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1.
J Biol Chem ; 300(8): 107528, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38960038

ABSTRACT

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.


Subject(s)
CD4 Antigens , Down-Regulation , Fluorescence Polarization , HIV-1 , High-Throughput Screening Assays , nef Gene Products, Human Immunodeficiency Virus , nef Gene Products, Human Immunodeficiency Virus/metabolism , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/genetics , CD4 Antigens/metabolism , CD4 Antigens/chemistry , Humans , Fluorescence Polarization/methods , HIV-1/metabolism , HIV-1/drug effects , High-Throughput Screening Assays/methods , Down-Regulation/drug effects , Adaptor Protein Complex 2/metabolism , Anti-HIV Agents/pharmacology , Anti-HIV Agents/chemistry , Protein Binding
2.
J Biol Chem ; 300(8): 107529, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38960039

ABSTRACT

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.


Subject(s)
Down-Regulation , Fluorescence Polarization , HIV-1 , High-Throughput Screening Assays , Histocompatibility Antigens Class I , nef Gene Products, Human Immunodeficiency Virus , nef Gene Products, Human Immunodeficiency Virus/metabolism , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/chemistry , HIV-1/drug effects , HIV-1/metabolism , Humans , Fluorescence Polarization/methods , High-Throughput Screening Assays/methods , Down-Regulation/drug effects , Histocompatibility Antigens Class I/metabolism , Anti-HIV Agents/pharmacology , Anti-HIV Agents/chemistry
3.
Biotechnol Lett ; 46(3): 315-354, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38403788

ABSTRACT

The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.


Subject(s)
AIDS Vaccines , HIV Infections , Interferon-gamma , Protein Subunit Vaccines , nef Gene Products, Human Immunodeficiency Virus , Animals , Humans , Mice , Adjuvants, Immunologic/pharmacology , AIDS Vaccines/immunology , AIDS Vaccines/chemistry , CD40 Ligand/immunology , CD40 Ligand/chemistry , Computer Simulation , Epitopes/immunology , Epitopes/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/chemistry , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1 , Interferon-gamma/metabolism , Interferon-gamma/immunology , Molecular Docking Simulation , nef Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/chemistry , Protein Subunit Vaccines/chemistry , Protein Subunit Vaccines/immunology
4.
Viruses ; 15(3)2023 02 28.
Article in English | MEDLINE | ID: mdl-36992361

ABSTRACT

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis.


Subject(s)
CD4 Antigens , CD4-Positive T-Lymphocytes , HIV-1 , Membrane Proteins , nef Gene Products, Human Immunodeficiency Virus , Humans , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Membrane Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , Amino Acid Substitution , HEK293 Cells , Jurkat Cells , HIV-1/pathogenicity , Amino Acid Sequence , Endocytosis , Clathrin , HIV Infections , CD4 Antigens/metabolism , Down-Regulation
5.
J Mol Biol ; 435(8): 168009, 2023 04 15.
Article in English | MEDLINE | ID: mdl-36773691

ABSTRACT

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.


Subject(s)
Cell Membrane , HIV-1 , Lipid Bilayers , nef Gene Products, Human Immunodeficiency Virus , Cell Membrane/chemistry , Cell Membrane/metabolism , HIV-1/chemistry , HIV-1/metabolism , Lipid Bilayers/metabolism , src Homology Domains , Protein Multimerization , Crystallography, X-Ray , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolism , Molecular Dynamics Simulation
6.
Retrovirology ; 18(1): 33, 2021 10 28.
Article in English | MEDLINE | ID: mdl-34711257

ABSTRACT

BACKGROUND: Autophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity. RESULTS: We found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag's central role in virion assembly and budding. We also identified residues T48 and A49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction. CONCLUSIONS: This study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.


Subject(s)
Autophagosomes/metabolism , HIV Infections/physiopathology , HIV-1/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acid Motifs , Autophagosomes/genetics , Autophagy , HIV Infections/metabolism , HIV Infections/virology , HIV-1/genetics , Humans , Lysosomes/metabolism , Proteolysis , Ubiquitin/metabolism , Ubiquitination , gag Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics
7.
Biochem J ; 478(8): 1525-1545, 2021 04 30.
Article in English | MEDLINE | ID: mdl-33787846

ABSTRACT

The Nef protein of human and simian immunodeficiency viruses boosts viral pathogenicity through its interactions with host cell proteins. By combining the polyvalency of its large unstructured regions with the binding selectivity and strength of its folded core domain, Nef can associate with many different host cell proteins, thereby disrupting their functions. For example, the combination of a linear proline-rich motif and hydrophobic core domain surface allows Nef to bind tightly and specifically to SH3 domains of Src family kinases. We investigated whether the interplay between Nef's flexible regions and its core domain could allosterically influence ligand selection. We found that the flexible regions can associate with the core domain in different ways, producing distinct conformational states that alter the way in which Nef selects for SH3 domains and exposes some of its binding motifs. The ensuing crosstalk between ligands might promote functionally coherent Nef-bound protein ensembles by synergizing certain subsets of ligands while excluding others. We also combined proteomic and bioinformatics analyses to identify human proteins that select SH3 domains in the same way as Nef. We found that only 3% of clones from a whole-human fetal library displayed Nef-like SH3 selectivity. However, in most cases, this selectivity appears to be achieved by a canonical linear interaction rather than by a Nef-like 'tertiary' interaction. Our analysis supports the contention that Nef's mode of hijacking SH3 domains is a virus-specific adaptation with no or very few cellular counterparts. Thus, the Nef tertiary binding surface is a promising virus-specific drug target.


Subject(s)
HIV-1/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Nuclear Proteins/chemistry , Proto-Oncogene Proteins c-fyn/chemistry , nef Gene Products, Human Immunodeficiency Virus/chemistry , Allosteric Site , Amino Acid Sequence , Cloning, Molecular , Computational Biology/methods , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Fetus , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HIV-1/genetics , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Host-Pathogen Interactions/genetics , Humans , Ligands , Molecular Dynamics Simulation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Thermodynamics , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolism
8.
Biotechnol Lett ; 43(3): 547-559, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33386500

ABSTRACT

OBJECTIVES: A potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323, and P248-151 epitopes was designed using bioinformatics tools. The pcDNA3.1-nef-vpr-gp160-p24 and pcDNA3.1-nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated inE. coli. These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice. RESULTS: The recombinant DNA constructs were confirmed as the ~ 867 bp and ~ 648 bp bands related tonef-vpr-gp160-p24 andnef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the ~ 32 kDa and ~ 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-γ and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses. CONCLUSIONS: The heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.


Subject(s)
AIDS Vaccines , Cell-Penetrating Peptides , Drug Delivery Systems/methods , Epitopes , nef Gene Products, Human Immunodeficiency Virus , AIDS Vaccines/chemistry , AIDS Vaccines/genetics , AIDS Vaccines/immunology , Adjuvants, Immunologic , Animals , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/immunology , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Female , HIV Antibodies/immunology , HIV-1/genetics , HIV-1/immunology , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/immunology
9.
Biotechnol Appl Biochem ; 68(3): 513-530, 2021 Jun.
Article in English | MEDLINE | ID: mdl-32447788

ABSTRACT

Intrinsically disordered proteins (IDPs) are highly flexible molecules that undergo disorder to order transition through their interaction with other molecules. IDPs play a vital role in several biological processes ranging from molecular recognition to several human diseases through the protein-protein interaction. The dynamic flexibility of IDPs and their implications in several human diseases enable these molecules to serve as novel therapeutic targets. However, the challenging task is to develop novel drugs against IDPs because of their lack of stable structures and the nature of high conformational flexibility. In this study, we have calculated the dynamic binding effect of the supramolecular tweezer CLR01 against the intrinsically disordered HIV-1 Nef by employing molecular docking and dynamics simulation approaches. From docking results, we predicted the strong binding affinity of the tweezer with the target residues of Nef. The docking results were further validated from the molecular dynamics simulation studies confirming the conformational stability of Nef upon tweezer binding. These findings provide useful insights into the development of potent inhibitors for targeting Nef protein functions.


Subject(s)
Bridged-Ring Compounds/chemistry , Organophosphates/chemistry , nef Gene Products, Human Immunodeficiency Virus/chemistry , Binding Sites/drug effects , Bridged-Ring Compounds/pharmacology , Humans , Models, Molecular , Molecular Structure , Organophosphates/pharmacology , Principal Component Analysis , Thermodynamics , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors
10.
J Biol Chem ; 295(46): 15540-15552, 2020 11 13.
Article in English | MEDLINE | ID: mdl-32873704

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.


Subject(s)
HIV-1/physiology , Membrane Proteins/metabolism , Transcription Factor AP-2/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Dimerization , Down-Regulation , Endocytosis , Endosomes/metabolism , HEK293 Cells , HIV Infections/metabolism , HIV Infections/pathology , HIV-1/metabolism , Humans , Jurkat Cells , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Transcription Factor AP-2/chemistry , Transcription Factor AP-2/genetics , Virus Internalization , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/genetics
11.
J Biol Chem ; 295(44): 15158-15171, 2020 10 30.
Article in English | MEDLINE | ID: mdl-32862141

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.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/enzymology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , CD4 Antigens/metabolism , Crystallography, X-Ray , Down-Regulation , HIV-1/pathogenicity , Immune Evasion , Major Histocompatibility Complex , Membrane Proteins/metabolism , Protein Kinases/drug effects , Protein Transport , Structure-Activity Relationship , nef Gene Products, Human Immunodeficiency Virus/chemistry
12.
Nat Struct Mol Biol ; 27(9): 822-828, 2020 09.
Article in English | MEDLINE | ID: mdl-32719457

ABSTRACT

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-Å crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef's activities and sensitize the virus to immune clearance.


Subject(s)
CD4 Antigens/metabolism , HIV Infections/metabolism , HIV-1/physiology , nef Gene Products, Human Immunodeficiency Virus/metabolism , Adaptor Protein Complex 2/chemistry , Adaptor Protein Complex 2/metabolism , CD4 Antigens/chemistry , Crystallography, X-Ray , HeLa Cells , Host-Pathogen Interactions , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , nef Gene Products, Human Immunodeficiency Virus/chemistry
13.
Protein Pept Lett ; 27(11): 1151-1158, 2020.
Article in English | MEDLINE | ID: mdl-32364062

ABSTRACT

BACKGROUND: There is no effective and safe preventive/therapeutics vaccine against HIV-1 worldwide. Different viral proteins such as Nef, and two regions of Env including; variable loop of gp120 (V3) and membrane proximal external region of gp41 (MPER) are particularly important for vaccine development in different strategies and they are also the primary targets of cellular and humoral immune responses. On the other side, LDP12 is a new cell-penetrating peptide (CPP) which is capable of therapeutic application and cargoes delivery across the cellular membrane. OBJECTIVE: In current study, we designed and produced Nef-MPER-V3 fusion protein harboring LDP12 that has the capability of being used in future vaccine studies. METHODS: The CPP-protein was expressed in E. coli Rosseta (DE3) strain and purified through Ni-NTA column. Characterization of cellular delivery and toxicity of the recombinant protein were evaluated by western blotting and MTT assay. RESULTS: Our results showed that the CPP-protein was successfully expressed and purified with high yield of 5 mg/L. Furthermore, non-cytotoxic effect was observed and specific band (~ 37 KDa) in western blotting indicated the capability of LDP12 to improve the rate of penetration into HEK-293T cells in comparison with a control sample. CONCLUSION: Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 Nef-MPER-V3 protein into eukaryotic cell lines without any toxicity and represented a valuable potential vaccine candidate and this guarantees the further evaluation towards the assessment of its immunogenicity in mice, which is currently under process.


Subject(s)
AIDS Vaccines , Cell-Penetrating Peptides , Drug Carriers , HIV Envelope Protein gp41 , HIV-1 , Oligopeptides , Recombinant Fusion Proteins , nef Gene Products, Human Immunodeficiency Virus , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , AIDS Vaccines/pharmacokinetics , AIDS Vaccines/pharmacology , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/immunology , Cell-Penetrating Peptides/pharmacokinetics , Cell-Penetrating Peptides/pharmacology , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Carriers/pharmacology , HEK293 Cells , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/immunology , HIV Envelope Protein gp41/pharmacokinetics , HIV Envelope Protein gp41/pharmacology , HIV-1/chemistry , HIV-1/immunology , Humans , Oligopeptides/chemistry , Oligopeptides/immunology , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/pharmacology , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/pharmacokinetics , nef Gene Products, Human Immunodeficiency Virus/pharmacology
14.
J Biol Chem ; 295(20): 6983-6991, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32269076

ABSTRACT

Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Lysophospholipids/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Histocompatibility Antigens Class I/immunology , Lipoylation/immunology , Lysophospholipids/immunology , Macaca mulatta , Peptides/chemistry , Peptides/immunology , Protein Structure, Quaternary , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/immunology
15.
Viruses ; 12(4)2020 04 18.
Article in English | MEDLINE | ID: mdl-32325729

ABSTRACT

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.


Subject(s)
Antigens, CD/metabolism , HIV Infections/metabolism , HIV Infections/virology , HIV-1/physiology , Macrophages/metabolism , Macrophages/virology , nef Gene Products, Human Immunodeficiency Virus/metabolism , Antigens, CD/genetics , Cell Line , Fluorescent Antibody Technique , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression , HIV Infections/immunology , Host-Pathogen Interactions , Humans , Macrophages/immunology , Protein Binding , Protein Interaction Domains and Motifs , nef Gene Products, Human Immunodeficiency Virus/chemistry
16.
J Biol Chem ; 295(15): 5163-5174, 2020 04 10.
Article in English | MEDLINE | ID: mdl-32144207

ABSTRACT

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.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Cell Membrane/metabolism , HIV Infections/immunology , HIV-1/immunology , Protein-Tyrosine Kinases/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , Agammaglobulinaemia Tyrosine Kinase/genetics , Antiviral Agents/pharmacology , HIV Infections/metabolism , HIV Infections/virology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Protein Multimerization , Protein-Tyrosine Kinases/genetics , Signal Transduction , Small Molecule Libraries/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolism
17.
Nat Methods ; 17(3): 279-282, 2020 03.
Article in English | MEDLINE | ID: mdl-32066961

ABSTRACT

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.


Subject(s)
Green Fluorescent Proteins/chemistry , Immunoglobulin Fragments/chemistry , Nanoparticles/chemistry , Single-Domain Antibodies/chemistry , Crystallography, X-Ray , DNA/chemistry , Databases, Protein , Escherichia coli , Fluorescence Resonance Energy Transfer , Gene Products, gag/chemistry , HEK293 Cells , HIV-1/chemistry , HeLa Cells , Humans , Kinetics , Ligands , Microscopy, Fluorescence , Mitosis , Protein Domains , nef Gene Products, Human Immunodeficiency Virus/chemistry
18.
J Virol ; 94(1)2019 12 12.
Article in English | MEDLINE | ID: mdl-31597760

ABSTRACT

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.


Subject(s)
HIV-1/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Protein Domains , Virion/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , COS Cells , Chlorocebus aethiops , Gene Expression , HEK293 Cells , HIV-1/metabolism , Humans , Immune Evasion , Jurkat Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mutation , Primary Cell Culture , Protein Binding , Sequence Alignment , Sequence Homology, Amino Acid , Virion/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/metabolism
19.
J Virol ; 93(24)2019 12 15.
Article in English | MEDLINE | ID: mdl-31578291

ABSTRACT

The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.


Subject(s)
HIV Infections/immunology , HIV Infections/metabolism , HIV-1/metabolism , Protein Transport/physiology , nef Gene Products, Human Immunodeficiency Virus/metabolism , Clathrin-Coated Vesicles , HIV Infections/virology , HIV-1/immunology , Host-Pathogen Interactions , Humans , Membrane Proteins/metabolism , Models, Molecular , Protein Conformation , Simian Immunodeficiency Virus/immunology , Viral Regulatory and Accessory Proteins/chemistry , nef Gene Products, Human Immunodeficiency Virus/chemistry
20.
Life Sci ; 224: 263-273, 2019 May 01.
Article in English | MEDLINE | ID: mdl-30902545

ABSTRACT

Human immunodeficiency type 1 virus accessory protein Nef is a key modulator of AIDS pathogenesis. With no enzymatic activity, Nef regulated functions in host cells largely depends on its ability to form multi-protein complex with the cellular proteins. Here, we identified Calcium (Ca2+)/Calmodulin dependent protein kinase II subunit delta (CAMKIIδ) as novel Nef interacting host protein. Further, we confirmed that Nef mediated [Ca2+]I promote formation of Nef-CAMKIIδ - apoptosis signal-regulating kinase (ASK-1) heterotrimeric complex. The assembly of Nef with CAMKIIδ - ASK-1 inhibits the downstream p38MAPK phosphorylation resulting in abrogation of apoptosis. Further, using competitive peptide inhibitors against Nef binding domains to CAMKIIδ, identified in the present study and ASK-1, individually blocked physical interaction of Nef with CAMKIIδ-ASK-1 complex and restored p38MAPK phosphorylation and apoptosis. Altogether, our study indicates that HIV-Nef modulates cytosolic [Ca2+]I and blocks CAMKIIδ - ASK-1 kinase activity to inhibit apoptosis of infected cells.


Subject(s)
Apoptosis , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , HIV Infections/metabolism , MAP Kinase Kinase Kinase 5/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/chemistry , HEK293 Cells , HIV Infections/virology , HIV-1/physiology , Humans , Jurkat Cells , MAP Kinase Kinase Kinase 5/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Signal Transduction , nef Gene Products, Human Immunodeficiency Virus/chemistry , p38 Mitogen-Activated Protein Kinases/metabolism
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