HIV-1 Tat Induces Dysregulation of PGC1-Alpha and Sirtuin 3 Expression in Neurons: The Role of Mitochondrial Biogenesis in HIV-Associated Neurocognitive Disorder (HAND).

During the antiretroviral era, individuals living with HIV continue to experience milder forms of HIV-associated neurocognitive disorder (HAND). Viral proteins, including Tat, play a pivotal role in the observed alterations within the central nervous system (CNS), with mitochondrial dysfunction emerging as a prominent hallmark. As a result, our objective was to examine the expression of genes associated with mitophagy and mitochondrial biogenesis in the brain exposed to the HIV-1 Tat protein. We achieved this by performing bilateral stereotaxic injections of 100 ng of HIV-1 Tat into the hippocampus of Sprague-Dawley rats, followed by immunoneuromagnetic cell isolation. Subsequently, we assessed the gene expression of Ppargc1a, Pink1, and Sirt1-3 in neurons using RT-qPCR. Additionally, to understand the role of Tert in telomeric dysfunction, we quantified the activity and expression of Tert. Our results revealed that only Ppargc1a, Pink1, and mitochondrial Sirt3 were downregulated in response to the presence of HIV-1 Tat in hippocampal neurons. Interestingly, we observed a reduction in the activity of Tert in the experimental group, while mRNA levels remained relatively stable. These findings support the compelling evidence of dysregulation in both mitophagy and mitochondrial biogenesis in neurons exposed to HIV-1 Tat, which in turn induces telomeric dysfunction.

Massively HIV-1-infected macrophages exhibit a severely hampered ability to differentiate into osteoclasts.

Introduction: Osteoclasts play a crucial role in bone resorption, and impairment of their differentiation can have significant implications for bone density, especially in individuals with HIV who may be at risk of altered bone health. The present study aimed to investigate the effects of HIV infection on osteoclast differentiation using primary human monocyte-derived macrophages as precursors. The study focused on assessing the impact of HIV infection on cellular adhesion, cathepsin K expression, resorptive activity, cytokine production, expression of co-receptors, and transcriptional regulation of key factors involved in osteoclastogenesis. Methods: Primary human monocyte-derived macrophages were utilized as precursors for osteoclast differentiation. These precursors were infected with HIV, and the effects of different inoculum sizes and kinetics of viral replication were analyzed. Subsequently, osteoclastogenesis was evaluated by measuring cellular adhesion, cathepsin K expression, and resorptive activity. Furthermore, cytokine production was assessed by monitoring the production of IL-1ß, RANK-L, and osteoclasts. The expression levels of co-receptors CCR5, CD9, and CD81 were measured before and after infection with HIV. The transcriptional levels of key factors for osteoclastogenesis (RANK, NFATc1, and DC-STAMP) were examined following HIV infection. Results: Rapid, massive, and productive HIV infection severely impaired osteoclast differentiation, leading to compromised cellular adhesion, cathepsin K expression, and resorptive activity. HIV infection resulted in an earlier production of IL-1ß concurrent with RANK-L, thereby suppressing osteoclast production. Infection with a high inoculum of HIV increased the expression of the co-receptor CCR5, as well as the tetraspanins CD9 and CD81, which correlated with deficient osteoclastogenesis. Massive HIV infection of osteoclast precursors affected the transcriptional levels of key factors involved in osteoclastogenesis, including RANK, NFATc1, and DC-STAMP. Conclusions: The effects of HIV infection on osteoclast precursors were found to be dependent on the size of the inoculum and the kinetics of viral replication. These findings underscore the importance of understanding the underlying mechanisms to develop novel strategies for the prevention and treatment of bone disorders in individuals with HIV.

Cerebrospinal fluid CD14++CD16+ monocytes in HIV-1 subtype C compared with subtype B.

CD14++CD16+ monocytes are susceptible to HIV-1 infection, and cross the blood-brain barrier. HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared to HIV-1B, which might influence monocyte trafficking into the CNS. We hypothesized that the proportion of monocytes in CSF in HIV-1C is lower than HIV-1B group. We sought to assess differences in monocyte proportions in cerebrospinal fluid (CSF) and peripheral blood (PB) between people with HIV (PWH) and without HIV (PWoH), and by HIV-1B and -C subtypes. Immunophenotyping was performed by flow cytometry, monocytes were analyzed within CD45 + and CD64 + gated regions and classified in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+). Among PWH, the median [IQR] CD4 nadir was 219 [32-531] cell/mm3; plasma HIV RNA (log10) was 1.60 [1.60-3.21], and 68% were on antiretroviral therapy (ART). Participants with HIV-1C and -B were comparable in terms of age, duration of infection, CD4 nadir, plasma HIV RNA, and ART. The proportion of CSF CD14++CD16+ monocytes was higher in participants with HIV-1C than those with HIV-1B [2.00(0.00-2.80) vs. 0.00(0.00-0.60) respectively, p = 0.03 after BH correction p = 0.10]. Despite viral suppression, the proportion of total monocytes in PB increased in PWH, due to the increase in CD14++CD16+ and CD14lowCD16+ monocytes. The HIV-1C Tat substitution (C30S31) did not interfere with the migration of CD14++CD16+ monocytes to the CNS. This is the first study to evaluate these monocytes in the CSF and PB and compare their proportions according to HIV subtype.

HIV-1 Gag specificity for PIP2 is regulated by macromolecular electric properties of both protein and membrane local environments.

HIV-1 assembly occurs at the plasma membrane, with the Gag polyprotein playing a crucial role. Gag association with the membrane is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with anionic lipids. Several pieces of evidence suggest that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP2) highly influences this binding. Furthermore, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP2-containing membranes. It is hypothesized that RNA has a chaperone function by interacting with the MA domain, preventing Gag from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focusing on the specificity for PIP2 and on the possible effects of a Gag N-terminal peptide on impairing the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but has no effect on the selectivity for PIP2. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negatively charged compositions, where MA alone does not discriminate between membranes with or without PIP2. Therefore, we propose that the specificity of MA for PIP2-containing membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario provides a new understanding of the regulation mechanism, with a macromolecular view, rather than considering molecular interactions within a ligand-receptor model.

Identification of Clinically Relevant HIV Vif Protein Motif Mutations through Machine Learning and Undersampling.

Human Immunodeficiency virus (HIV) and its clinical entity, the Acquired Immunodeficiency Syndrome (AIDS) continue to represent an important health burden worldwide. Although great advances have been made towards determining the way viral genetic diversity affects clinical outcome, genetic association studies have been hindered by the complexity of their interactions with the human host. This study provides an innovative approach for the identification and analysis of epidemiological associations between HIV Viral Infectivity Factor (Vif) protein mutations and four clinical endpoints (Viral load and CD4 T cell numbers at time of both clinical debut and on historical follow-up of patients. Furthermore, this study highlights an alternative approach to the analysis of imbalanced datasets, where patients without specific mutations outnumber those with mutations. Imbalanced datasets are still a challenge hindering the development of classification algorithms through machine learning. This research deals with Decision Trees, Naïve Bayes (NB), Support Vector Machines (SVMs), and Artificial Neural Networks (ANNs). This paper proposes a new methodology considering an undersampling approach to deal with imbalanced datasets and introduces two novel and differing approaches (MAREV-1 and MAREV-2). As theses approaches do not involve human pre-determined and hypothesis-driven combinations of motifs having functional or clinical relevance, they provide a unique opportunity to discover novel complex motif combinations of interest. Moreover, the motif combinations found can be analyzed through traditional statistical approaches avoiding statistical corrections for multiple tests.

Biotinylation Eliminates the Intermediate State of Top7 Designed with an HIV-1 Epitope.

Broadly neutralizing antibodies against HIV-1 are rare with the 2F5 antibody being one of the most protective. Insertion of an antibody epitope into a stable and small protein scaffold overcomes many of the obstacles found to produce antibodies. However, the design leads to grafting of epitopes that may cause protein aggregation. Here, I investigated the 2F5 epitope grafted into the Top7 as the scaffold in which the resulting immunoreactive protein precipitates along the storage time, as opposed to its completely soluble biotinylated version. Molecular dynamics showed that biotinylation eliminates the intermediate state of the scaffold-epitope Top7-2F5 by switching a noncooperative to a cooperative folding. The aggregation propensity of the Top7-designed proteins is examined in light of thermodynamic cooperativity and kinetic traps along the decreasing depth of the intermediate ensemble in the free energy landscape. This protocol may predict stable and soluble scaffold-epitopes with the purpose of composing novel therapeutic and diagnostic platforms.

Epitranscriptomic regulation of HIV-1 full-length RNA packaging.

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

Endocytic Motif on a Biotin-Tagged HIV-1 Env Modulates the Co-Transfer of Env and Gag during Cell-to-Cell Transmission.

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell-cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell-cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

STING and cGAS gene expressions were downregulated among HIV-1-infected persons after antiretroviral therapy.

BACKGROUND: The HIV-1 epidemic is still considered a global public health problem, but great advances have been made in fighting it by antiretroviral therapy (ART). ART has a considerable impact on viral replication and host immunity. The production of type I interferon (IFN) is key to the innate immune response to viral infections. The STING and cGAS proteins have proven roles in the antiviral cascade. The present study aimed to evaluate the impact of ART on innate immunity, which was represented by STING and cGAS gene expression and plasma IFN-α level. METHODS: This cohort study evaluated a group of 33 individuals who were initially naïve to therapy and who were treated at a reference center and reassessed 12 months after starting ART. Gene expression levels and viral load were evaluated by real-time PCR, CD4+ and CD8+ T lymphocyte counts by flow cytometry, and IFN-α level by enzyme-linked immunosorbent assay. RESULTS: From before to after ART, the CD4+ T cell count and the CD4+/CD8+ ratio significantly increased (p < 0.0001), the CD8+ T cell count slightly decreased, and viral load decreased to undetectable levels in most of the group (84.85%). The expression of STING and cGAS significantly decreased (p = 0.0034 and p = 0.0001, respectively) after the use of ART, but IFN-α did not (p = 0.1558). Among the markers evaluated, the only markers that showed a correlation with each other were STING and CD4+ T at the time of the first collection. CONCLUSIONS: ART provided immune recovery and viral suppression to the studied group and indirectly downregulated the STING and cGAS genes. In contrast, ART did not influence IFN-α. The expression of STING and cGAS was not correlated with the plasma level of IFN-α, which suggests that there is another pathway regulating this cytokine in addition to the STING-cGAS pathway.

CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function.

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1ß/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Design, synthesis, in vitro and in silico studies of novel 4-oxoquinoline ribonucleoside derivatives as HIV-1 reverse transcriptase inhibitors.

Human immunodeficiency virus type 1 (HIV-1) is a public health problem that affects over 38 million people worldwide. Although there are highly active antiretroviral therapies, emergence of antiviral resistant strains is a problem which leads to almost a million death annually. Thus, the development of new drugs is necessary. The viral enzyme reverse transcriptase (RT) represents a validated therapeutic target. Because the oxoquinolinic scaffold has substantial biological activities, including antiretroviral, a new series of 4-oxoquinoline ribonucleoside derivatives obtained by molecular hybridization were studied here. All synthesized compounds were tested against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and 9a and 9d displayed the highest antiviral activities, with IC50 values of 1.4 and 1.6 µM, respectively. These compounds were less cytotoxic than AZT and showed CC50 values of 1486 and 1394 µM, respectively. Molecular docking studies showed that the most active compounds bound to the allosteric site of the enzyme, suggesting a low susceptibility to the development of antiviral resistance. In silico pharmacokinetic and toxicological evaluations reinforced the potential of the active compounds as anti-HIV candidates for further exploration. Overall, this work showed that compounds 9a and 9d are promising scaffold for future anti-HIV-1 RT drug design.

Association of IL-6, IL-10 and CXCL10 serum concentrations with visceral Kaposi's sarcoma in people living with HIV/AIDS.

Human gammaherpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), one of the most common cancers in people living with HIV/AIDS. It is believe that the course of both HIV and HHV-8 infection is associated with the imbalance of anti- and/or pro-inflammatory cytokines. Here, we evaluated the IL-6, TNF-α, IL-10, CCL2 and CXCL10 serum concentrations in HIV- and HIV/HHV-8 (without KS) individuals, and in patients with cutaneous or visceral AIDS-KS. Serum concentrations of IL-6, IL-10 and CXCL10 were significantly higher in the AIDS-KS group compared to HIV and HIV/HHV-8 individuals. Similarly, the concentrations of theses cytokines were higher in patients with visceral than in those with cutaneous AIDS-KS. The TNF-α concentration was significantly higher in the HIV group compared to HIV/HHV-8 (with and without KS) individuals, and CCL2 levels did not present significant difference among the groups. The HIV viral load was undetectable in all patients from the HIV and HIV/HHV-8 groups. On the other hand, in the AIDS-KS group, most patients had detectable HIV viral load. In this context, we believe that the cytokine levels in AIDS-KS may be result of a complex interaction between HIV, HHV-8 and immunity.

New Challenges of HIV-1 Infection: How HIV-1 Attacks and Resides in the Central Nervous System.

Acquired immunodeficiency syndrome (AIDS) has become one of the most devastating pandemics in recorded history. The main causal agent of AIDS is the human immunodeficiency virus (HIV), which infects various cell types of the immune system that express the CD4 receptor on their surfaces. Today, combined antiretroviral therapy (cART) is the standard treatment for all people with HIV; although it has improved the quality of life of people living with HIV (PLWH), it cannot eliminate the latent reservoir of the virus. Therefore HIV/AIDS has turned from a fatal disease to a chronic disease requiring lifelong treatment. Despite significant viral load suppression, it has been observed that at least half of patients under cART present HIV-associated neurocognitive disorders (HAND), which have been related to HIV-1 infection and replication in the central nervous system (CNS). Several studies have focused on elucidating the mechanism by which HIV-1 can invade the CNS and how it can generate the effects seen in HAND. This review summarizes the research on HIV-1 and its interaction with the CNS with an emphasis on the generation of HAND, how the virus enters the CNS, the relationship between HIV-1 and cells of the CNS, and the effect of cART on these cells.

Histidine-Rich Glycoprotein Inhibits HIV-1 Infection in a pH-Dependent Manner.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCE Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA.

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.

The alpha7-nicotinic receptor contributes to gp120-induced neurotoxicity: implications in HIV-associated neurocognitive disorders.

Currently, there are no specific therapies to treat HIV-1 associated neurocognitive disorders (HAND). The HIV-1 envelope, gp120, induces neuropathological changes similar to those in HAND patients; furthermore, it triggers an upregulation of the α7-nicotinic acetylcholine receptor (α7-nAChR), facilitating intracellular calcium overload and neuronal cell death. Using a gp120IIIB-transgenic mouse (gp120-tgm) model, we demonstrate that α7-nAChRs are upregulated on striatal neurons. Activation of α7-nAChRs leads to an increase in both intracellular calcium and percentage of apoptotic cells, which can be abrogated by antagonizing the receptor, suggesting a role for α7-nAChRs in gp120-induced neurotoxicity. Moreover, we demonstrate for the first time that gp120-tgm have learning deficiencies on a striatum-dependent behavioral task. They also show locomotor deficiencies, which improved with α7-nAChR antagonists, further supporting a role for this receptor in gp120-induced neurotoxicity. Together, these results uncover a new mechanism through which gp120-induced modulation of α7-nAChRs in the striatum can contribute to HAND development.

Pace of Coreceptor Tropism Switch in HIV-1-Infected Individuals after Recent Infection.

HIV-1 entry into target cells influences several aspects of HIV-1 pathogenesis, including viral tropism, HIV-1 transmission and disease progression, and response to entry inhibitors. The evolution from CCR5- to CXCR4-using strains in a given human host is still unpredictable. Here we analyzed timing and predictors for coreceptor evolution among recently HIV-1-infected individuals. Proviral DNA was longitudinally evaluated in 66 individuals using Geno2pheno[coreceptor] Demographics, viral load, CD4+ and CD8+ T cell counts, CCR5Δ32 polymorphisms, GB virus C (GBV-C) coinfection, and HLA profiles were also evaluated. Ultradeep sequencing was performed on initial samples from 11 selected individuals. A tropism switch from CCR5- to CXCR4-using strains was identified in 9/49 (18.4%) individuals. Only a low baseline false-positive rate (FPR) was found to be a significant tropism switch predictor. No minor CXCR4-using variants were identified in initial samples of 4 of 5 R5/non-R5 switchers. Logistic regression analysis showed that patients with an FPR of >40.6% at baseline presented a stable FPR over time whereas lower FPRs tend to progressively decay, leading to emergence of CXCR4-using strains, with a mean evolution time of 27.29 months (range, 8.90 to 64.62). An FPR threshold above 40.6% determined by logistic regression analysis may make it unnecessary to further determine tropism for prediction of disease progression related to emergence of X4 strains or use of CCR5 antagonists. The detection of variants with intermediate FPRs and progressive FPR decay over time not only strengthens the power of Geno2pheno in predicting HIV tropism but also indirectly confirms a continuous evolution from earlier R5 variants toward CXCR4-using strains.IMPORTANCE The introduction of CCR5 antagonists in the antiretroviral arsenal has sparked interest in coreceptors utilized by HIV-1. Despite concentrated efforts, viral and human host features predicting tropism switch are still poorly understood. Limited longitudinal data are available to assess the influence that these factors have on predicting tropism switch and disease progression. The present study describes longitudinal tropism evolution in a group of recently HIV-infected individuals to determine the prevalence and potential correlates of tropism switch. We demonstrated here that a low baseline FPR determined by the Geno2pheno[coreceptor] algorithm can predict tropism evolution from CCR5 to CXCR4 coreceptor use.

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking.

The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or µ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.