HIV-1 Nef Protein Research Articles

Involvement of Human Cellular Proteins and Structures in Realization of the HIV Life Cycle: A Comprehensive Review, 2024.

Human immunodeficiency virus (HIV) continues to be a global health challenge, with over 38 million people infected by the end of 2022. HIV-1, the predominant strain, primarily targets and depletes CD4+ T cells, leading to immunodeficiency and subsequent vulnerability to opportunistic infections. Despite the progress made in antiretroviral therapy (ART), drug resistance and treatment-related toxicity necessitate novel therapeutic strategies. This review delves into the intricate interplay between HIV-1 and host cellular proteins throughout the viral life cycle, highlighting key host factors that facilitate viral entry, replication, integration, and immune evasion. A focus is placed on actual findings regarding the preintegration complex, nuclear import, and the role of cellular cofactors such as FEZ1, BICD2, and NPC components in viral transport and genome integration. Additionally, the mechanisms of immune evasion via HIV-1 proteins Nef and Vpu, and their interaction with host MHC molecules and interferon signaling pathways, are explored. By examining these host-virus interactions, this review underscores the importance of host-targeted therapies in complementing ART, with a particular emphasis on the potential of genetic research and host protein stability in developing innovative treatments for HIV/AIDS.

HIV Protein Nef Induces Cardiomyopathy Through Induction of Bcl2 and p21.

HIV-associated cardiovascular diseases remain a leading cause of death in people living with HIV/AIDS (PLWHA). Although antiretroviral drugs suppress the viral load, they fail to remove the virus entirely. HIV-1 Nef protein is known to play a role in viral virulence and HIV latency. Expression of Nef protein can be detected in different organs, including cardiac tissue. Despite the established role of Nef protein in HIV-1 replication, its impact on organ function inside the human body is not clear. To understand the effect of Nef at the organ level, we created a new Nef-transgenic (Nef-TG) mouse that expresses Nef protein in the heart. Our study found that Nef expression caused inhibition of cardiac function and pathological changes in the heart with increased fibrosis, leading to heart failure and early mortality. Further, we found that cellular autophagy is significantly inhibited in the cardiac tissue of Nef-TG mice. Mechanistically, we found that Nef protein causes the accumulation of Bcl2 and Beclin-1 proteins in the tissue, which may affect the cellular autophagy system. Additionally, we found Nef expression causes upregulation of the cellular senescence marker p21 and senescence-associated β-galactosidase expression. Our findings suggest that the Nef-mediated inhibition of autophagy and induction of senescence markers may promote aging in PLWHA. Our mouse model could help us to understand the effect of Nef protein on organ function during latent HIV infection.

Extracellular vesicle-based anti-HOXB7 CD8+ T cell-specific vaccination strengthens antitumor effects induced by vaccination against Her2/neu

We previously developed an innovative strategy to induce CD8+ T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+ T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two “self” tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.

Targeting ADA and HIV-1 Nef Protein with Protease Inhibitors: A Repurposing Strategy through Molecular Networking and an In silico Approach for Integrated Management of HIV Co-infected with Abdominal Tuberculosis

Background: Background: The co-infection of HIV and abdominal TB poses a worldwide danger to humanity. This is because there are more strains of bacteria that are resistant to many classes of currently available medications. According to current findings, repurposing existing available medications will result in more effective functioning than using newly designed medications. Objective: Based on this fact, we hypothesised that the PI could be repurposed; we used Food and Drug Administration (FDA)-approved PI drugs to treat HIV co-infected patients with abdominal TB, and a computational study has been conducted. Methods: This comprises network analysis models to find their protein drug interaction (PDI) through a search tool for interacting chemicals (STICH) module of Cytoscape network analysis model followed by the screening of these drugs for their ADMET prediction and binding affinity with adenosine deaminase (ADA), a protein responsible for abdominal TB, and the HIV-1 Nef protein, responsible for the regulation of immune function (CD4+). Results: The network analysis showed 13 nearest binding drugs of these proteins of interest. The ADMET study result showed the pharmacologically relevant parameters that have a significant effect on the binding affinity, bioavailability, and toxicity of PI. The top three scores achieved by PI against adenosine deaminase enzyme activity (PDB ID: 1A4M) are viz., -23.7919, -23.3529, and -22.6773 for Ritonavir, Tipranavir, and Atazanavir, respectively. The top three scores achieved by PI against HIV-1 Nef protein activity (PDB ID: 6URI) are viz., -28.7321, -28.4987, and -28.3155 for Atazanavir, Tipranavir, and Simeprevir, respectively. The active site of ADA and HIV-1 Nef proteins comprises amino acid residues such as for Tipranavir: Arene-Cation interaction (Phenyl and Pyridine)- Arg B1081, and Lys B1033 (1A4M) and Arene-Cation interaction (Pyridine and Phenyl)- Lys D11 and Arg D33; Sidechain acceptor Thr B40; Sidechain donor- Asp D30; Backbone donor- Ala B37 (6URI). Atazanavir: Arene-Cation interaction (Phenyl)- Lys A254 and Lys B1033; Sidechain acceptor - Arg A251 (1A4M). Conclusion: Thus, from the computational studies carried out, we could obtain hints for optimising the molecular selectivity of the PI to provide help in the design of new compounds via the repurposing strategy of the FDA-approved PI for effective treatment of co-morbidity with HIV and abdominal TB. However, further pharmacokinetics, pharmacodynamics, preclinical, and clinical studies permit the design of the new agents without undesirable interactions.

In-silico Study of Secondary Metabolites as Potential Inhibitors of NEF and P24 Protein of HIV-1

Introduction: Acquired immune deficiency syndrome (HIV/AIDS) has been a major global health concern for over 38 years. No safe and effective preventive or therapeutic vaccine has been developed although many products have been investigated. Methods: This computational study was conducted on plant-based active compounds against HIV-1 NEF and p24 protein to obtain and complexes with high binding scores were used for two-dimensional interaction studies via Ligplot to explore hydrogen bond and hydrophobic bond formation. ADMET analysis for best phytocompounds was performed using DruLito, ALOGPS, and PROTOX II. Results: According to the study conducted, phytocompounds like, Protostrychnine, Isostrychnine, Pseudo-Alpha-Colubrine, Alpha-Colubrine, Camptothecin, Benzo[f]quinoline, and (+) -Camptothecin are safe to be considered as a potential drug candidate after experimental validation against NEF and p24 proteins of HIV-1. While, Picrasidine M, Chaetochromin, 3’,3’-Binaringenin, and Sequoiaflavone displayed high binding scores of -10.8, -8.2, -9.5, -9.2 and -9.0, -8.8, -10.6, -9.0 respectively for NEF and p24 protein. All drugs belong to the toxicity class of either 4 or 5. They are inactive for hepatotoxicity and carcinogenicity but active for immunogenicity. Conclusion: For further validation of the results the phytocompounds can be extracted through solvent extraction method and tested on cell lines or animal models for their effectiveness.

Structural and Functional Dysregulation of the Brain Endothelium in HIV Infection and Substance Abuse.

Blood-brain barrier (BBB) injury and dysfunction following infection with the human immunodeficiency virus (HIV) enables viral entry into the brain, infection of resident brain cells, neuronal injury and subsequent neurodegeneration leading to HIV-associated neurocognitive disorders (HAND). Although combination antiretroviral therapy has significantly reduced the incidence and prevalence of acquired immunodeficiency syndrome and increased the life expectancy of people living with HIV, the prevalence of HAND remains high. With aging of people living with HIV associated with increased comorbidities, the prevalence of HIV-related central nervous system (CNS) complications is expected to remain high. Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on the pathobiology of the brain endothelium structural and functional dysregulation in HIV infection, including in the presence of HIV-1 and viral proteins (gp120, Tat, Nef, and Vpr). We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms. We further summarize evidence of synergy or lack thereof between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV- or viral protein-induced BBB injury and dysfunction.

Abstract We125: HIV-1 Nef protein induces aging and cardiac dysfunction through autophagy dysregulation

Background: Despite highly active antiretroviral therapy (ART), HIV-associated cardiovascular disease and accelerated aging cause increased mortality in people living with HIV/AIDS (PLWHA). Nef protein has been detected in HIV patients even with ART therapy. We hypothesized that the Nef protein may compromise heart function and accelerate aging by dysregulation of autophagy. Methods: We generated a Nef transgenic (Nef-TG) mouse model by cloning the HIV-Nef gene in the CAG-Lox-CAT vector. We used Western blotting to determine protein expression in cardiac tissue. Heart function was evaluated by echocardiography. Nef protein function was also determined by plasmid DNA transfection in the HEK293 cells. Results: Nef-TG mouse shows cardiac dysfunction, increased cardiac fibrosis, and early mortality. Expression of autophagy marker LC3II was significantly decreased in the heart of Nef-TG mice. The data suggest Nef inhibits autophagy in the cell. Mechanistically, we found that senescence-associated markers p21, Bcl2, and Beclin1 levels significantly increased in Nef-TG mice's hearts, which may negatively regulate heart function and promote aging. Conclusion: Our new Nef-TG mouse model revealed the cause of HIV-associated aging in PLWHA. Our mouse model can also be used to elucidate the role of Nef protein on other organ systems and test novel therapeutics for HIV.

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

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

HIV1-Nef perturbs the integrity of blood testis barrier in rat model

ABSTRACT The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

Guiding a language-model based protein design method towards MHC Class-I immune-visibility targets in vaccines and therapeutics

Proteins have an arsenal of medical applications that include disrupting protein interactions, acting as potent vaccines, and replacing genetically deficient proteins. While therapeutics must avoid triggering unwanted immune-responses, vaccines should support a robust immune-reaction targeting a broad range of pathogen variants. Therefore, computational methods modifying proteins’ immunogenicity without disrupting function are needed. While many components of the immune-system can be involved in a reaction, we focus on Cytotoxic T-lymphocytes (CTLs). These target short peptides presented via the MHC Class I (MHC-I) pathway. To explore the limits of modifying the visibility of those peptides to CTLs within the distribution of naturally occurring sequences, we developed a novel machine learning technique, CAPE-XVAE. It combines a language model with reinforcement learning to modify a protein’s immune-visibility. Our results show that CAPE-XVAE effectively modifies the visibility of the HIV Nef protein to CTLs. We contrast CAPE-XVAE to CAPE-Packer, a physics-based method we also developed. Compared to CAPE-Packer, the machine learning approach suggests sequences that draw upon local sequence similarities in the training set. This is beneficial for vaccine development, where the sequence should be representative of the real viral population. Additionally, the language model approach holds promise for preserving both known and unknown functional constraints, which is essential for the immune-modulation of therapeutic proteins. In contrast, CAPE-Packer, emphasizes preserving the protein’s overall fold and can reach greater extremes of immune-visibility, but falls short of capturing the sequence diversity of viral variants available to learn from. Source code: https://github.com/hcgasser/CAPE (Tag: v1.1)

Transcriptionally Active Defective HIV-1 Proviruses and Their Association With Immunological Nonresponse to Antiretroviral Therapy.

A subset of antiretroviral therapy-treated persons with human immunodeficiency virus (HIV), referred to as immunological nonresponders (INRs), fails to normalize CD4+ T-cell numbers. In a case-control study involving 26 INRs (CD4 < 250 cells/µL) and 25 immunological responders (IRs; CD4 ≥ 250 cells/µL), we evaluated the potential contribution of transcriptionally competent defective HIV-1 proviruses to poor CD4+ T-cell recovery. Compared to the responders, the INRs had higher levels of cell-associated HIV RNA (P = .034) and higher percentages of HLA-DR+ CD4+ T cells (P < .001). While not encoding replication-competent viruses, the RNA transcripts frequently encoded HIV-1 Gag-p17 and Nef proteins. These transcripts and/or resulting proteins may activate pathway(s) leading to the immunological nonresponse phenotype.

SMR peptide antagonizes Staphylococcus aureus biofilm formation.

The emergence and international dissemination of multi-drug resistant Staphylococcus aureus (S. aureus) strains challenge current antibiotic-based therapies, representing an urgent threat to public health worldwide. In the U.S. alone, S. aureus infections are responsible for 11,000 deaths and 500,000 hospitalizations annually. Biofilm formation is a major contributor to antibiotic tolerance and resistance-induced delays in empirical therapy with increased infection severity, frequency, treatment failure, and mortality. Developing novel treatment strategies to prevent and disrupt biofilm formation is imperative. In this article, we test the Secretion Modification Region (SMR) peptides for inhibitory effects on resistant S. aureus biofilm-forming capacity by targeting the molecular chaperone DnaK. The dose effect of SMR peptides on biofilm formation was assessed using microtiter plate methods and confocal microscopy. Interaction between the antagonist and DnaK was determined by immune precipitation with anti-Flag M2 Affinity and Western blot analysis. Increasing SMR peptide concentrations exhibited increasing blockade of S. aureus biofilm formation with significant inhibition found at 18 µM, 36 µM, and 72 µM. This work supports the potential therapeutic benefit of SMR peptides in reducing biofilm viability and could improve the susceptibility to antimicrobial agents.IMPORTANCEThe development of anti-biofilm agents is critical to restoring bacterial sensitivity, directly combating the evolution of resistance, and overall reducing the clinical burden related to pervasive biofilm-mediated infections. Thus, in this study, the SMR peptide, a novel small molecule derived from the HIV Nef protein, was preliminarily explored for anti-biofilm properties. The SMR peptide was shown to effectively target the molecular chaperone DnaK and inhibit biofilm formation in a dose-dependent manner. These results support further investigation into the mechanism of SMR peptide-mediated biofilm formation and inhibition to benefit rational drug design and the identification of therapeutic targets.

In Silico and In vitro Evaluations of the Antibacterial Activities of HIV-1 Nef Peptides against Pseudomonas aeruginosa.

One of the burning issues facing healthcare organizations is multidrug-resistant (MDR) bacteria.P. aeruginosais an MDR opportunistic bacterium responsible for nosocomial and fatal infections in immunosuppressed individuals. According to previous studies,efflux pump activityand biofilm formation are the most commonresistance mechanisms in P. aeruginosa. The aim of this study was to propose new antimicrobial peptides (AMPs) that target P. aeruginosa and can effectively address these resistance mechanisms through in silico and in vitro assessments. Since AMPs are an attractive alternative to antibiotics, in vitro experiments were carried out along with bioinformatics analyses on 19 Nef peptides (derived from the HIV-1 Nef protein) in the current study. Several servers, including Dbaasps, Antibp2, CLASSAMP2, ToxinPred, dPABBs and ProtParam were used to predict Nef peptides as AMPs. To evaluate the binding affinities, a molecular docking analysis was performed with the HADDOCK web server for all Nef peptide models against two effective proteins ofP. aeruginosa(MexB and PqsR) that play a role in efflux and quorum sensing. Moreover, the antibacterial and antibiofilm activity of the Nef peptides was investigated in a resistant strain ofP. aeruginosa. The results of molecular docking revealed that all Nef peptides have a significant binding affinity to the abovementioned proteins. Nef-Peptide-19 has the highest affinity to the active sites of MexB and PqsR with the HADDOCK scores of -136.1 ± 1.7 and -129.4 ± 2, respectively. According to the results ofin vitroevaluation, Nef peptide 19 showed remarked activity againstP. aeruginosawith minimum inhibitory and bactericidal concen-trations (MIC and MBC) of 10 µM and 20 µM, respectively. In addition, biofilm inhibitory activity was observed at a concentration of 20 µM. Finally, Nef peptide 19 is proposed as a new AMP againstP. aeruginosa.

1530. Reactivation of Latent HIV in CD4 T Cells Procured from Patients on ART by Autologous CD8 T cells or Fas Ligand

Abstract Background HIV persists in a long-lived infected CD4 T cell reservoir, which harbors an integrated transcriptionally latent virus. We reported that cytotoxic CD8 T cells conjugate with, and kill, autologous HIV-infected CD4 T cells isolated from all stages of HIV infection including reservoir cells of patients under antiretroviral therapy (ART) (image 1). This killing is attenuated by HIV Nef protein. We also previously reported that FasL presented by the cytotoxic T cells interacts with Fas on the surface of the target cells, resulting in the apoptosis of the target cells. Wajant demonstrated that Fas/FasL interaction can result in the target cell activation through NFkB, whereas NFkB binding site is present in the Long Terminal Repeat of HIV. Therefore, we hypothesize that the interaction of FasL and autologous CD8 T cells with CD4 T cells can result in the reactivation of latent HIV. Conjugation and apoptosis of CD4 T cells by autologous CD8 T cells procured from patients on ART and analyzed by ImageStream. The CD4 T cells in apoptosis are positive for activated caspase 3 (yellow) and DNA fragmentation/ TUNEL assay (orange). CD4 T cells (green), CD8 T cells (red), DAPI (blue). Methods CD4 T cells and CD8 T cells were procured from the PBMC of 20 HIV-infected patients on ART with undetectable viral load and 10 healthy volunteers. The cells were isolated using magnetic beads. Resting memory CD4 T cells (CD25-, CD69- and HLA-DR-) were isolated using a two-step bead depletion purification procedure. These cells were then incubated with soluble FasL (sFasL) and autologous CD8 T cells, for 2 and 16 hours. P24 was looked for in the supernatant by ELISA, and the expression of viral proteins was looked for inside CD4 T cells using immunohistochemistry and confocal microscopy Results CD4 T cells and resting memory CD4 T cells, procured from PBMC of HIV-infected patients on ART, showed HIV reactivation after incubation with sFasL and autologous CD8 T cells. This reactivation was demonstrated by the appearance of P24 in the supernatant (figure 1). HIV proteins p24, gp120, and Nef appeared inside the CD4 T cells (Image 2). HIV reactivation was not demonstrated in the CD4 T cells procured from HIV-infected patients that were incubated without sFasL or autologous CD8 T cells for 18 hours (figure 1). Immunohistochemistry and confocal microscopy of CD4 T cells incubated with sFasL for 18 hours. The cells were then incubated with a primary fluorescent antibody against HIV proteins (P24, Nef, GP120) and a secondary antibody ALEXA594 (red). The cells were also marked with DAPI (blue). We can see the expression of HIV proteins inside the CD4 T cells (red arrows). ELISA for the expression of P24 in the supernatant of CD4 T cells procured from HIV-infected patients, following different manipulations On the left, we note positive ELISA for P24 in the supernatant of the CD4 T cells of patients 11 to 16 (pg/ml). On the right, we note positive ELISA for P24 in the supernatant of the latent CD4 T cells of patients 17-20 (pg/ml). Conclusion HIV manipulates the cellular immune system in two ways; First, HIV attenuates CD8 T cells killing of HIV-infected CD4 T cells by the HIV-Nef protein. Second, reactivation of latent HIV by CD8 T cells and sFasL, that results in further infection of additional CD4 T cells. Disclosures All Authors: No reported disclosures

Abstract 14459: Endothelial Cell Specific Expression of HIV-Nef Induces Endothelial Senescence and Reduced Cardiac Output

Introduction: People living with HIV experience increased cardiovascular comorbidities despite immediate prescription of combined antiviral therapy (ART) upon HIV diagnosis. Previously, we reported that the virally encoded HIV-Nef protein persists in extracellular vesicles (EV) of aviremic HIV patients on ART and endothelial-specific transgenic expression of HIV-Nef in murine models causes increased endothelial surface expression of the pro-apoptotic protein EMAPII. Co-culture with HIV-Nef expressing T cells induced endothelial senescence which could be completely abrogated by using EMAPII-neutralizing antibodies. Hypothesis: Here we hypothesize that endothelial Nef expression in transgenic mice induces EMAPII-associated endothelial senescence after intermittent exposure to hypoxia. Methods: Female (n=5) transgenic mice with endothelium-specific Nef expression using VE-Cadherin promoter were challenged with 4 weeks of mild hypoxia (10% O 2 ) followed by 8 weeks of normoxia and then evaluated for cardiac function using non-invasive echocardiography with INVEVO2100. Results: We observed decreased cardiac output (Nef= 15.10 ul/min vs WT= 21.46 ul/min, p&lt;0.05). Subsequently, we observed a 3.5-fold increased frequency of senescent endothelial cells using the senescence-associated marker p16INK4α (Nef= 0.54% vs WT= 0.15%, p&lt;0.05) from the heart using flow cytometry. Furthermore, we also observed a 3.5-fold increase in the frequency of EMAPII+/P16INK4α+ cells (Nef= 0.50% vs WT= 0.14%, p&lt;0.05). Conclusions: Transgenic endothelial HIV-Nef expression leads to upregulation of endothelial cellular senescence and EMAPII expression. EMAPII-neutralizing antibodies and senolytic agents are likely tools to reduce HIV-Nef-induced senescence. Endothelial cell senescence could affect cardiac circulation to impair heart function. Future studies will evaluate sex differences in HIV-Nef induces cardiac output at various ages.

An active HIV reservoir during ART is associated with maintenance of HIV-specific CD8+ T cell magnitude and short-lived differentiation status

Before initiation of antiretroviral therapy (ART), HIV-specific CD8+ Tcells are dysfunctional and short lived. To better understand the relationship between the HIV reservoir in CD4+ Tcells and the magnitude and differentiation status of HIV-specific CD8+ Tcells, we investigated these cells from acute and chronic HIV-infected individuals after 2 years of ART. Although both the HIV reservoir and the CD8+ Tcell responses declined significantly after 2 years of ART, sustained HIV-specific CD8+ Tcell responses correlated with a greater reduction of integrated HIV provirus. However, the magnitude of CD8+ Tcells specific for HIV Gag, Pol, Nef, and Vif proteins positively associated with the active reservoir size during ART, measured as cell-associated RNA. Importantly, high HIV DNA levels strongly associate with maintenance of short-lived HIV-specific CD8+ Tcells, regardless of ART initiation time. Our data suggest that the active reservoir maintains HIV-specific CD8+ Tcell magnitude but prevents their differentiation into functional cells.

Computer-aided de novo design and optimization of novel potential inhibitors of HIV-1 Nef protein

Nef is a small accessory protein pivotal in the HIV-1 viral replication cycle. It is a multifunctional protein and its interactions with kinases in host cells have been well characterized through many in vitro and structural studies. Nef forms a homodimer to activate the kinases and subsequently the phosphorylation pathways. The disruption of its homodimerization represents a valuable approach in the search for novel classes of antiretroviral. However, this research avenue is still underdeveloped as just a few Nef inhibitors have been reported so far, with very limited structural information about their mechanism of action. To address this issue, we have employed an in silico structure-based drug design strategy that combines de novo ligand design with molecular docking and extensive molecular dynamics simulations. Since the Nef pocket involved in homodimerization has high lipophilicity, the initial de novo-designed structures displayed poor drug-likeness and solubility. Taking information from the hydration sites within the homodimerization pocket, structural modifications in the initial lead compound have been introduced to improve the solubility and drug-likeness, without affecting the binding profile. We propose lead compounds that can be the starting point for further optimizations to deliver long-awaited, rationally designed Nef inhibitors.

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

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

Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes.

In recent years, dendritic cells (DCs)-based vaccines have been developed to combat HIV-1 infection in preclinical and clinical trials. In this study, mice bone marrow cells-derived DCs were pulsed with the recombinant Nef, heat shock protein 27 (Hsp27) and Hsp27-Nef proteins, and also green fluorescent protein (GFP) as a positive control. Then, new platforms of DCs loaded with HIV-1 Nef and Hsp27-Nef proteins (i.e., DC prime/DC boost, DNA prime/DC boost, and DC prime/protein boost) were used to evaluate immune responses in BALB/c mice. Finally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was investigated to secret cytokines in vitro. Our data indicated that the recombinant Nef (∼30kDa), Hsp27 (∼27kDa), GFP (∼27kDa), and Hsp27-Nef (∼53kDa) proteins were greatly generated in E. coli. Moreover, the modified DCs with the recombinant proteins were prepared in large scale. The results of mice immunization showed the highest levels of antibodies, cytokines, and Granzyme B in heterologous DC prime/protein boost regimen using Hsp27-Nef antigen (DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen). The levels of IFN-γ and IL-10 cytokines in splenocytes isolated from mice immunized with DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen were higher than those in other regimens after exposure to SCR virions. These findings demonstrated the importance of Hsp27 as an adjuvant and heterologous DC prime/ protein boost regimen in improvement of immune responses. Indeed, DC Hsp27-Nef prime/ protein Hsp27-Nef boost regimen can be utilized as a promising candidate for HIV-1 vaccine development.

Restriction of Influenza A Virus by SERINC5.

Serine incorporator 5 (Ser5), a transmembrane protein, has recently been identified as a host antiviral factor against human immunodeficiency virus (HIV)-1 and gammaretroviruses like murine leukemia viruses (MLVs). It is counteracted by HIV-1 Nef and MLV glycogag. We have investigated whether it has antiviral activity against influenza A virus (IAV), as well as retroviruses. Here, we demonstrated that Ser5 inhibited HIV-1-based pseudovirions bearing IAV hemagglutinin (HA); as expected, the Ser5 effect on this glycoprotein was antagonized by HIV-1 Nef protein. We found that Ser5 inhibited the virus-cell and cell-cell fusion of IAV, apparently by interacting with HA proteins. Most importantly, overexpressed and endogenous Ser5 inhibited infection by authentic IAV. Single-molecular fluorescent resonance energy transfer (smFRET) analysis further revealed that Ser5 both destabilized the pre-fusion conformation of IAV HA and inhibited the coiled-coil formation during membrane fusion. Ser5 is expressed in cultured small airway epithelial cells, as well as in immortal human cell lines. In summary, Ser5 is a host antiviral factor against IAV which acts by blocking HA-induced membrane fusion. IMPORTANCE SERINC5 (Ser5) is a cellular protein which has been found to interfere with the infectivity of HIV-1 and a number of other retroviruses. Virus particles produced in the presence of Ser5 are impaired in their ability to enter new host cells, but the mechanism of Ser5 action is not well understood. We now report that Ser5 also inhibits infectivity of Influenza A virus (IAV) and that it interferes with the conformational changes in IAV hemagglutinin protein involved in membrane fusion and virus entry. These findings indicate that the antiviral function of Ser5 extends to other viruses as well as retroviruses, and also provide some information on the molecular mechanism of its antiviral activity.