and >9.5, respectively. The CCR5, CCR2, SDF, MBL, and HIV nef genes were amplified and digested with restriction enzymes, except for the nef gene amplicon. Restrictions fragment length polymorphism (RFLP) was used to determine whether allelic variations were present in the digested host gene products, while sequencing was done for HIV nef amplicons without digestion. CCR5 delta 32 heterozygous variants were present in two samples from the HAND group. Three samples with HAND showed SDF-1 3' heterozygous allelic variant, while the MBL-2 gene presented with a homozygous mutant allele (D/D) in codon 52, heterozygous mutant allele (A/B) in codon 54, and codon 57 (A/C) for all samples except IHDS-2 irrespective of dementia status. Furthermore, amino acid alignment of Nef sequences confirmed the heterogeneity, while prediction of the human leukocyte antigen binding epitopes further explored its effect on functional motifs with variable binding efficiency such as epitopes GAFDLSFFL (aa 83) and LTFGWCFKL (aa 138) binding with HLA molecules at 60% and 80%, respectively. Thus, host genetics evidently influence predisposition to HIV infection and HAND. The genetic variability in the nef gene from both groups resulted in altering the functionality of specific domains and showing its impact on the progression of the disease, which needs to be explored.

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity.

BACKGROUND: Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. RESULTS: No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. CONCLUSIONS: These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins.

Our recent data established that HIV-1 Nef is pivotal in determining the fate of cellular proteins by modulating ubiquitination. However, it is unknown which proteins are ubiquitinated in the presence of Nef, a question critical for understanding the proliferation/restriction strategies of HIV-1 in infected cells. To identify cellular proteins ubiquitinated by Nef, we conducted a proteomic analysis of cellular proteins in the presence and absence of Nef. Proteomic analysis in HEK293T cells indicated that 93 proteins were upregulated and 232 were downregulated in their ubiquitination status by Nef. Computational analysis classified these proteins based on molecular function, biological process, subcellular localization, and biological pathway. Of those proteins, we found a majority of molecular functions to be involved in binding and catalytic activity. With respect to biological processes, a significant portion of the proteins identified were related to cellular and metabolic processes. Subcellular localization analysis showed the bulk of proteins to be localized to the cytosol and cytosolic compartments, which is consistent with the known function and location of Nef during HIV-1 infection. As for biological pathways, the wide range of affected proteins was denoted by the multiple modes to fulfill function, as distinguished from a strictly singular means, which was not detected. Among these ubiquitinated proteins, six were found to directly interact with Nef, wherein two were upregulated and four downregulated. We also identified 14 proteins involved in protein stability through directly participating in the Ubiquitin Proteasome System (UPS)-mediated proteasomal degradation pathway. Of those proteins, we found six upregulated and eight downregulated. Taken together, these analyses indicate that HIV-1 Nef is integral to regulating the stability of various cellular proteins via modulating ubiquitination. The molecular mechanisms directing Nef-triggered regulation of cellular protein ubiquitination are currently under investigation.

HIV-1 release requires Nef-induced caspase activation.

HIV infection remains incurable to date and there are no compounds targeted at the viral release. We show here HIV viral release is not spontaneous, rather requires caspases activation and shedding of its adhesion receptor, CD62L. Blocking the caspases activation caused virion tethering by CD62L and the release of deficient viruses. Not only productive experimental HIV infections require caspases activation for viral release, HIV release from both viremic and aviremic patient-derived CD4 T cells also require caspase activation, suggesting HIV release from cellular viral reservoirs depends on apoptotic shedding of the adhesion receptor. Further transcriptomic analysis of HIV infected CD4 T cells showed a direct contribution of HIV accessory gene Nef to apoptotic caspases activation. Current HIV cure focuses on the elimination of latent cellular HIV reservoirs that are resistant to infection-induced cell death. This has led to therapeutic strategies to stimulate T cell apoptosis in a "kick and kill" approach. Our current work has shifted the paradigm on HIV-induced apoptosis and suggests such approach would risk to induce HIV release and thus be counter-productive. Instead, our study supports targeting of viral reservoir release by inhibiting of caspases activation.

Viral competition assay to assess the role of HIV-1 proteins in immune evasion.

CD8+ T lymphocytes can recognize and eliminate cells infected by viruses. However, the human immunodeficiency virus (HIV-1) has developed mechanisms to evade CD8+ T-cell-mediated clearance. Here, we describe a protocol to assess the role of the HIV-1 protein Nef in immune evasion. The viral competition assay reveals the preferential killing of HIV-1-infected cells unable to express Nef. This methodology can be extended to study HIV-1 proteins involved in immune evasion and viral variants encoding cytotoxic T lymphocyte escape mutations. For complete details on the use and execution of this protocol, please refer to Duette et al. (2022).1.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers.

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.