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A primary function of HIV-1 Nef is the enhancement of viral infectivity and replication. Whether counteraction of the antiretroviral proteins SERINC3 and SERINC5 is the cause of this positive influence on viral growth-rate and infectivity remains unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays nef-related infectivity and growth-rate phenotypes. Viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc5 alone or of both serinc5 and serinc3 together failed to restore the infectivity of HIV1ΔNef virions produced from infected CEM cells. Our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 does not fully explain the virology of HIV-1 lacking Nef.

Cross-recognized public TCRs against HIV epitopes have been proposed to be important for the control of AIDS disease progression and HIV variants. The overlapping Nef138-8 and Nef138-10 peptides from the HIV Nef protein are HLA-A24–restricted immunodominant T cell epitopes, and an HIV mutant strain with a Y139F substitution in Nef protein can result in immune escape and is widespread in Japan. Here, we identified a pair of public TCRs specific to the HLA-A24–restricted Nef-138-8 epitope using PBMCs from White and Japanese patients, respectively, namely TD08 and H25-11. The gene use of the variable domain for TD08 and H25-11 is TRAV8-3, TRAJ10 for the α-chain and TRBV7-9, TRBD1*01, TRBJ2-5 for the β-chain. Both TCRs can recognize wild-type and Y2F-mutated Nef138-8 epitopes. We further determined three complex structures, including TD08/HLA-A24-Nef138-8, H25-11/HLA-A24-Nef138-8, and TD08/HLA-A24-Nef138-8 (2F). Then, we revealed the molecular basis of the public TCR binding to the peptide HLA, which mostly relies on the interaction between the TCR and HLA and can tolerate the mutation in the Nef138-8 peptide. These findings promote the molecular understanding of T cell immunity against HIV epitopes and provide an important basis for the engineering of TCRs to develop T cell–based immunotherapy against HIV infection.

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.

HIV-1 Nef regulates several cellular functions in an infected cell which results in viral persistence and AIDS pathogenesis. The currently understood molecular mechanism(s) underlying Nef-dependent cellular function(s) are unable to explain how events are coordinately regulated in the host cell. Intracellular membranous trafficking maintains cellular homeostasis and is regulated by Rab GTPases - a member of the Ras superfamily.In the current study, we tried to decipher the role of Nef on the Rab GTPases-dependent complex and vesicular trafficking. Expression profiling of Rabs in Nef-expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell-specific manner. Further analysis of Rabs in HIV-1This study demonstrates that Nef differentially regulates the expression of Rab proteins in HIV-1 infected cells to hijack the host intracellular trafficking, which augments viral replication and HIV-1 pathogenesis.Our study emphasized the indispensable role of HIV-1 protein Nef on various aspects of the intracellular trafficking regulated by Rabs GTPases, which explained how HIV-1 Nef may hijack membrane trafficking pathways in infected cells.

HIV-1 escapes by acquiring mutations that differentially influence the course of infection. Unlike HIV-1 structural and enzymatic proteins, it remains elusive what extent the host immune-mediated selection pressure influences the variability of the accessory (Vif, Vpu, Vpr, and Nef) and regulatory (Tat and Rev) proteins. To address this, we analyzed the viral sequences encoding accessory and regulatory proteins from 446 human leukocyte antigen (HLA)-typed, chronically HIV-1 subtype B-infected, and treatment-naive individuals in Japan. We observed that Vpu and Vpr were the most and least polymorphic proteins with the average Shannon entropy scores of 0.63 and 0.38, respectively. Phylogenetically corrected methods identified a total of 161 HLA-associated polymorphisms; whereby Nef and Vpu had the highest (26.6%) and lowest (1.2%) proportion of amino acid sites associated with HLA-class I alleles, respectively. These results add further insight on the role of HLA-mediated selection pressure on HIV-1 sequence polymorphisms of HIV-1 accessory and regulatory proteins.

HIV-associated neurocognitive disorders (HAND) is a term used to describe a variety of neurological impairments observed in HIV-infected individuals. The pathogenic mechanisms of HAND and of its connection to HIV infection remain unknown, but one of the considered hypotheses suggests that HIV infection accelerates the development of Alzheimer’s disease. Previous studies suggested that HIV-1 Nef may contribute to HAND by inhibiting cholesterol efflux, increasing the abundance of lipid rafts, and affecting their functionality. Our comparative analysis of postmortem brain samples demonstrated a trend toward the decreased abundance of cholesterol transporter ABCA1 in samples from HIV-infected ART-treated individuals relative to samples from uninfected controls, and a reverse correlation between ABCA1 and flotillin 1, a marker for lipid rafts, in all analyzed samples. The brain samples from HIV-infected individuals, both with and without HAND, were characterized by the increased abundance of p-Tau217 peptide, which correlated with the abundance of flotillin 1. HIV-1 Nef was analyzed in samples from HAND-affected individuals by Western blot with 4 different antibodies and by LC–MS/MS, producing a Nef-positivity score. A significant correlation was found between this score and the abundance of flotillin 1, the abundance of p-Tau217, and the severity of HAND. These results highlight the contribution of Nef and Nef-dependent impairment of cholesterol efflux to HAND pathogenesis and support a connection between the pathogenesis of HAND and Alzheimer’s disease.

Long interspersed element type 1 (LINE-1) is the only known type of retroelement that can replicate autonomously, and its retrotransposition activity can trigger interferon (IFN) production. IFN production suppresses the infectivity of exogenous viruses, such as human immunodeficiency virus (HIV). As a counteraction, HIV has been reported to use multiple proteins and mechanisms to suppress LINE-1 replication. However, the mechanisms of HIV-mediated LINE-1 regulation are not fully understood. In this study, we discovered that Nef protein, which is expressed by HIV and is important for HIV pathogenesis, inhibits LINE-1 retrotransposition. Two distinct mechanisms have been uncovered for Nef-induced LINE-1 suppression. Without direct interaction with LINE-1 DNA, Nef potently inhibits the promoter activity of the LINE-1 5'-untranslated region (5'-UTR) and reduces the expression levels of LINE-1 RNA and proteins. Alternatively, although Nef does not bind to the LINE-1 open reading frame 1 protein (ORF1p) or LINE-1 RNA, it significantly compromises the ORF1p-LINE-1 RNA interaction, which is essential for LINE-1 retrotransposition. Both mechanisms can be suppressed by the G2A mutation, which abolishes myristoylation of Nef, suggesting that membrane attachment is essential for Nef to suppress LINE-1. Consequently, through LINE-1 inhibition, Nef downregulates IFN production in host cells. Therefore, our data revealed that Nef is a potent LINE-1 suppressor and an effective innate immune regulator, which not only provides new information on the intricate interaction between HIV, LINE-1, and IFN signaling systems but also strengthens the importance of Nef in HIV infection and highlights the potential of designing novel Nef-targeting anti-HIV drugs.

HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.

Clinically used opioids, such as morphine, activate the mu opioid receptor (MOR) encoded by Opioid Receptor Mu 1 (OPRM1) gene. Examination of the opioid receptor genes showed that the human OPRM1 pre-mRNA undergoes extensive alternative splicing events and capable of expressing 21 isoforms. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR-1) has been extensively studied. Compounding this issue is the increasing significance of intravenous drug abuse in HIV neuropathogenesis. Here, we investigated the molecular impact of morphine and HIV-1 on regulation of OPRM1 pre-mRNA splicing in in vitro and in vivo models. Our results suggested that morphine treatment specifically induces the alternative splicing of MOR-1X isoform among the other isoforms analyzed in neuronal cells. Interestingly, alternative splicing and expression of MOR-1X isoform was also induced in postmortem brain tissues obtained from people with HIV (PWH). Additionally, treatment of control rats with morphine induced alternative splicing of MOR-1X in the brain regions involved in the reward pathways. More interestingly, HIV-1 transgenic (HIV-1Tg) rats, showed an additive induction of MOR-1X isoform with the exposure to morphine. To further assess the possible role of HIV secretory proteins in alternative splicing of OPRM1 gene, we analyzed the impact of HIV-1 Tat, gp120 and Nef proteins on alternative splicing of MOR-1X isoform. While the Tat and gp120 had no visible effects, treatment of neurons with Nef induced MOR-1X alternative splicing that was comparable to treatment with morphine. Altogether, our results suggest that HIV-1 may alter MOR isoform expression with Nef protein by amplifying the rate of MOR-1X alternative splicing induced by morphine.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro 327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

HIV-1 mediated neurotoxicity is thought to be associated with HIV-1 viral proteins activating astrocytes and microglia by inducing inflammatory cytokines leading to the development of HIV-associated neurocognitive disorder (HAND). In the current study, we observe how HIV-1 Nef upregulates the levels of IL-6, IP-10, and TNF-α around 6.0fold in normal human astrocytes (NHAs) compared to cell and empty vector controls. Moderate downregulation in the expression profile of inflammatory cytokines was observed due to RNA interference. Furthermore, we determine the impact of inflammatory cytokines in the upregulation of kynurenine pathway metabolites, such as indoleamine 2,3-dioxygenase (IDO), and 3-hydroxyanthranilic acid oxygenase (HAAO) in NHA, and found the same to be 3.0- and 3.2-fold, respectively. Additionally, the variation in the level of nitric oxide before and after RNA interference was significant. The upregulated cytokines and pathway-specific metabolites could be linked with the neurotoxic potential of HIV-1 Nef. Thus, the downregulation in cytokines and kynurenine metabolites observed after siRNA-Nef interference indicates the possibility of combining the RNA interference approach with current antiretroviral therapy to prevent neurotoxicity development.

Osteoporosis is related to lncRNA-neighboring enhancer of FOXA2 (NEF) and inversely correlated to ankylosing spondylitis (AS), implying that lncRNA-NEF might also relate to AS. Thus, the study was carried out to investigate the involvement of lncRNA-NEF in AS.The study included 60 AS patients and 60 healthy controls. LncRNA-NEF expression in synovial fluid samples was analyzed by reverse transcription quantitative real-time polymerase chain reaction. Disease activity of the 60 AS patients was determined using the Ankylosing Spondylitis Disease Activity Score (ASDAS) 1-4 and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Western blot was carried out to investigate the effects of lncRNA-NEF on inflammatory factors in human fibroblast-like synovial (HFLS) cells. A 3-year follow-up was performed to analyze the role of lncRNA-NEF in the prediction of the recurrence of AS.Our study observed that lncRNA-NEF expression was upregulated in synovial fluid of AS patients and significantly correlated with the ASDAS 1-4, BASDAI, erythrocyte sedimentation rate (ESR), and C-reactive protein level (p .05). Treatment with nonsteroidal anti-inflammatory drugs significantly downregulated lncRNA-NEF expression (p .01). A 3-year follow-up showed that patients with high lncRNA-NEF levels had a high recurrence rate (hazard ratio = 2.266). In addition, lncRNA-NEF was found to regulate the expression of inflammatory factors in HFLS cells.Therefore, lncRNA-NEF upregulation can predict recurrence and poor treatment outcomes of AS and has a great potential to serve as a predictive biomarker factor for the recurrent AS.

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of the HIV-1 Nef gene show extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in a eukaryotic expression vector and further characterized with respect to their ability to downregulate cell surface expression of CD4 and MHC-1 molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 downregulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising from amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

Objective: Despite successful antiviral therapy, the recovery of CD4+ T cells may not be complete in certain HIV-1-infected individuals. In our previous work with humanized mice infected with CXCR4-tropic HIV-1LAI (LAI), viral protein Nef was found the major factor determining rapid loss of both CD4+ T cells and CD4+CD8+ thymocytes but its effect on early T-cell development is unknown. The objective of this study is to investigate the influence of LAI Nef on the development of hematopoietic stem/progenitor cells (HSPCs) into T lymphoid cells. Design: HSPC-OP9-DL1 cell co-culture and humanized mouse model was used to investigate the objective of our study in vitro and in vivo. RNA-seq was exploited to study the change of gene expression signature after nef expression in HSPCs. Results: Nef expression in HSPCs was found to block their development into T lymphoid cells both in vitro and in the mice reconstituted with nef-expressing HSPCs derived from human cord blood. More surprisingly, in humanized mice nef expression preferentially suppressed the production of CD4+ T cells. This developmental defect was not the result of CD34+ cell loss. RNA-seq analysis revealed that Nef affected the expression of 176 genes in HSPCs, including those involved in tumor necrosis factor, Toll-like receptor, and nucleotide-binding oligomerization domain-like receptor signaling pathways that are important for hematopoietic cell development. Conclusion: Our results demonstrate that Nef compromises the development of HSPCs into T lymphoid cells, especially CD4+ T cells. This observation suggests that therapeutics targeting Nef may correct HIV-1-associated hematopoietic abnormalities, especially defects in T-cell development.

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.

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.

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 (Leu(112), Tyr(115), and Phe(121)) 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.

Significance This study describes the discovery of a class of drug that can help the immune system eliminate hard-to-kill cells infected with HIV. HIV establishes a persistent infection for which there is no cure, necessitating the development of new approaches to enhance the clearance of HIV-infected cells. HIV encodes Nef, which down-regulates MHC-I expression in infected cells to impair immune-mediated clearance by cytotoxic T lymphocytes. We identified the plecomacrolide family of natural products as potent inhibitors of Nef, and concanamycin A restored MHC-I and enhanced the clearance of HIV-infected primary cells by cytotoxic T lymphocytes. Concanamycin A counteracted Nef from diverse clades of HIV targeting multiple allotypes of MHC-I, indicating the potential for broad therapeutic utility., Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Human Immunodeficiency Virus-1 (HIV-1) Nef promotes p53 protein degradation to protect HIV-1 infected cells from p53 induced apoptosis. We found that Nef mediated p53 degradation is accomplished through ubiquitin proteasome pathway in an Mdm2-independent manner. By GST pulldown and immunoprecipitation assays, we have shown that Nef interacts with E3 ubiquitin ligase E6AP in both Nef transfected HEK-293T cells and HIV-1 infected MOLT3 cells. The p53 ubiquitination and degradation was found to be enhanced by Nef with E6AP but not by Nef with E6AP-C843A, a dominant negative E6AP mutant. We show that Nef binds with E6AP and promotes E6AP dependent p53 ubiquitination. Further, Nef inhibits apoptosis of p53 null H1299 cells after exogenous expression of p53 protein. The p53 dependent apoptosis of H1299 cells was further reduced after the expression of Nef with E6AP. However, Nef mediated reduction in p53 induced apoptosis of H1299 cells was restored when Nef was co-expressed with E6AP-C843A. Thus, Nef and E6AP co-operate to promote p53 ubiquitination and degradation in order to suppress p53 dependent apoptosis. CHME3 cells, which are a natural host of HIV-1, also show p53 ubiquitination and degradation by Nef and E6AP. These results establish that Nef induces p53 degradation via cellular E3 ligase E6AP to inhibit apoptosis during HIV-1 infection.

Background: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens’ features before applying them. Objective: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1. Methods: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction. Results: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression. Conclusion: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.

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.

Objectives Certain human leucocyte antigen (HLA)-B alleles (protective alleles) associate with durable immune control of HIV-1, but with substantial heterogeneity in the level of control. It remains elusive whether viral factors including Nef-mediated immune evasion function diminish protective allele effect on viral control. Design The naturally occurring non-Ser variant at position 9 of HIV-1 subtype C Nef has recently exhibited an association with enhanced HLA-B downregulation function and decreased susceptibility to recognition by CD8 T cells. We therefore hypothesized this Nef genotype leads to diminished immune control mediated by protective HLA alleles. Methods Nef sequences were isolated from HIV-1 subtype C-infected patients harboring protective alleles and several Nef functions including downregulation of HLA-A, HLA-B, CD4, and SERINC5 were examined. Association between Nef non-Ser9 and plasma viral load was examined in two independent South African and Botswanan treatment-naive cohorts. Results Nef clones isolated from protective allele individuals encoding Nef non-Ser9 variant exhibited greater ability to downregulate HLA-B when compared with the Ser9 variant, while other Nef functions including HLA-A, CD4, and SERINC5 downregulation activity were unaltered. By analyzing a cohort of South African participants chronically infected with subtype C HIV-1, Nef non-Ser9 associated with higher plasma viral load in patients harboring protective alleles. Corroboratively, the Nef non-Ser9 correlated with higher plasma viral load in an independent cohort in Botswana. Conclusion Taken together, our study identifies the Nef genotype, non-Ser9 that subverts host immune control in HIV-1 subtype C infection.

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.

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.

Antiretroviral therapy can control human immunodeficiency virus type 1 (HIV-1) replication in people living with HIV; however, these treatments are not curative and no practical approach for an HIV-1 cure has yet shown success in clinical trials. Counteracting the multiple barriers HIV-1 presents against a practical cure is a direct means to functionalize these curative approaches in vivo. Pharmacological inhibition of the HIV-1 accessory protein, Nef, represents a particularly promising and ambitious approach, with Nef inhibitors holding the potential to reverse HIV-1-related defects in T cell receptor and kinase signaling, apoptosis, autophagy and most importantly, antigen presentation. Together, the capacity for Nef inhibitors to restore these activities underscores their potential as supportive agents in a practical HIV-1 cure. In this review, we outline a rationale for pharmacologically targeting Nef and review the progress made in the identification and development of Nef inhibitors.

Efforts to cure HIV have focused on reactivating latent proviruses to enable elimination by CD8

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4(+) T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4(+) T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4(+) T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

Despite the clinical importance of latent human immunodeficiency virus type 1 (HIV-1) infection, our understanding of the biomolecular processes involved in HIV-1 latency control is still limited. This study was designed to address whether interactions between viral proteins, specifically HIV Nef, and the host cell could affect latency establishment. The study was driven by three reported observations. First, early reports suggested that human immunodeficiency virus type 2 (HIV-2) infection in patients produces a lower viral RNA/DNA ratio than HIV-1 infection, potentially indicating an increased propensity of HIV-2 to produce latent infection. Second, Nef, an early viral gene product, has been shown to alter the activation state of infected cells in a lentiviral lineage-dependent manner. Third, it has been demonstrated that the ability of HIV-1 to establish latent infection is a function of the activation state of the host cell at the time of infection. Based on these observations, we reasoned that HIV-2 Nef may have the ability to promote latency establishment. We demonstrate that HIV-1 latency establishment in T cell lines and primary T cells is indeed differentially modulated by Nef proteins. In the context of an HIV-1 backbone, HIV-1 Nef promoted active HIV-1 infection, while HIV-2 Nef strongly promoted latency establishment. Given that Nef represents the only difference in these HIV-1 vectors and is known to interact with numerous cellular factors, these data add support to the idea that latency establishment is a host cell-virus interaction phenomenon, but they also suggest that the HIV-1 lineage may have evolved mechanisms to counteract host cell suppression. IMPORTANCE Therapeutic attempts to eliminate the latent HIV-1 reservoir have failed, at least in part due to our incomplete biomolecular understanding of how latent HIV-1 infection is established and maintained. We here address the fundamental question of whether all lentiviruses actually possess a similar capacity to establish latent infections or whether there are differences between the lentiviral lineages driving differential latency establishment that could be exploited to develop improved latency reversal agents. Research investigating the viral RNA/DNA ratio in HIV-1 and HIV-2 patients could suggest that HIV-2 indeed has a much higher propensity to establish latent infections, a trait that we found, at least in part, to be attributable to the HIV-2 Nef protein. Reported Nef-mediated effects on host cell activation thus also affect latency establishment, and HIV-1 vectors that carry different lentiviral nef genes should become key tools to develop a better understanding of the biomolecular basis of HIV-1 latency establishment.

Antiviral NK cell activity is regulated through the interaction of activating and inhibitory NK cell receptors with their ligands on infected cells. HLA class I molecules serve as ligands for most killer cell immunoglobulin-like receptors (KIRs), but no HLA class I ligands for the inhibitory NK cell receptor KIR2DL5 have been identified to date. Using a NK cell receptor/ligand screening approach, we observed no strong binding of KIR2DL5 to HLA class I or class II molecules, but confirmed that KIR2DL5 binds to the poliovirus receptor (PVR, CD155). Functional studies using primary human NK cells revealed a significantly decreased degranulation of KIR2DL5+NK cells in response to CD155-expressing target cells. We subsequently investigated the role of KIR2DL5/CD155 interactions in HIV-1 infection, and showed that multiple HIV-1 strains significantly decreased CD155 expression levels on HIV-1-infected primary human CD4+T cells via a Nef-dependent mechanism. Co-culture of NK cells with HIV-1-infected CD4+T cells revealed enhanced anti-viral activity of KIR2DL5+NK cells against wild-type versus Nef-deficient viruses, indicating that HIV-1-mediated downregulation of CD155 renders infected cells more susceptible to recognition by KIR2DL5+NK cells. These data show that CD155 suppresses the antiviral activity of KIR2DL5+NK cells and is downmodulated by HIV-1 Nef protein as potential trade-off counteracting activating NK cell ligands, demonstrating the ability of NK cells to counteract immune escape mechanisms employed by HIV-1.

Significance Lentiviruses encode accessory proteins to manipulate their host cells in order to efficiently replicate and evade antiviral defenses. Interestingly, most lentiviral Nefs down-regulate CD3 from the surface of infected T cells to perturb immune responses. However, for reasons that are incompletely understood, HIV-1 and its simian immunodeficiency virus ancestors lack this function. Here, we report that engineering HIV-1 for Nef-mediated down-regulation of CD3 reduces Env-dependent HIV-1 infectivity, resulting in less efficient cell-to-cell spread and replication. Our data suggest that HIV-1 may have evolved to lose the CD3 down-modulation function of Nef in order to allow T cell activation and to boost viral replication, possibly at the cost of less effective immune evasion and increased pathogenicity., 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.

To enhance infection, enveloped viruses exploit adhesion molecules expressed on the surface of host cells. Specifically, phosphatidylserine (PS) receptors - including members of the human T cell immunoglobulin and mucin domain (TIM)-family - have gained attention for their ability to mediate the entry of many enveloped viruses. However, recent evidence that TIM-1 can restrict viral release reveals a new role for these PS receptors. Additionally, viral factors such as the HIV-1 accessory protein Nef can antagonize this antiviral activity of TIM-1 while host restriction factors such as SERINC5 can enhance it. In this review, we examine the various roles of PS receptors, specifically TIM-family proteins, and the intricate relationship between host and viral factors. Elucidating the multifunctional roles of PS receptors in virus-host interaction is important for understanding viral pathogenesis and developing novel antiviral therapeutics.

Classical antiviral restriction factors promote cellular immunity by their ability to interfere with virus replication and induction of their expression by proinflammatory cytokines such as interferons. The serine incorporator proteins SERINC3 and SERINC5 potently reduce the infectivity of HIV-1 particles when overexpressed, and RNA interference or knockout approaches in T cells have indicated antiviral activity also of the endogenous proteins. Due to lack of reagents for detection of endogenous SERINC proteins, it is still unclear whether SERINC3/5 are expressed to functionally relevant levels in different primary target cells of HIV infection and how the expression levels of these innate immunity factors are regulated. In the current study, analysis of SERINC3/5 mRNA steady-state levels in primary lymphoid and monocyte-derived cells revealed selective induction of their expression upon differentiation of myeloid cells. Contrary to classical antiviral restriction factors, various antiviral α-interferon subtypes and proinflammatory interleukins had no effect on SERINC levels, which were also not dysregulated in CD4+ T cells and monocytes isolated from patients with chronic HIV-1 infection. Notably, HIV-1 particles produced by terminally differentiated monocyte-derived macrophages with high SERINC5 expression, but not by low-expressing monocytes, showed a Nef-dependent infectivity defect. Overall, these findings suggest endogenous expression of SERINC5 to antivirally active levels in macrophages. Our results classify SERINC5 as an unconventional HIV-1 restriction factor whose expression is specifically induced upon differentiation of cells towards the myeloid lineage.

Combination antiretroviral therapy has dramatically changed the outcome of HIV infection, turning it from a death sentence to a manageable chronic disease. However, comorbidities accompanying HIV infection, such as metabolic and cardio-vascular diseases, as well as cognitive impairment, persist despite successful virus control by combination antiretroviral therapy and pose considerable challenges to clinical management of people living with HIV. These comorbidities involve a number of pathological processes affecting a variety of different tissues and cells, making it challenging to identify a common cause(s) that would link these different diseases to HIV infection. In this article, we will present evidence that impairment of cellular cholesterol metabolism may be a common factor driving pathogenesis of HIV-associated comorbidities. Potential implications for therapeutic approaches are discussed.

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.

Rationale:Even in antiretroviral therapy-treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on antiretroviral therapy. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis.Objective:To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models.Methods and Results:Extracellular vesicles derived from both peripheral blood mononuclear cells and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma-derived extracellular vesicles from antiretroviral therapy+ patients who were HIV-Nef+ induced significantly greater endothelial apoptosis compared with HIV-Nef-plasma extracellular vesicles. Both HIV-Nef expressing T cells and HIV-Nef-induced extracellular vesicles increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells, thereby resulting in endothelial colony forming cell premature senescence and endothelial nitric oxide synthase downregulation. These Rac1-dependent activities were characterized by NOX2-mediated reactive oxygen species production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely because of the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced reactive oxygen species formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5 mg/kg atorvastatin.Conclusions:These studies establish a mechanism by which HIV-Nef persistence despite antiretroviral therapy could contribute to ongoing HIV-related vascular dysfunction, which may then be ameliorated by statin treatment.

Aim HIV-1 Nef downregulates surface MHC-I to protect the infected cells from CTLs-mediated killing. Although MHC-I downregulation has been extensively studied, the Nef-dependent assembly of the multi-protein complex and subsequent pathways activation has not yet been well explored. The present study is aimed for the identification of Nef-mediated sequential recruitment of cellular proteins that constitute the functional multi-protein complex, required for the downregulation of MHC-I. Main methods Different Cellular protein complexes were identified by co-immunoprecipitation in Nef or NefE4A mutant-expressing Jurkat T, and THP-1 cells followed by exposure to Nef-specific peptides 24 h post infection. The MHC-I downregulation was analyzed by confocal microscopy and flow cytometry. Key findings We found the association of Nef with PACS-2, GCC185, PI3K, AP-1, SFK, and MHC-I proteins that probably constitute a functional multi-protein complex. Furthermore, the immunoprecipitations with PACS-2 and GCC185 in the presence or absence of Nef, Nef E4A mutant and Nef with CP-inhibitor divide the functional complex of Nef into Nef-dependent (AP-1 and PI3K) and GCC185-dependent complex (MHC-I and SFK). The molecular mechanisms for activation of cellular pathways have been deciphered on the basis of these interactions that are brought in close proximity through Nef-GCC185 interaction. Knockdown of GCC185 using siRNA in Jurkat T cells showed a direct relationship between the assembly of functional multi-protein complex and MHC-I accumulation at GCC185. Significance Overall, our study elucidates that GCC185 is a focal point for the assembly of the Nef-mediated multi-protein complex at TGN.

Functional characterisation of different HIV-1 subtypes may improve understanding of viral pathogenesis and spread. Here, we evaluated the ability of 345 unique HIV-1 Nef clones representing subtypes A, B, C and D to inhibit NFAT signalling following TCR stimulation. The contribution of this Nef function to disease progression was also assessed in 211 additional Nef clones isolated from unique subtype C infected individuals in early or chronic infection. On average, subtype A and C Nef clones exhibited significantly lower ability to inhibit TCR-mediated NFAT signalling compared to subtype B and D Nef clones. While this observation corroborates accumulating evidence supporting relative attenuation of subtypes A and C that may paradoxically contribute to their increased global prevalence and spread, no significant correlations between Nef-mediated NFAT inhibition activity and clinical markers of HIV-1 infection were observed, indicating that the relationship between Nef function and pathogenesis is complex.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that blocks virus entry but is antagonized by three unrelated retroviral accessory proteins. The S2 protein from equine infectious anemia virus (EIAV) has been reported to reduce SERINC5 expression at steady-state levels likely via the endocytic pathway; however, the precise mechanism is still unclear. Here, we investigated how EIAV S2 protein down-regulates SERINC5 compared with down-regulation induced by Nef from HIV-1 and glycoMA proteins from murine leukemia virus (MLV). Using bimolecular fluorescence complementation (BiFC) assay and immunoprecipitation (IP), we detected an interaction between S2 and SERINC5. We found that this interaction relies on the S2 myristoylation site, indicating that it may occur on the plasma membrane. S2 internalized SERINC5 via receptor-mediated endocytosis and targeted it to endosomes and lysosomes, resulting in a ubiquitination-dependent decrease in SERINC5 expression at steady-state levels. Both BiFC and IP detected a glycoMA–SERINC5 interaction, but a Nef–SERINC5 interaction was detected only by BiFC. Moreover, S2 and glycoMA down-regulated SERINC5 more effectively than did Nef. We further show that unlike Nef, both S2 and glycoMA effectively down-regulate SERINC2 and also SERINC5 from Xenopus tropicalis (xSERINC5). Moreover, we detected expression of the equine SERINC5 (eSERINC5) protein and observed that its expression is much weaker than expression levels of SERINC5 from other species. Nonetheless, eSERINC5 had a strong antiviral activity that was effectively counteracted by S2. We conclude that HIV-1, EIAV, and MLV share a similar mechanism to antagonize viral restriction by host SERINC5.

A possible explanation for chronic inflammation in HIV-infected individuals treated with anti-retroviral therapy is hyperreactivity of myeloid cells due to a phenomenon called "trained immunity." Here, we demonstrate that human monocyte-derived macrophages originating from monocytes initially treated with extracellular vesicles containing HIV-1 protein Nef (exNef), but differentiating in the absence of exNef, release increased levels of pro-inflammatory cytokines after lipopolysaccharide stimulation. This effect is associated with chromatin changes at the genes involved in inflammation and cholesterol metabolism pathways and upregulation of the lipid rafts and is blocked by methyl-β-cyclodextrin, statin, and an inhibitor of the lipid raft-associated receptor IGF1R. Bone-marrow-derived macrophages from exNef-injected mice, as well as from mice transplanted with bone marrow from exNef-injected animals, produce elevated levels of tumor necrosis factor α (TNF-α) upon stimulation. These phenomena are consistent with exNef-induced trained immunity that may contribute to persistent inflammation and associated co-morbidities in HIV-infected individuals with undetectable HIV load.

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.

HIV-1 must counteract various host restrictions to establish productive infection. SERINC5 is a potent restriction factor that blocks HIV-1 entry from virions, but its activity is counteracted by Nef. The SERINC5 and Nef activities are both initiated from the plasma membrane, where SERINC5 is packaged into virions for viral inhibition or downregulated by Nef via lysosomal degradation. However, it is still unclear how SERINC5 is localized to and how its expression is regulated on the plasma membrane. We now report that Cullin 3-KLHL20, a trans-Golgi network (TGN)-localized E3 ubiquitin ligase, polyubiquitinates SERINC5 at lysine 130 via K33/K48-linked ubiquitination. The K33-linked polyubiquitination determines SERINC5 expression on the plasma membrane, and the K48-linked polyubiquitination contributes to SERINC5 downregulation from the cell surface. Our study reveals an important role of K130 polyubiquitination and K33/K48-linked ubiquitin chains in HIV-1 infection by regulating SERINC5 post-Golgi trafficking and degradation.

Serine incorporator 5 (SERINC5) reduces the infectivity of progeny HIV-1 virions by incorporating into the outer host-derived viral membrane during egress. To counter SERINC5, the HIV-1 accessory protein Nef triggers SERINC5 internalization by engaging the adaptor protein 2 (AP-2) complex using the [D/E]xxxL[L/I](167) Nef dileucine motif. Nef also engages AP-2 via its dileucine motif to downregulate the CD4 receptor. Although these two Nef functions are related, the mechanisms governing SERINC5 downregulation are incompletely understood. Here, we demonstrate that two primary Nef isolates, referred to as 2410 and 2391 Nef, acquired from acutely HIV-1 infected women from Zimbabwe, both downregulate CD4 from the cell surface. However, only 2410 Nef retains the ability to downregulate cell surface SERINC5. Using a series of Nef chimeras, we mapped the region of 2391 Nef responsible for the functional uncoupling of these two antagonistic pathways to the dileucine motif. Modifications of the first and second x positions of the 2410 Nef dileucine motif to asparagine and aspartic acid residues, respectively (ND(164)), impaired cell surface SERINC5 downregulation, which resulted in reduced infectious virus yield in the presence of SERINC5. The ND(164) mutation additionally partially impaired, but did not completely abrogate, Nef-mediated cell surface CD4 downregulation. Furthermore, the patient infected with HIV-1 encoding 2391 Nef had stable CD4(+) T cell counts, whereas infection with HIV-1 encoding 2410 Nef resulted in CD4(+) T cell decline and disease progression. IMPORTANCE A contributing factor to HIV-1 persistence is evasion of the host immune response. HIV-1 uses the Nef accessory protein to evade the antiviral roles of the adaptive and intrinsic innate immune responses. Nef targets SERINC5, a restriction factor which potently impairs HIV-1 infection by triggering SERINC5 removal from the cell surface. The molecular determinants underlying this Nef function remain incompletely understood. Recent studies have found a correlation between the extent of Nef-mediated SERINC5 downregulation and the rate of disease progression. Furthermore, single-residue polymorphisms outside the known Nef functional motifs can modulate SERINC5 downregulation. The identification of a naturally occurring Nef polymorphism impairing SERINC5 downregulation in this study supports a link between Nef downregulation of SERINC5 and the rate of plasma CD4(+) T cell decline. Moreover, the observed functional impairments of this polymorphism could provide clues to further elucidate unknown aspects of the SERINC5 antagonistic pathway via Nef.

SERINC5 restricts nef-defective HIV-1 by affecting early steps of the virus life cycle. Distantly related retroviruses with a wide host range encode virulent factors in response to challenge by SERINC5. However, the evolutionary origins of this antiretroviral activity, its prevalence among the paralogs, and its ability to target retroviruses remain understudied. In agreement with previous studies, we found that four human SERINC paralogs inhibit nef-defective HIV-1, with SERINC2 being an exception. Here, we demonstrate that this lack of activity in human SERINC2 is associated with its post-whole-genome duplication (post-WGD) divergence, as evidenced by the ability of pre-WGD orthologs from Saccharomyces cerevisiae and flies and a post-WGD-proximate SERINC2 from coelacanths to inhibit the virus. Intriguingly, Nef is unable to counter coelacanth SERINC2, indicating that such activity was directed toward other retroviruses found in coelacanths (like foamy viruses). However, foamy virus-derived vectors are intrinsically resistant to the action of SERINC2, and we show that the foamy virus envelope confers this resistance by affecting its steady-state levels. Our study highlights an ancient origin of antiretroviral activity in SERINCs and a hitherto-unknown interaction with a foamy virus. IMPORTANCE SERINC5 constitutes a critical barrier to the propagation of retroviruses, as highlighted by parallel emergence of anti-SERINC5 activities among distant retroviral lineages. Therefore, understanding the origin and evolution of these host factors will provide key information about virus-host relationships that can be exploited for future drug development. Here, we show that SERINC5-mediated nef-defective HIV-1 infection inhibition is evolutionarily conserved. SERINC2 from coelacanth restricts HIV-1, and it was functionally adapted to target foamy viruses. Our findings provide insights into the evolutionary origin of antiretroviral activity in the SERINC gene family and uncover the role of SERINCs in shaping the long-term conflicts between retroviruses and their hosts.

This paper presents a compact DDA-based fully-differential CMOS instrumentation amplifier dedicated for micro-power ECG monitoring. Only eight transistors are employed to realize a power-efficient current-sharing DDA. A RC network (using MOS pseudo resistors and poly capacitors) forms feedback loops around the DDA creating an ac-only amplification. The proposed amplifier is dc-coupled via gate terminals of the p-channel input transistors. It thus achieves sufficiently high input impedance over the entire ECG frequency range. Fabricated in a 0.35-μm CMOS process, the proposed amplifier occupies 0.0712 mm

Ubiquitination is a process that acts upon every step of the HIV replication cycle. The activity, subcellular localization, and stability of HIV dependency factors as well as negative modulators can be affected by ubiquitination. These modifications consequently have an impact on the progression and outcome of infection. Additionally, recent findings suggest new roles for ubiquitination in the interplay between HIV and the cellular environment, specifically in the interactions between HIV, autophagy and apoptosis. On one hand, autophagy is a defense mechanism against HIV that promotes the degradation of the viral protein Gag, likely through ubiquitination. Gag is an essential structural protein that drives virion assembly and release. Interestingly, the ubiquitination of Gag is vital for HIV replication. Hence, this post-translational modification in Gag represents a double-edged sword: necessary for virion biogenesis, but potentially detrimental under conditions of autophagy activation. On the other hand, HIV uses Nef to circumvent autophagy-mediated restriction by promoting the ubiquitination of the autophagy inhibitor BCL2 through Parkin/PRKN. Although the Nef-promoted ubiquitination of BCL2 occurs in both the endoplasmic reticulum (ER) and mitochondria, only ER-associated ubiquitinated BCL2 arrests the progression of autophagy. Importantly, both mitochondrial BCL2 and PRKN are tightly connected to mitochondrial function and apoptosis. Hence, by enhancing the PRKN-mediated ubiquitination of BCL2 at the mitochondria, HIV might promote apoptosis. Moreover, this effect of Nef might account for HIV-associated disorders. In this article, we outline our current knowledge and provide perspectives of how ubiquitination impacts the molecular interactions between HIV, autophagy and apoptosis.

Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable. Strikingly, among the numerous Src-family downstream effectors, serine/threonine kinase Raf-1 was found to be necessary and alone sufficient to trigger the secretion of TACE into EVs. These data reveal the involvement of Raf-1 in regulation of unconventional protein secretion and highlight the importance of Raf-1 as a cellular effector of Nef, thereby suggesting a novel rationale for testing pharmacological inhibitors of the Raf-MAPK pathway to treat HIV-associated immune activation. IMPORTANCE Chronic immune activation contributes to the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and is associated with poor recovery of the immune system despite potent antiretroviral therapy, which is observed in 10% to 40% drug-treated patients depending on the definition of immune reconstitution. We have previously shown that the HIV pathogenicity factor Nef can promote loading of the proinflammatory protease TACE into extracellular vesicles (EVs), and the levels of such TACE-containing EVs circulating in the blood correlate with low CD4 lymphocyte counts in HIV patients receiving antiretroviral therapy. Here, we show that Nef promotes uploading of TACE into EVs by triggering unconventional secretion via activation of the Hck/Raf/mitogen-activated protein kinase (MAPK) cascade. We find that several pharmaceutical inhibitors of these kinases that are currently in clinical use for other diseases can potently suppress this pathogenic deregulation and could thus provide a novel strategy for treating HIV-associated immune activation.

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.

HIV-1 infection poses a major threat to the public health worldwide. The antiretroviral agents that are currently used to treat HIV-1 infection target viral reverse transcriptase, integrase and protease, or block the fusion of viral envelop and cell membrane. Studies have shown that the HIV-1 encoded protein Nef plays an important role in the pathogenesis of viral infection. Nef ensures efficient counterattack against host immune responses as well as long-term evasion of immune surveillance. In addition, Nef, expressing at a high level early in the viral life cycle, is required for maintaining a high viral load in the persistent infection in vivo and for full pathologic potential. Therefore, Nef may be an excellent target to treat HIV-1 infection. In this manuscript, we reviewed five potential Nef inhibitors, namely, DLC27-14, t ightly bound hydroxypyrazole HIV-1 Nef inhibitor B9, 2c-like inhibitors, N-(3-aminoquinoxalin-2-yl)-4-chlorobenzenesulfonamide and compound 1[(7-oxo-7H-benzo[anthracene]-3-yl)amino]anthraquinone, and their working mechanisms. These drugs may be further developed into new regimens for the treatment of HIV-1 infection.

Various approaches have been investigated to prevent or eliminate HIV-1 since 1981. However, the virus has been affecting human population worldwide with no effective vaccine yet. The conserved regions among the viral genes are suitable targets in mutable viruses to induce the immune responses via an effective delivery platform. In this study, we aimed at evaluation of p24 and nef in two forms of full and truncated genes as two fusion antigenic forms according to our previous bioinformatics analysis. The designed antigens were then transferred through ex vivo generated dendritic cells and also proteins in BALB/c to assess and compare immunogenicity. p24 and Nef amino acid sequences were aligned, then, the most conserved regions were selected and two fusion forms as the truncated (p24:80-231aa-Nef:120-150aa) and the full from (p24-Nef) were cloned and expressed in prokaryotic and eukaryotic systems. Lentiviral vectors were applied to generate recombinant virions harboring the genes of interest to transduce generated murine dendritic cells. BALB/c mice received the recombinant DCs or recombinant proteins according to the defined schedule. IgG development was assessed to determine humoral immune activity and cellular immune responses were evaluated by IL-5 and IFN-y induction. Granzyme B secretion was also investigated to determine CTL activity in different immunized groups. The data showed high induction of cellular immune responses in dendritic cell immunization specifically in immunized mice with the truncated form of the p24 and Nef by high secretion of IFN-y and strong CTL activity. Moreover, protein/ DC prime-boost formulation led to stronger Th1 pathway and strong CTL activation in comparison with other formulations. The generated recombinant dendritic cells expressing p24-Nef induced humoral and cellular immunity in a Th1 pathway specifically with the in silico predicted truncated antigen which could be of high value as a dendritic cell therapeutic vaccine candidate against HIV-1.