HIV-1 Gp120 Research Articles

Retraction: Comparative immunogenicity of HIV-1 gp160, gp140 and gp120 expressed by live attenuated newcastle disease virus vector.

Retraction: Comparative immunogenicity of HIV-1 gp160, gp140 and gp120 expressed by live attenuated newcastle disease virus vector.

The V2 loop of HIV gp120 delivers costimulatory signals to CD4+ T cells through Integrin α4β7 and promotes cellular activation and infection

Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4+ T cells. Trafficking of α4β7-expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α4β7 that is expressed on gut endothelial cells. MAdCAM signaling through α4β7 costimulates CD4+ T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α4β7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α4β7 resulting in CD4+ T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α4β7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α4β7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis.

HIV-1 gp120 enhances Cryptococcus neoformans-mediated blood-brain barrier disruption in an in vitro human brain microvascular endothelial cell model

HIV-1 gp120 enhances Cryptococcus neoformans-mediated blood-brain barrier disruption in an in vitro human brain microvascular endothelial cell model

Possible transmission of HIV infection due to human bite

Background: The potential risk of HIV-1 infection following human bite although epidemiologically insignificant, but it is biologically possible. There are anecdotal reports of HIV transmission by human bites particularly if saliva is mixed with blood. The oral tissues support HIV replication and may serve as a previously unrecognized HIV reservoir. The case presented here is of a primary HIV infections following a human bite where in the saliva was not blood stained but it got smeared on a raw nail bed of a recipient. Case description: The samples were analyzed for confirmation of HIV DNA by amplifying C2-V3 Region of HIV1C envelope gene and further confirmed by PCR and sequencing. PBMCs was isolated by Ficoll-hypaque method. DNA extraction by Qiagen DNA mini kit. The C2-V3 region of env gene was amplified by nested PCR by heteroduplex mobility assay using primers obtained from NIH AIDS Research and Reference Reagent Programme. The virological and immunological parameters were measured The HIV-1 viral load in the blood and saliva was estimated using the MagNa pure Compact Nucleic Acid Automated System (Roche) with Cobas TaqMan 48 Real time PCR. Discussion: The C2-V3 region of second round product was sequenced. Sequence revealed 91% homology between Mr. A and Mr. X The presence of single NLG site at V3 region of HIV 1C env gene showed in both the individuals suggesting the possible usages of CCR5 tropic virus. The blood and saliva of the source and blood of the recipient showed a detectable viral load with 91% sequence homology of C2-V3 region of HIV gp120 between the two individuals. Conclusion: The recipient did not receive PEP as his family physician was unaware of salivary transmission. Our observations revealed transmission of HIV infection from the smear of non-contaminated saliva with blood potentially transmit the virus to conclude, the family physician should have taken PEP decision after proper evaluation of the severe and bleeding bite. Hence it is necessary to treat the HIV infected human bites with post exposure prophylaxis.

Assessing donor suitability for blood donation: Utility of Geenius HIV 1/2 confirmatory assay

BackgroundBlood donor care and blood safety require a quick and accurate decision on the presence or absence of Human Immunodeficiency Virus (HIV) infection, based on the proper selection of blood donors, serological and molecular HIV testing as well as western blot test. The aim was investigating the possibility of inclusion of Geenius HIV 1/2 Confirmatory Assay in blood donor testing algorithm in order to shorten test time and decrease the number of indeterminate results. MethodsA total of 75 archived serum/plasma samples were tested. Their previous serological and molecular HIV results were: 3 negative samples, 7 positive samples, 65 serological indeterminate or positive but confirmatory testing and NAT negative samples. ResultsGeenius assay confirmed the presence of antibodies in all blood donors with HIV positive serology and Nucleic Acid Testing (NAT). HIV-1 gp160 and gp41 antibodies were detected in these donors, while p31 and p24 antibodies were not detected in two and three donors, respectively. HIV-2 antibodies gp36 and gp140 were not found. Blood donor with HIV indeterminate or positive serology but negative confirmatory testing and NAT, were negative in Geenius assay.Conclusion The results obtained are consistent with western blot results. The assay proved simple and quick to perform. Studies have confirmed the possibility of introducing Bio-Rad Geenius into a routine blood donor testing protocol.

HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China

BackgroundHuman immunodeficiency virus (HIV) increasing molecular diversity and emergence of drug resistant mutants remain a major concern in China. Enfuvirtide (ENF/T-20) is the first entry inhibitor used in patients failing highly active antiretroviral therapy (HAART). However, data on HIV-1 gp41genetic diversity and primary ENF resistance-associated mutations among treatment-naïve patients in China is limited. The objective was to identify molecular diversity and ENF resistance patterns of HIV-1 in southern China, using envelope (env) gp41 sequences and bioinformatics tools, which may help optimize antiretroviral therapy. MethodsFrom November 2018 to January 2019, 439 blood plasma samples from ENF-naïve patients were collected from Shenzhen, Wuhan and Chongqing, of which 396 HIV env regions were sequenced and subtyped, and were performed the analysis of drug resistance-associated mutations (DRMs). ResultsThe main subtypes were circulating recombinant form (CRF) 01_AE (30.6 %) and CRF07_BC (48.7 %). CRF55_01B had been the fourth subtype in the study, and many rare CRFs were observed. Notably, CRF02_AG and CRF_BF strains typically found in Africa and US respectively were identified amongst Chinese patients. Known DRMs were detected in 27.5 % (109/396) of ENF treatment-naïve patients. One major DRM (L44 M), many secondary DRMs (including N126 K, E137 K, S138A), and lots of polymorphisms were found in the study, which have been proved to elevate resistance to ENF. ConclusionsHIV-1 molecular diversity was observed in the study, which indicating that HIV-1 variability is becoming increasingly complex in southern China. A diverse set of primary DRMs discovered in this study described the serious threat to ART, which reminds us the urgent need of timely surveillance of HIV-1 viral diversity and drug resistance in China.

Concentration-Dependent Structural Transition of the HIV-1 gp41 MPER Peptide into α-Helical Trimers.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

Neutralizing Antibodies Targeting HIV-1 gp41.

HIV-1 vaccine research has obtained an enormous boost since the discovery of many broadly neutralizing antibodies (bnAbs) targeting all accessible sites on the HIV-1 envelope glycoprotein (Env). This in turn facilitated high-resolution structures of the Env glycoprotein in complex with bnAbs. Here we focus on gp41, its highly conserved heptad repeat region 1 (HR1), the fusion peptide (FP) and the membrane-proximal external region (MPER). Notably, the broadest neutralizing antibodies target MPER. Both gp41 HR1 and MPER are only fully accessible once receptor-induced conformational changes have taken place, although some studies suggest access to MPER in the close to native Env conformation. We summarize the data on the structure and function of neutralizing antibodies targeting gp41 HR1, FP and MPER and we review their access to Env and their complex formation with gp41 HR1, MPER peptides and FP within native Env. We further discuss MPER bnAb binding to lipids and the role of somatic mutations in recognizing a bipartite epitope composed of the conserved MPER sequence and membrane components. The problematic of gp41 HR1 access and MPER bnAb auto- and polyreactivity is developed in the light of inducing such antibodies by vaccination.

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.

Antibody Neutralization of HIV-1 Crossing the Blood-Brain Barrier.

HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. Here, we examined whether antibodies targeting the HIV-1 envelope glycoproteins interfere with the transcytosis of virions across the human BBB endothelium. We found that although the viral envelope spike gp160 is required for optimal endothelial cell endocytosis, no anti-gp160 antibodies blocked the BBB transcytosis of HIV-1 in vitro Instead, both free viruses and those in complex with antibodies transited across endothelial cells in the BBB model, as observed by confocal microscopy. HIV-1 infectious capacity was considerably altered by the transcytosis process but still detectable, even in the presence of nonneutralizing antibodies. Only virions bound by neutralizing antibodies lacked posttranscytosis infectivity. Overall, our data support the role of neutralizing antibodies in protecting susceptible brain cells from HIV-1 infection despite their inability to inhibit viral BBB endocytic transport.IMPORTANCE HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. The HIV-1 envelope spike gp160 is partially required for viral transcytosis across the BBB endothelium. But do antibodies developing in infected individuals and targeting the HIV-1 gp160 glycoproteins block HIV-1 transcytosis through the BBB? We addressed this issue and discovered that anti-gp160 antibodies do not block HIV-1 transport; instead, free viruses and those in complex with antibodies can transit across BBB endothelial cells. Importantly, we found that only neutralizing antibodies could inhibit posttranscytosis viral infectivity, highlighting their ability to protect susceptible brain cells from HIV-1 infection.

DNA adjuvant Amiloride conjunct long immunization interval promote higher antibody responses to HIV-1 gp41 and gp140 immunogens

Recent studies have revealed that the interface of gp120 and gp41 and some parts of gp41 are also critical epitopes for elicitation of broadly neutralizing antibodies. Therefore, potential trimeric gp41 or gp140 immunogen candidates are needed. Previously, we developed a trimer motif MTQ and demonstrated that it could help formation of trimeric gp120 and gp140 proteins. In the present study, we immunized Balb/c mice using trimeric gp41-expressing plasmid for prime and monomeric gp41 or trimeric gp140 protein as well as a mutant (Q577A) for boost. The antibody responses in the context of regimens with various immunization intervals and DNA adjuvants including praziquantel (PZQ), cimetidine (CIM), and amiloride (AML) were evaluated. We found that these three adjuvants were not enough to elicit remarkable specific Abs after gp41 DNA immunization, while AML could significantly promote humoral immune responses after protein boosts. Long immunization interval could induce the specific binding Abs earlier and higher and maintain a high level of Abs in the following 27 weeks after final protein boost. Moreover, two times of protein boosts with DNA adjuvant and a longer time interval achieved a higher titer of specific Abs than three times of protein boosts with a shorter time interval. Q577A mutant was benefit for trimeric gp140 boost in the production of binding Abs but harmful to inducing neutralizing Abs, while this mutant in monomeric gp41 presented the opposite trend which may be associated with the immunogen structures. This study highlights the significance of DNA adjuvant Amiloride and long immunization interval in promoting antibody responses and provides new insights into effective HIV immunization regimen design in the future.

Human Immunodeficiency Virus Type 1 and Methamphetamine-Mediated Mitochondrial Damage and Neuronal Degeneration in Human Neurons.

Methamphetamine, a potent psychostimulant, is a highly addictive drug commonly used by persons living with HIV (PLWH), and its use can result in cognitive impairment and memory deficits long after its use is discontinued. Although the mechanism(s) involved with persistent neurological deficits is not fully known, mitochondrial dysfunction is a key component in methamphetamine neuropathology. Specific mitochondrial autophagy (mitophagy) and mitochondrial fusion and fission are protective quality control mechanisms that can be dysregulated in HIV infection, and the use of methamphetamine can further negatively affect these protective cellular mechanisms. Here, we observed that treatment of human primary neurons (HPNs) with methamphetamine and HIV gp120 and Tat increase dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and neuronal degeneration. Methamphetamine and HIV proteins increased microtubule-associated protein 1 light chain 3 beta-II (LC3B-II) lipidation and induced sequestosome 1 (SQSTM1, p62) translocation to damaged mitochondria. Additionally, the combination inhibited autophagic flux, increased reactive oxygen species (ROS) production and mitochondrial damage, and reduced microtubule-associated protein 2 (MAP2) dendrites in human neurons. N-Acetylcysteine (NAC), a strong antioxidant and ROS scavenger, abrogated DRP1-dependent mitochondrial fragmentation and neurite degeneration. Thus, we show that methamphetamine combined with HIV proteins inhibits mitophagy and induces neuronal damage, and NAC reverses these deleterious effects on mitochondrial function.IMPORTANCE Human and animal studies show that HIV infection, combined with the long-term use of psychostimulants, increases neuronal stress and the occurrence of HIV-associated neurocognitive disorders (HAND). On the cellular level, mitochondrial function is critical for neuronal health. In this study, we show that in human primary neurons, the combination of HIV proteins and methamphetamine increases oxidative stress, DRP1-mediated mitochondrial fragmentation, and neuronal injury manifested by a reduction in neuronal network and connectivity. The use of NAC, a potent antioxidant, reversed the neurotoxic effects of HIV and methamphetamine, suggesting a novel approach to ameliorate the effects of HIV- and methamphetamine-associated cognitive deficits.

Roles of variable linker length in dual acting virucidal entry inhibitors on HIV-1 potency via on-the-fly free energy molecular simulations.

The Dual-Acting Virolytic Entry Inhibitors, or DAVEI's, are a class of recombinant chimera fusion proteins consisting of a lectin, a flexible polypeptide linker, and a fragment of the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEIs trigger virolysis of HIV-1 virions through interactions with the trimeric envelope glycoprotein complex (Env), though the details of these interactions are not fully determined as yet. The purpose of this work was to use structural modeling to rationalize a dependence of DAVEI potency on the molecular length of the linker connecting the two components. We used temperature accelerated molecular dynamics and on-the-fly parameterization to compute free energy versus end-to-end distance for two different linker lengths, DAVEI L0 (His6 ) and DAVEI L2 ([Gly4 Ser]2 His6 ). Additionally, an envelope model was created based on a cryo-electron microscopy-derived structure of a cleaved, soluble Env construct, with high-mannose glycans added which served as putative docking locations for the lectin, along with MPER added that served as a putative docking location for the MPER region of DAVEI (MPERDAVEI ). Using MD simulation, distances between the lectin C-terminus and Env gp41 MPER were measured. We determined that none of the glycans were close enough to gp41 MPER to allow DAVEI L0 to function, while one, N448, will allow DAVEI L2 to function. These findings are consistent with the previously determined dependence of lytic function on DAVEI linker lengths. This supports the hypothesis that DAVEI's engage Env at both glycans and the Env MPER in causing membrane poration and lysis.

Small molecule modulation of the p75 neurotrophin receptor suppresses age- and genotype-associated neurodegeneration in HIV gp120 transgenic mice

The persistence of HIV in the central nervous system leads to cognitive deficits in up to 50% of people living with HIV even with systemic suppression by antiretroviral treatment. The interaction of chronic inflammation with age-associated degeneration places these individuals at increased risk of accelerated aging and other neurodegenerative diseases and no treatments are available that effectively halt these processes. The adverse effects of aging and inflammation may be mediated, in part, by an increase in the expression of the p75 neurotrophin receptor (p75NTR) which shifts the balance of neurotrophin signaling toward less protective pathways. To determine if modulation of p75NTR could modify the disease process, we treated HIV gp120 transgenic mice with a small molecule ligand designed to engage p75NTR and downregulate degenerative signaling. Daily treatment with 50 mg/kg LM11A-31 for 4 months suppressed age- and genotype-dependent activation of microglia, increased microtubule associated protein-2 (MAP-2), reduced dendritic varicosities and slowed the loss of parvalbumin immunoreactive neurons in the hippocampus. An age related accumulation of microtubule associated protein Tau was identified in the hippocampus in extracellular clusters that co-expressed p75NTR suggesting a link between Tau and p75NTR. Although the significance of the relationship between p75NTR and Tau is unclear, a decrease in Tau-1 immunoreactivity as gp120 mice entered old age (>16 months) suggests that the Tau may transition to more pathological modifications; a process blocked by LM11A-31. Overall, the effects of LM11A-31 are consistent with strong neuroprotective and anti-inflammatory actions that have significant therapeutic potential.

Single Cell RNA-Seq Analysis Reveals Compartment-Specific Heterogeneity and Plasticity of Microglia

Microglia are ubiquitous CNS-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single cell RNA-seq (scRNA-seq) to compare microglial subtypes in the cortex and the spinal cord. A multi-way comparative analysis was carried out on samples from C57/BL and HIV gp120 transgenic mice at two, four, and eight months of age. The results revealed overlapping but distinct microglial populations in the cortex and the spinal cord. The differential heterogeneity of microglia in these CNS regions was further suggested by their disparity of plasticity in response to life span progression and HIV-1 pathogenic protein gp120. Our findings indicate that microglia in different CNS compartments are adapted to their local environments to fulfil region-specific biological functions.

Selection and immune recognition of HIV-1 MPER mimotopes

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by several neutralizing antibodies (NAbs) and is of interest for vaccine design. In this study, we identified novel MPER peptide mimotopes and evaluated their reactivity with HIV + plasma antibodies to characterize the diversity of the immune responses to MPER during natural infection. We utilized phage display technology to generate novel mimotopes that fit antigen-binding sites of MPER NAbs 4E10, 2F5 and Z13. Plasma antibodies from 10 HIV + patients were mapped by phage immunoprecipitation, to identify unique patient MPER binding profiles that were distinct from, and overlapping with, those of MPER NAbs. 4E10 mimotope binding profiles correlated with plasma neutralization of HIV-2/HIV-1 MPER chimeric virus, and with overall plasma neutralization breadth and potency. When administered as vaccines, 4E10 mimotopes elicited low titer NAb responses in mice. HIV mimotopes may be useful for detailed analysis of plasma antibody specificity.

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8

Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.

Establishment of a gp120 transgenic mouse model with α7 nAChR knockout

To construct a HIV-1 gp120 transgenic mouse model (gp120+) with α7 nicotinic acetylcholine receptor (α7nAChR) gene knockout. The α7nAChR gene knockout mice (α7R-/-) were crossed with HIV-1gp120 transgenic mice (gp120+) to generate F1 generation mice. We selected the F1 mice with the genotype of α7R+/-/gp120+ to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and α7nAChR inhibitor, and the expressions of IL-1β and TNF-α were detected using ELISA. The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (α7R-/-/gp120+) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express α7 nAChR but with high gp120 protein expression. In the in vitro cell experiment, treatment with gp120 promoted the secretion of IL-1β and TNF-α in BV2 cells, while inhibition of α7nAChR significantly decreased the expression of IL-1β and TNF-α (P < 0.001). By mating gp120 Tg mice with α7R-/- mice, we obtained gp120 transgenic mice with α7nAChR gene deletion, which serve as a new animal model for exploring the role of α7nAChR in gp120-induced neurotoxicity.

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis.

HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens.IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

HIV X4 Variants Increase Arachidonate 5-Lipoxygenase in the Pulmonary Microenvironment and are associated with Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH.