HIV-1 Nef Research Articles

SIV Infection and the HIV Proteins Tat and Nef Induce Senescence in Adipose Tissue and Human Adipose Stem Cells, Resulting in Adipocyte Dysfunction.

Background: Aging is characterized by adipose tissue senescence, inflammation, and fibrosis, with trunk fat accumulation. Aging HIV-infected patients have a higher risk of trunk fat accumulation than uninfected individuals—suggesting that viral infection has a role in adipose tissue aging. We previously demonstrated that HIV/SIV infection and the Tat and Nef viral proteins were responsible for adipose tissue fibrosis and impaired adipogenesis. We hypothesized that SIV/HIV infection and viral proteins could induce adipose tissue senescence and thus lead to adipocyte dysfunctions. Methods: Features of tissue senescence were evaluated in subcutaneous and visceral adipose tissues of SIV-infected macaques and in human adipose stem cells (ASCs) exposed to Tat or Nef for up to 30 days. Results: p16 expression and p53 activation were higher in adipose tissue of SIV-infected macaques than in control macaques, indicating adipose tissue senescence. Tat and Nef induced higher senescence in ASCs, characterized by higher levels of senescence-associated beta-galactosidase activity, p16 expression, and p53 activation vs. control cells. Treatment with Tat and Nef also induced oxidative stress and mitochondrial dysfunction. Prevention of oxidative stress (using N-acetyl-cysteine) reduced senescence in ASCs. Adipocytes having differentiated from Nef-treated ASCs displayed alterations in adipogenesis with lower levels of triglyceride accumulation and adipocyte marker expression and secretion, and insulin resistance. Conclusion: HIV/SIV promotes adipose tissue senescence, which in turn may alter adipocyte function and contribute to insulin resistance.

HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane

The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2-inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.

A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen "arms race" is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5 Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef.

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

Loss of Nef-mediated CD3 down-regulation in the HIV-1 lineage increases viral infectivity and spread

Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef- and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-to-cell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread.

Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.

HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

ABSTRACT Macroautophagy/autophagy is an auto-digestive pro-survival pathway activated in response to stress to target cargo for lysosomal degradation. In recent years, autophagy has become prominent as an innate antiviral defense mechanism through multiple processes, such as targeting virions and viral components for elimination. These exciting findings have encouraged studies on the ability of autophagy to restrict HIV. However, the role of autophagy in HIV infection remains unclear. Whereas some reports indicate that autophagy is detrimental for HIV, others have claimed that HIV deliberately activates this pathway to increase its infectivity. Moreover, these contrasting findings seem to depend on the cell type investigated. Here, we show that autophagy poses a hurdle for HIV replication, significantly reducing virion production. However, HIV-1 uses its accessory protein Nef to counteract this restriction. Previous studies have indicated that Nef affects autophagy maturation by preventing the fusion between autophagosomes and lysosomes. Here, we uncover that Nef additionally blocks autophagy initiation by enhancing the association between BECN1 and its inhibitor BCL2, and this activity depends on the cellular E3 ligase PRKN. Remarkably, the ability of Nef to counteract the autophagy block is more frequently observed in pandemic HIV-1 and its simian precursor SIVcpz infecting chimpanzees than in HIV-2 and its precursor SIVsmm infecting sooty mangabeys. In summary, our findings demonstrate that HIV-1 is susceptible to autophagy restriction and define Nef as the primary autophagy antagonist of this antiviral process. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin, beta; ATG16L1: autophagy related 16 like 1; BCL2: bcl2 apoptosis regulator; BECN1: beclin 1; cDNA: complementary DNA; EGFP: enhanced green fluorescence protein; ER: endoplasmic reticulum; Gag/p55: group-specific antigen; GFP: green fluorescence protein; GST: glutathione S transferase; HA: hemagglutinin; HIV: human immunodeficiency virus; IP: immunoprecipitation; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Nef: negative factor; PRKN: parkin RBR E3 ubiquitin ligase; PtdIns3K: phosphatidylinositol 3 kinase; PtdIns3P: phosphatidylinositol 3 phosphate; PTM: post-translational modification; RT-qPCR: reverse transcription followed by quantitative PCR; RUBCN: rubicon autophagy regulator; SEM: standard error of the mean; SERINC3: serine incorporator 3; SERINC5: serine incorporator 5; SIV: simian immunodeficiency virus; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; UVRAG: UV radiation resistance associated gene; VSV: vesicular stomatitis virus; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Pregnancy Gestation Impacts on HIV-1-Specific Granzyme B Response and Central Memory CD4 T Cells.

Pregnancy induces alterations in peripheral T-cell populations with both changes in subset frequencies and anti-viral responses found to alter with gestation. In HIV-1 positive women anti-HIV-1 responses are associated with transmission risk, however detailed investigation into both HIV-1-specific memory responses associated with HIV-1 control and T-cell subset changes during pregnancy have not been undertaken. In this study we aimed to define pregnancy and gestation related changes to HIV-1-specific responses and T-cell phenotype in ART treated HIV-1 positive pregnant women. Eleven non-pregnant and 24 pregnant HIV-1 positive women were recruited, peripheral blood samples taken, fresh cells isolated, and compared using ELISpot assays and flow cytometry analysis. Clinical data were collected as part of standard care, and non-parametric statistics used. Alterations in induced IFNγ, IL-2, IL-10, and granzyme B secretion by peripheral blood mononuclear cells in response to HIV-1 Gag and Nef peptide pools and changes in T-cell subsets between pregnant and non-pregnant women were assessed, with data correlated with participant clinical parameters and longitudinal analysis performed. Cross-sectional comparison identified decreased IL-10 Nef response in HIV-1 positive pregnant women compared to non-pregnant, while correlations exhibited reversed Gag and Nef cytokine and protease response associations between groups. Longitudinal analysis of pregnant participants demonstrated transient increases in Gag granzyme B response and in the central memory CD4 T-cell subset frequency during their second trimester, with a decrease in CD4 effector memory T cells from their second to third trimester. Gag and Nef HIV-1-specific responses diverge with pregnancy time-point, coinciding with relevant T-cell phenotype, and gestation associated immunological adaptations. Decreased IL-10 Nef and both increased granzyme B Gag response and central memory CD4 T cells implies that amplified antigen production is occurring, which suggests a period of compromised HIV-1 control in pregnancy.

Defective HIV-1 proviruses produce viral proteins

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice

Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.

Proteasomal degradation of human SERINC4: A potent host anti-HIV-1 factor that is antagonized by nef

The serine incorporator (SERINC) protein family has five paralogous members with 9–11 transmembrane domains. SERINC5 is a potent host restriction factor and antagonized by HIV-1 Nef and two other retroviral accessory proteins via the lysosomal degradation pathway. Here, we investigated human SERINC4 expression and antiviral mechanisms. Unlike its four paralogs, human SERINC4 is subjected to proteasome-mediated turnover, resulting in ~250-fold lower expression than SERINC5. However, when expression was normalized, human SERINC4 restricted HIV-1 replication as effectively as SERINC5, and SERINC4 was also antagonized by Nef via the lysosomal pathway. Although SERINC4 proteins are conserved within primates or rodents, their N-terminal regions are highly variable across species. Interestingly, unlike human SERINC4, murine SERINC4 was stably expressed but had a very poor antiviral activity. We created stable SERINC4 chimeras by replacing the N-terminal region and found that the 1–34 and 35–92 amino acids determine SERINC4 antiviral activity or protein expression, respectively. Using these chimeras, we demonstrate that SERINC4 is incorporated into HIV-1 virions and restricts Tier 1 HIV-1 more effectively than Tier 3 HIV-1. Importantly, SERINC4 increases HIV-1 sensitivity to broadly neutralizing antibodies. Thus, human SERINC4 strongly restricts HIV-1 replication when it is overexpressed, which reflects a potential antiviral activity of this gene product under physiological conditions.

MVA-B COMO CANDIDATO VACUNAL FRENTE AL VIH/SIDA: DE LA INVESTIGACIÓN BÁSICA A LOS ENSAYOS CLÍNICOS PROFILÁCTICO Y TERAPÉUTICO

The highly attenuated poxvirus strain modified vaccinia virus Ankara (MVA) has reached maturity as antigen delivery system and as a vaccine candidate against a broad spectrum of infectious diseases. This has been largely recognized from research on virus–host cell interactions, gene expression profiling, virus distribution and immunological studies in preclinical and clinical trials. This review includes our main contributions from the basic knowledge of the biology of the MVA vector, both in vitro and in vivo, in comparison with the attenuated NYVAC strain, to its evaluation as a vaccine candidate against HIV/AIDS in clinical trials. We will detail the generation and characterization of the recombinant poxvirus vector MVA expressing the HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B) and review the preclinical data that supported the evaluation of MVA-B as the first in human HIV-1 prophylactic and therapeutic vaccine in Spain. In addition, we will assess the results of clinical trials and discuss the research projects we are currently working on considering the latest scientific advances in the HIV vaccine field.

Dendritic Cells Focus CTL Responses Toward Highly Conserved and Topologically Important HIV Epitopes

Background: During early HIV Infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV epitopes, using HIV-infected individuals from the Thailand RV254/SEARH010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). Methods: Autologous MDC1 were used as peptide antigen presenting cells to induce ex vivo CTL responses against HIV Gag, Pol, Env and Nef as determined by flow cytometry and ELISPOT assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. Findings: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-specific T cells specific to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens, but higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically important Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. Interpretation: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward potentially fitness-constrained regions of HIV as a means to prevent immune escape and control HIV-1 infection. Funding Statement: This study was supported by the NIH, National Institute of Allergy and Infectious Diseases grants R21-AI131763, R21-AI138716, UM1-AI126603, and U01-AI35041. Declaration of Interests: The authors have nothing to disclose. Ethics Approval Statement: The Chulalongkorn University Institutional Review Board and the Walter Reed Army Institute of Research, USA, approved this study.

Analysis of HIV-1 (Human immunodeficiency virus-1, Lentivirus, Orthoretrovirinae, Retroviridae) Nef protein polymorphism of variants circulating in the former USSR countries.

The human immunodeficiency virus (HIV) Nef protein is one of the key factors determining the infectivity and replicative properties of HIV. With the ability to interact with numerous proteins of the host cell, this protein provides the maximum level of virus production and protects it from the immune system. The main activities of Nef are associated with a decrease in the expression of the CD4 receptor and major histocompatibility complex class I molecules (MHC-I), as well as the rearrangement of the cytoskeleton. These properties of the protein are determined by the structure of several motifs in the structure of the nef gene encoding it, which is quite variable. The main goal of the work was to analyze the characteristics of Nef protein of HIV-1 variant A6, which dominates in the countries of the former USSR. The objective of the work was a comparative analysis of natural polymorphisms in the nef gene of HIV-1 sub-subtypes A6 and A1 and subtype B. The sequences of the HIV-1 genome obtained during the previous work of the laboratory were used, as well as the reference sequence from GenBank. In this work, Sanger sequencing and new generation sequencing methods, as well as bioinformation analysis methods were used. The existence of noticeable differences in the prevalence of Nef natural polymorphisms (A32P, E38D, I43V, A54D, Q104K, H116N, Y120F, Y143F, V168M, H192T, V194R, R35Q, D108E, Y135F, E155K, E182M, R184K and F191L), some of which are characteristic mutations for variant A6, was shown. Characteristic substitutions were found in the Nef structure, potentially capable of weakening the replicative properties of HIV-1 variant A6.

Multifunctional Roles of the N-Terminal Region of HIV-1SF2Nef Are Mediated by Three Independent Protein Interaction Sites.

HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1SF2Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread.IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1SF2Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.

Structure of HIV-2 Nef Reveals Features Distinct from HIV-1 Involved in Immune Regulation

SummaryThe human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation.

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with KD values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC50 values in the 1-10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure-activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

Infusion of HIV-1 Nef-expressing astrocytes into the rat hippocampus induces enteropathy and interstitial pneumonitis and increases blood-brain-barrier permeability.

Even though HIV-1 replication can be suppressed by combination antiretroviral therapy (cART) inflammatory processes still occur, contributing to comorbidities. Comorbidities are attributed to variety of factors, including HIV-1 mediated inflammation. Several HIV-1 proteins mediate central nervous system (CNS) inflammation, including Nef. Nef is an early HIV-1 protein, toxic to neurons and glia and is sufficient to cause learning impairment similar to some deficits observed in HIV-1 associated neurocognitive disorders. To determine whether hippocampal Nef expression by astrocytes contributes to comorbidities, specifically peripheral inflammation, we infused Sprague Dawley rats with GFP- (control) or Nef-transfected astrocytes into the right hippocampus. Brain, lung, and ileum were collected postmortem for the measurement of inflammatory markers. Increased blood-brain-barrier permeability and serum IL-1β levels were detected in the Nef-treated rats. The lungs of Nef-treated rats demonstrated leukocyte infiltration, macrophage upregulation, and enhanced vascular permeability. Ileal tissue showed reactive follicular lymphoid hyperplasia, increased permeability and macrophage infiltration. The intracerebroventricular application of IL-1 receptor antagonist reduced infiltration of immune cells into ileum and lung, indicating the important role of IL-1β in mediating the spread of inflammation from the brain to other tissues. This suggests that localized expression of a single viral protein, HIV-1 Nef, can contribute to a broader inflammatory response by upregulation of IL-1β. Further, these results suggest that Nef contributes to the chronic inflammation seen in HIV patients, even in those whose viremia is controlled by cART.

HIV-1 Impairment via UBE3A and HIV-1 Nef Interactions Utilizing the Ubiquitin Proteasome System.

Molecular basis of HIV-1 life cycle regulation has thus far focused on viral gene stage-specificity, despite the quintessence of post-function protein elimination processes in the virus life cycle and consequent pathogenesis. Our studies demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), suggesting that interaction between Nef and UBE3A is integral to regulation of viral and cellular protein decay and thereby the competing HIV-1 and host cell survivals. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thus significantly inhibiting HIV-1 replication in Jurkat T cells only in the presence of Nef, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. Mechanistic study showed that Nef and UBE3A were specific and antagonistic to each other in regulating proteasome activity and ubiquitination of cellular proteins in general, wherein specific domains of Nef overlapping with the long terminal repeat (LTR) were essential for the observed actions. Further, Nef itself reduced the level of intracellular Gag by degrading a cardinal transcription regulator, Tat, demonstrating a broad role for Nef in the regulation of the HIV-1 life cycle. Taken together, these data demonstrated that the Nef and UBE3A complex plays a crucial role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.