Human Immunodeficiency Virus Glycoprotein Research Articles

Sirtuin 3 Mediated by Spinal cMyc-Enhancer of Zeste Homology 2 Pathway Plays an Important Role in Human Immunodeficiency Virus-Related Neuropathic Pain Model.

Clinical data demonstrate that chronic use of opioid analgesics increases neuropathic pain in people living with human immunodeficiency virus (HIV). Therefore, it is important to elucidate the molecular mechanisms of HIV-related chronic pain. In this study, we investigated the role of the transcription factor cMyc, epigenetic writer enhancer of zeste homology 2 (EZH2), and sirtuin 3 (Sirt3) pathway in HIV glycoprotein gp120 with morphine (gp120M)-induced neuropathic pain in rats. Neuropathic pain was induced by intrathecal administration of recombinant gp120 with morphine. Mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. Spinal expression of cMyc, EZH2, and Sirt3 were measured using Western blots. Antinociceptive effects of intrathecal administration of antisense oligodeoxynucleotide against cMyc, a selective inhibitor of EZH2, or recombinant Sirt3 were tested. In the spinal dorsal horn, gp120M upregulated expression of cMyc (ratio of gp120M versus control, 1.68 ± 0.08 vs 1.00 ± 0.14, P = .0132) and EZH2 (ratio of gp120M versus control, 1.76 ± 0.05 vs 1.00 ± 0.16, P = .006), and downregulated Sirt3 (ratio of control versus gp120M, 1.00 ± 0.13 vs 0.43 ± 0.10, P = .0069) compared to control. Treatment with intrathecal antisense oligodeoxynucleotide against cMyc, GSK126 (EZH2 selective inhibitor), or recombinant Sirt3 reduced mechanical allodynia and thermal hyperalgesia in this gp120M pain model. Knockdown of cMyc reduced spinal EZH2 expression in gp120M treated rats. Chromatin immunoprecipitation (ChIP) assay showed that enrichment of cMyc binding to the ezh2 gene promoter region was increased in the gp120M-treated rat spinal dorsal horn, and that intrathecal administration of antisense ODN against cMyc (AS-cMyc) reversed the increased enrichment of cMyc. Enrichment of trimethylation of histone 3 on lysine residue 27 (H3K27me3; an epigenetic mark associated with the downregulation of gene expression) binding to the sirt3 gene promoter region was upregulated in the gp120M-treated rat spinal dorsal horn; that intrathecal GSK126 reversed the increased enrichment of H3K27me3 in the sirt3 gene promoter. Luciferase reporter assay demonstrated that cMyc mediated ezh2 gene transcription at the ezh2 gene promoter region, and that H3K27me3 silenced sirt3 gene transcription at the gene promoter region. These results demonstrated that spinal Sirt3 decrease in gp120M-induced neuropathic pain was mediated by cMyc-EZH2/H3K27me3 activity in an epigenetic manner. This study provided new insight into the mechanisms of neuropathic pain in HIV patients with chronic opioids.

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices.

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high affinity phage-displayed antibodies against virus glycoproteins, including Human Immunodeficiency Virus-1 (HIV-1) glycoprotein (gp120) or the Ebola Virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high affinity phages were recovered at channels proximal to the application site, whereas low affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high affinity ligands displayed in phages. This article is protected by copyright. All rights reserved.

Identification of human immunodeficiency virus -1 E protein-targeting lead compounds by pharmacophore based screening

To identify potential compounds by seeking the knowledge of molecular interactions between human immunodeficiency virus (HIV) glycoprotein (gp) 120 protein and anti-HIV drug (BMS-488043). This study is a computational structure-based drug design study, carried out at University of Taif, Saudi Arabia and African Genome Centre (AGC), Mohammed VI Polytechnic University, Benguerir, Morocco from January 2021 to March 2022. Initially, using the docked structure of gp120 with BMS-488043, a structure-based pharmacophore model was created. The generated model was utilized for virtual screening of the ZINC and ChemBridge database in order to identify hit compounds. To further assess the time-dependent stability of the selected complexes, computer simulation was performed. From pharmacophore-based screening, 356 hits were obtained from both the database. The docking studies of the retrieved hit compounds reveal that all the compounds fit into the binding site of gp120. However, based on the significant interactions with the crucial residues and docking scores four compounds were suggested as potential hits. MD simulation of ChemBridge14695864 and ZINC06893293 in complex with gp120 suggested that both compounds significantly stabilized the receptor. These findings could aid in the design of effective drugs against HIV by inhibiting the interaction between gp120 and CD4.

ANALYSIS OF HUMAN IMMUNODEFICIENCY VIRUS GLYCOPROTEIN 160 AS A VACCINE CANDIDATE FOR B CELL EPITOPE OF ACQUIRED IMMUNODEFICIENCY SYNDROME

An in silico study was conducted to analyze the potential of Human Immunodeficiency Virus (HIV) glycoprotein 160 as a candidate source of B cell subunit (epitope) vaccine for Acquired Immunodeficiency Syndrome (AIDS). The method used was immunoinformatics. The tools used were a set of laptop device, uniprot application, IEDB AR application and ToxinPred application. The materials used were glycoprotein sequences. The analysis results showed that there were 4 candidate peptides for B cell subunit (epitope) vaccines for AIDS, namely MRVMGIQ (Methionine, Arginine, Valine, Methionine, Glycine, Isoleucine, Glutamine, NMWKNNM (Asparagine, Methionine, Tryptophan, Lysine, Asparagine, Asparagine, Methionine), EIMGDIR (Glutamate, Isoleucine, Methionine, Glycine, Aspartic Acid, Isoleucine, and Arginine) and MWQRVGQ (Methionine, Tryptophan, Glutamine, Arginine, Valine, Glycine and Glutamine). All peptides are non-toxic so they are safe for the body

Japanese Kampo Medicine Juzentaihoto Improves Antiviral Cellular Immunity in Tumour-Bearing Hosts.

Global and antigen-independent immunosuppression by growing tumours can cause life-threating damage when concurrent with an infection in tumour-bearing hosts. In the present study, we investigated whether the oral administration of the Japanese traditional herbal (Kampo) medicine, juzentaihoto (JTT), plays a role in the improvement of antiviral cellular immunity in tumour-bearing hosts. Female BALB/c mice subcutaneously injected with murine colorectal cancer CT26 cells fed a control or JTT diet were inoculated with recombinant vaccinia virus expressing human immunodeficiency virus-1 glycoprotein 160 (vSC25). At 7 days postinfection, anti-vSC25 cellular immunity was evaluated by measuring the abundance of splenic virus-specific CD8+ T cells. JTT had no impact on CT26 tumour growth in vivo. Surprisingly, JTT augmented anti-vSC25 cellular immunity in CT26-bearing mice. Depletion of either CD25+ regulatory T (Treg) cells or myeloid-derived suppressor cells (MDSCs) also enhanced anti-vSC25 cellular immunity in tumour-bearing mice but had no therapeutic benefit against tumour growth. However, JTT had no impact on the abundance of these immunosuppressive cells. Overall, our data indicates that JTT contributes to the improvement of anti-vSC25 cellular immunity in tumour-bearing hosts possibly via a mechanism independent of CD25+ Treg cells and MDSCs, suggesting that this Kampo medicine can act as a promising antiviral adjuvant in an immunosuppressive state caused by tumours.

HIV and FIV glycoproteins increase cellular tau pathology via cGMP-dependent kinase II activation.

As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically improves the lifespan of individuals with HIV, many are now entering the prime age when Alzheimer's disease (AD)-like symptoms begin to manifest. It has been shown that hyperphosphorylated tau, a known AD pathological characteristic, is prematurely increased in the brains of HIV-infected individuals as early as in their 30s and that its levels increase with age. This suggests that HIV infection might lead to accelerated AD phenotypes. However, whether HIV infection causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have previously revealed that the viral glycoproteins HIV gp120 and feline immunodeficiency virus (FIV) gp95 induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII; also known as PRKG2) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons to demonstrate that the presence of HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Taken together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.

Unsaturated Lipid Accelerates Formation of Oligomeric β-Sheet Structure of GP41 Fusion Peptide in Model Cell Membrane.

Membrane fusion of the viral and host cell membranes is the initial step of virus infection and is catalyzed by fusion peptides. Although the β-sheet structure of fusion peptides has been proposed to be the most important fusion-active conformation, it is still very challenging to experimentally identify different types of β-sheet structures at the cell membrane surface in situ and in real time. In this work, we demonstrate that the interface-sensitive amide II spectral signals of protein backbones, generated by the sum frequency generation vibrational spectroscopy, provide a sensitive probe for directly capturing the formation of oligomeric β-sheet structure of fusion peptides. Using human immunodeficiency virus (HIV) glycoprotein GP41 fusing peptide (FP23) as the model, we find that formation speed of oligomeric β-sheet structure depends on lipid unsaturation. The unsaturated lipid such as POPG can accelerate formation of oligomeric β-sheet structure of FP23. The β-sheet structure is more deeply inserted into the hydrophobic region of the POPG bilayer than the α-helical segment. This work will pave the way for future researches on capturing intermediate structures during membrane fusion processes and revealing the fusion mechanism.

Ryanodine Receptor to Mitochondrial Reactive Oxygen Species Pathway Plays an Important Role in Chronic Human Immunodeficiency Virus gp120MN-Induced Neuropathic Pain in Rats.

Chronic pain is one of the most common complaints in patients with human immunodeficiency virus (HIV)-associated sensory neuropathy. Ryanodine receptor (RyR) and mitochondrial oxidative stress are involved in neuropathic pain induced by nerve injury. Here, we investigated the role of RyR and mitochondrial superoxide in neuropathic pain induced by repeated intrathecal HIV glycoprotein 120 (gp120) injection. Recombinant HIV glycoprotein gp120MN was intrathecally administered to induce neuropathic pain. Mechanical threshold was tested using von Frey filaments. Peripheral nerve fiber was assessed by the quantification of the intraepidermal nerve fiber density in the skin of the hindpaw. The expression of spinal RyR was examined using Western blots. Colocalization of RyR with neuronal nuclei (NeuN; neuron marker), glial fibrillary acidic protein (GFAP; astrocyte marker), or ionizing calcium-binding adaptor molecule 1 (Iba1; microglia marker) in the spinal cord was examined using immunohistochemistry. MitoSox-positive profiles (a mitochondrial-targeted fluorescent superoxide indicator) were examined. The antiallodynic effects of intrathecal administration of RyR antagonist, dantrolene (a clinical drug for malignant hyperthermia management), or selective mitochondrial superoxide scavenger, Mito-Tempol, were evaluated in the model. We found that repeated but not single intrathecal injection of recombinant protein gp120 induced persistent mechanical allodynia. Intraepidermal nerve fibers in repeated gp120 group was lower than that in sham at 2 weeks, and the difference in means (95% confidence interval) was 8.495 (4.79-12.20), P = .0014. Repeated gp120 increased expression of RyR, and the difference in means (95% confidence interval) was 1.50 (0.504-2.495), P = .007. Repeated gp120 also increased mitochondrial superoxide cell number in the spinal cord, and the difference in means (95% confidence interval) was 6.99 (5.99-8.00), P < .0001. Inhibition of spinal RyR or selective mitochondrial superoxide scavenger dose dependently reduced mechanical allodynia induced by repeated gp120 injection. RyR and mitochondrial superoxide were colocalized in the neuron, but not glia. Intrathecal injection of RyR inhibitor lowered mitochondrial superoxide in the spinal cord dorsal horn in the gp120 neuropathic pain model. These data suggest that repeated intrathecal HIV gp120 injection induced an acute to chronic pain translation in rats, and that neuronal RyR and mitochondrial superoxide in the spinal cord dorsal horn played an important role in the HIV neuropathic pain model. The current results provide evidence for a novel approach to understanding the molecular mechanisms of HIV chronic pain and treating chronic pain in patients with HIV.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vector pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High Five™ insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after immunizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90% and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune responses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and immunogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates. Key words: Human immunodeficiency virus-1; Insect cell expression system; Glycoprotein; gp120; Immune response

Methamphetamine potentiates HIV-1gp120-induced microglial neurotoxic activity by enhancing microglial outward K+ current.

Methamphetamine (Meth) abuse not only increases the risk of human immunodeficiency virus-1 (HIV-1) infection, but exacerbates HIV-1-associated neurocognitive disorders (HAND) as well. The mechanisms underlying the co-morbid effect are not fully understood. Meth and HIV-1 each alone interacts with microglia and microglia express voltage-gated potassium (KV) channel KV1.3. To understand whether KV1.3 functions an intersecting point for Meth and HIV-1, we studied the augment effect of Meth on HIV-1 glycoprotein 120 (gp120)-induced neurotoxic activity in cultured rat microglial cells. While Meth and gp120 each alone at low (subtoxic) concentrations failed to trigger microglial neurotoxic activity, Meth potentiated gp120-induced microglial neurotoxicity when applied in combination. Meth enhances gp120 effect on microglia by enhancing microglial KV1.3 protein expression and KV1.3 current, leading to an increase of neurotoxin production and resultant neuronal injury. Pretreatment of microglia with a specific KV1.3 antagonist 5-(4-Phenoxybutoxy)psoralen (PAP) or a broad spectrum KV channel blocker 4-aminopyridine (4-AP) significantly attenuated Meth/gp120-treated microglial production of neurotoxins and resultant neuronal injury, indicating an involvement of KV1.3 in Meth/gp120-induced microglial neurotoxic activity. Meth/gp120 activated caspase-3 and increased caspase-3/7 activity in microglia and inhibition of caspase-3 by its specific inhibitor significantly decreased microglial production of TNF-α and iNOS and attenuated microglia-associated neurotoxic activity. Moreover, blockage of KV1.3 by specific blockers attenuated Meth/gp120 enhancement of caspase-3/7 activity. Taking together, these results suggest an involvement of microglial KV1.3 in the mediation of Meth/gp120 co-morbid effect on microglial neurotoxic activity via caspase-3 signaling.

Identification of interaction between HIV-1 glycoprotein 41 and integrase.

Human immunodeficiency virus-1 (HIV-1) encodes 15 viral proteins. Protein-protein interactions play a large role in the function of these proteins. In this study, we attempted to identify novel interactions between the HIV-1 proteins to better understand the role played by viral protein-protein interactions in the life cycle of HIV-1. Genes encoding the 15 viral proteins from the HIV-1 strain AD8 were inserted into the plasmids of a yeast two-hybrid system. By screening 120 pairs of proteins, interactions between seven pairs were found. This led to the discovery of an interaction between the HIV-1 proteins integrase (IN) and glycoprotein 41 (gp41), which was confirmed by both co-immunoprecipitation (Co-IP) assays and fluorescence resonance energy transfer (FRET) imaging in live cells. In addition, it was found that the amino acids at positions 76-100 of gp41 are required for it to bind to IN. Deletion of this region from gp41 prevented its interaction with IN and reduced the production of HIV-1 in 293T cells. This study provides new information on HIV-1 protein-protein interactions which improves the understanding of the biological functions of gp41 and IN during the virus life cycle.

An HIV gp120-CD4 Immunogen Does Not Elicit Autoimmune Antibody Responses in Cynomolgus Macaques.

A promising concept for human immunodeficiency virus (HIV) vaccines focuses immunity on the highly conserved transition state structures and epitopes that appear when the HIV glycoprotein gp120 binds to its receptor, CD4. We are developing chimeric antigens (full-length single chain, or FLSC) in which gp120 and CD4 sequences are flexibly linked to allow stable intrachain complex formation between the two moieties (A. DeVico et al., Proc Natl Acad Sci U S A 104:17477-17482, 2007, doi:10.1073/pnas.0707399104; T. R. Fouts et al., J Virol 74:11427-11436, 2000, doi:10.1128/JVI.74.24.11427-11436.2000). Proof of concept studies with nonhuman primates show that FLSC elicited heterologous protection against simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) (T. R. Fouts et al., Proc Natl Acad Sci U S A 112:E992-E999, 2016, doi:10.1073/pnas.1423669112), which correlated with antibodies against transition state gp120 epitopes. Nevertheless, advancement of any vaccine that comprises gp120-CD4 complexes must consider whether the CD4 component breaks tolerance and becomes immunogenic in the autologous host. To address this, we performed an immunotoxicology study with cynomolgus macaques vaccinated with either FLSC or a rhesus variant of FLSC containing macaque CD4 sequences (rhFLSC). Enzyme-linked immunosorbent assay (ELISA) binding titers, primary CD3(+) T cell staining, and temporal trends in T cell subset frequencies served to assess whether anti-CD4 autoantibody responses were elicited by vaccination. We find that immunization with multiple high doses of rhFLSC did not elicit detectable antibody titers despite robust responses to rhFLSC. In accordance with these findings, immunized animals had no changes in circulating CD4(+) T cell counts or evidence of autoantibody reactivity with cell surface CD4 on primary naive macaque T cells. Collectively, these studies show that antigens using CD4 sequences to stabilize transition state gp120 structures are unlikely to elicit autoimmune antibody responses, supporting the advancement of gp120-CD4 complex-based antigens, such as FLSC, into clinical testing.

Antibody recognition of carbohydrate epitopes†.

Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.

Alteration of the Langerin Oligomerization State Affects Birbeck Granule Formation

Langerin, a trimeric C-type lectin specifically expressed in Langerhans cells, has been reported to be a pathogen receptor through the recognition of glycan motifs by its three carbohydrate recognition domains (CRD). In the context of HIV-1 (human immunodeficiency virus-1) transmission, Langerhans cells of genital mucosa play a protective role by internalizing virions in Birbeck Granules (BG) for elimination. Langerin (Lg) is directly involved in virion binding and BG formation through its CRDs. However, nothing is known regarding the mechanism of langerin assembly underlying BG formation. We investigated at the molecular level the impact of two CRD mutations, W264R and F241L, on langerin structure, function, and BG assembly using a combination of biochemical and biophysical approaches. Although the W264R mutation causes CRD global unfolding, the F241L mutation does not affect the overall structure and gp120 (surface HIV-1 glycoprotein of 120 kDa) binding capacities of isolated Lg-CRD. In contrast, this mutation induces major functional and structural alterations of the whole trimeric langerin extracellular domain (Lg-ECD). As demonstrated by small-angle x-ray scattering comparative analysis of wild-type and mutant forms, the F241L mutation perturbs the oligomerization state and the global architecture of Lg-ECD. Correlatively, despite conserved intrinsic lectin activity of the CRD, avidity property of Lg-ECD is affected as shown by a marked decrease of gp120 binding. Beyond the change of residue itself, the F241L mutation induces relocation of the K200 side chain also located within the interface between protomers of trimeric Lg-ECD, thereby explaining the defective oligomerization of mutant Lg. We conclude that not only functional CRDs but also their correct spatial presentation are critical for BG formation as well as gp120 binding.

Hyper IgE, Dermatitis and Staphylococcus aureus in an HIV-Positive Man

A variety of mucocutaneous disorders are seen in patients infected with Human immunodeficiency virus (HIV) [1]. While some of these conditions like Kaposi’s sarcoma are considered acquired immunodeficiency syndrome (AIDS)-defining illnesses, most HIV patients suffer from common infectious and inflammatory skin disorders like folliculitis and psoriasis [2]. Psoriasis in HIV patients presents more severely and is associated with a worse outcome [3]. The skin cytokine expression in HIV patients is altered [4] and elevated levels of IgE, a hallmark of atopic disorders, were observed in HIVinfected adults and children [5-7]. Studies have shown that HIV glycoproteins can act as viral superantigens and contribute to the release of IgE and histamine, leading to a shift of the T cell response towards the Th2 phenotype [8]. The skewing towards Th2 cytokines is thought to predispose HIV-infected patients to atopic dermatitis and account for the increased incidence of atopic dermatitis observed in HIV patients [2].

Report of Rare Urachal Carcinoma Presenting with Thrombocytopenia and Microangiopathic Hemolytic Anemia

Introduction:Thrombotic microangiopathy (TMA) is clinical syndrome characterized by microvascular thrombosis, consumptive thrombocytopenia and microangiopathic hemolytic anemia (MAHA). It is a feature of a number of clinical conditions, most prominently thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) and rarely an initial manifestation of systemic malignancy.Case:A previously healthy 31-year-old black woman presented to the emergency department with a 2 week history of lower back pain and hematuria and 1 week history of dyspnea on exertion and dizziness. She denied diarrhea, hematochezia, fever, or abdominal pain. Physical findings were unremarkable with normal blood pressure. Initial laboratory results revealed a white blood cell count of 4.4 x103/µL, hemoglobin of 4.4 g/dL, hematocrit 12.9%, platelets 43 x103/µL, absolute reticulocyte count 259.8 x103/µL, reticulocyte percentage 23.1%, haptoglobin <10 L mg/dL, LDH 1424 units/L, creatinine 0.7 mg/dL, total bilirubin 1.6 mg/dL, negative pregnancy test, and 10-15 white blood cells and >50 red blood cells per high powered field on microscopic urinalysis. Coombs test results were negative, the coagulation profile was normal, and peripheral blood smear revealed evidence of thrombocytopenia and MAHA (figure 1a). Diagnosis of TTP was made and treatment with daily therapeutic plasma exchange (TPE) was initiated. On hospital day 4, ADAMTS13 level drawn at diagnosis before initiation of plasma exchange returned at 94%, prompting further investigation as to the cause of her TMA. Serologic tests for hepatitis B & C virus, human immunodeficiency virus, antinuclear antibody (ANA), cardiolipin and beta-2 glycoprotein antibodies and computerized tomography (CT) scan of the abdomen and pelvis were all negative. Despite daily TPE for 6 days, she remained thrombocytopenic and continued to have ongoing hemolysis. Therefore, on hospital day 6, bone marrow aspiration and biopsy (figure 1b) was performed which showed a normocellular bone marrow for age (cellularity 70%) with full-spectrum trinlineage hematopoiesis, involved by metastatic signet-cell ring carcinoma. Immunostains were positive for CK7, CK20, CK 8/18, CDX2, pancytokeratin, and negative for PAX-8 and mammoglobin. TPE was discontinued and she was supported with packed red blood cell and platelet transfusions.Positron emission tomography–computed tomography (PET-CT) revealed a heterogeneously enhancing mass in the midline superior anterior wall of the bladder measuring 2.7 x 5.1 cm, but without abnormal uptake on PET. She underwent cystourethroscopy and a transurethral resection of bladder tumor which revealed a 6 cm solid sessile mass. Final pathology confirmed the diagnosis of moderate to poorly differentiated invasive urachal adenocarcinoma with mucinous and signet-ring cell features.Given the presence of metastatic disease, she was treated with a chemotherapy regimen consisting of cisplatin, fluorouracil, and leucovorin on days 1 through 5. Gemcitabine was held due to elevated liver transaminases. On day 1 of treatment, platelet count was 24 x103/µL and hemoglobin 8.1 g/dL. By treatment day 7, platelets had risen to 79 x103/µL. She last required a PRBC transfusion on treatment day 4.Discussion:TMA can occur in a variety of disorders, including TTP, HUS, malignant hypertension, scleroderma, antiphospholipid antibody syndrome, systemic lupus erythematosus, preeclampsia, HIV infection, and disseminated malignancy. Patients presenting with evidence of TMA and thrombocytopenia should elicit a high clinical suspicion for TTP and be urgently treated with plasma exchange. Deficiency of the von Willebrand factor-cleaving metalloproteinase ADAMTS13, or the presence of antibodies to ADAMTS13 plays a key role in the pathogenesis of primary TTP, although these findings are not necessary to make the diagnosis. However, patients with a high or normal ADAMTS13 level and without antibodies to ADAMTS13, especially those who do not improve with plasma exchange, should prompt an investigation for other causes of TMA. To our knowledge, we report the first case of metastatic urachal carcinoma presenting with features of MAHA and thrombocytopenia.Conclusion:TMA associated with malignancy is a rare, but potentially fatal condition. Recognition of this rare entity is critical to the initiation of appropriate treatment of underlying malignancy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The cholesterol-binding motif of the HIV-1 glycoprotein gp41 regulates lateral sorting and oligomerization.

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

Disulfide Reduction in CD4 Domain 1 or 2 Is Essential for Interaction with HIV Glycoprotein 120 (gp120), which Impairs Thioredoxin-driven CD4 Dimerization

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.

Thirty years on: HIV receptor gymnastics and the prevention of infection

During 30 years of research on human immunodeficiency virus (HIV), our knowledge of its cellular receptors - CD4, CCR5 and CXCR4 - has illuminated aspects of the pathogenesis of the acquired immune deficiency syndrome (AIDS). Studying how the HIV envelope glycoproteins interact with the receptors led to anti-retroviral drugs based on blocking the docking or fusion of virus to the host cell. Genetic polymorphisms of CCR5 determine resistance to HIV infection and the rate of progression to AIDS. Eliciting neutralizing antibodies to the sites of receptor interaction on HIV glycoproteins is a promising approach to HIV vaccine development.

Cell-To-Cell Variability in Plasma Membrane Lipid Rafts

In recent years compelling evidence pointed to an important role of cholesterol enriched microdomains, so-called lipid rafts in miscellaneous cellular functions. Supposably, this lateral sub-compartmentalization facilitates selective protein-protein interactions by a local enrichment of the components involved. To study lipid rafts in the context of virus assembly, we produced several variants of two viral proteins, the Influenza virus transmembrane protein hemagglutinin and the Human Immunodeficiency Virus glycoprotein gp41. Fluorescence lifetime imaging microscopy was used to report Forster Resonance Energy Transfer (FRET) between a raft marker labelled with a cyan fluorescent protein and viral chimeras in living cells. Since it is highly distance dependent, occurring FRET indicates a co-clustering of both fluorescent protein species in membrane microdomains. Both viral proteins were found to be associated with plasma membrane lipid rafts and interestingly we observed a significant cell-to-cell variability in our samples exhibiting two distinct cell-populations with clearly differing raft related properties. To verify this intercellular lipid raft heterogeneity we used fluorescence polarization microscopy, displaying local enrichment of GPI-anchored proteins as a complementary approach. Furthermore, we utilized the environment-sensitive fluorescence lifetime of NBD-PC to further study membrane order on a single-cell level. We surmise, that the cell-to-cell variability detected is representative of a context-dependence of cellular plasma membrane organization. Cell synchronization and cell cycle analysis were conducted to elucidate a potential correlation of plasma membrane condition and the position of the cells in the cell cycle.Considering the high spatial and temporal dynamics of plasma membrane lipid rafts and the sophisticated methods that are necessary to study them it is conceivable, that the additional complexity of the membrane organization described here has not been detected in previous investigations. Nevertheless, it might be representative of cellular mechanisms that enables adoption to environmental demands or proliferative progression.