Envelope Glycoprotein Gp120 Research Articles

Mannose-Specific Lectins from Marine Algae: Diverse Structural Scaffolds Associated to Common Virucidal and Anti-Cancer Properties.

To date, a number of mannose-specific lectins have been isolated and characterized from seaweeds, especially from red algae. In fact, man-specific seaweed lectins consist of different structural scaffolds harboring a single or a few carbohydrate-binding sites which specifically recognize mannose-containing glycans. Depending on the structural scaffold, man-specific seaweed lectins belong to five distinct structurally-related lectin families, namely (1) the griffithsin lectin family (β-prism I scaffold); (2) the Oscillatoria agardhii agglutinin homolog (OAAH) lectin family (β-barrel scaffold); (3) the legume lectin-like lectin family (β-sandwich scaffold); (4) the Galanthus nivalis agglutinin (GNA)-like lectin family (β-prism II scaffold); and, (5) the MFP2-like lectin family (MFP2-like scaffold). Another algal lectin from Ulva pertusa, has been inferred to the methanol dehydrogenase related lectin family, because it displays a rather different GlcNAc-specificity. In spite of these structural discrepancies, all members from the five lectin families share a common ability to specifically recognize man-containing glycans and, especially, high-mannose type glycans. Because of their mannose-binding specificity, these lectins have been used as valuable tools for deciphering and characterizing the complex mannose-containing glycans from the glycocalyx covering both normal and transformed cells, and as diagnostic tools and therapeutic drugs that specifically recognize the altered high-mannose N-glycans occurring at the surface of various cancer cells. In addition to these anti-cancer properties, man-specific seaweed lectins have been widely used as potent human immunodeficiency virus (HIV-1)-inactivating proteins, due to their capacity to specifically interact with the envelope glycoprotein gp120 and prevent the virion infectivity of HIV-1 towards the host CD4+ T-lymphocyte cells in vitro.

Flexible HIV-1 Biosensor Based on the Au/MoS2 Nanoparticles/Au Nanolayer on the PET Substrate.

An electrochemical flexible biosensor composed of gold (Au), molybdenum disulfide nanoparticles (MoS2 NPs), and Au (Au/MoS2/Au nanolayer) on the polyethylene terephthalate (PET) substrate is developed to detect envelope glycoprotein GP120 (gp120), the surface protein of HIV-1. To fabricate the nanolayer on the PET substrate, Au is sputter coated on the flexible PET substrate and MoS2 NPs are spin coated on Au, which is sputter coated once again with Au. The gp120 antibody is then immobilized on this flexible electrode through cysteamine (Cys) modified on the surface of the Au/MoS2/Au nanolayer. Fabrication of the biosensor is verified by atomic force microscopy, scanning electron microscopy, and cyclic voltammetry. A flexibility test is done using a micro-fatigue tester. Detection of the gp120 is measured by square wave voltammetry. The results indicate that the prepared biosensor detects 0.1 pg/mL of gp120, which is comparable with previously reported gp120 biosensors prepared even without flexibility. Therefore, the proposed biosensor supports the development of a nanomaterial-based flexible sensing platform for highly sensitive biosensors with flexibility for wearable device application.

Mechanisms of Blood-Retinal Barrier Disruption by HIV-1.

It has been found that human immunodeficiency virus (HIV)-1 RNA or antigens can be detected in the intraocular tissues of HIV-1 patients even under effective highly active anti-retroviral therapy (HAART). In vivo, blood-retinal barrier (BRB) establishes a critical, physiological guardian against microbial invasion of the eye, but may be compromised in the presence of HIV-1. The envelope glycoprotein gp120 is exposed on the surface of the HIV envelope, essential for virus entry into cells by the attachment to specific cell surface receptors. The BRB disruption by glycoprotein gp120 has been widely recognized, which is toxic to human retinal epithelial cells (RPE) and umbilical vein endothelial cells (HUVEC). The present review elaborates on various mechanisms of BRB disruption induced by HIV gp120, which may represent potential targets for the prevention of ocular HIV complications in the future.

Dimeric C34 Derivatives Linked through Disulfide Bridges as New HIV-1 Fusion Inhibitors.

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH2 CONH2 )-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors.

Phylogenetic measures of indel rate variation among the HIV-1 group M subtypes

The transmission fitness and pathogenesis of HIV-1 is disproportionately influenced by evolution in the five variable regions (V1–V5) of the surface envelope glycoprotein (gp120). Insertions and deletions (indels) are a significant source of evolutionary change in these regions. However, the rate and composition of indels has not yet been quantified through a large-scale comparative analysis of HIV-1 sequences. Here, we develop and report results from a phylogenetic method to estimate indel rates for the gp120 variable regions across five major subtypes and two circulating recombinant forms (CRFs) of HIV-1 group M. We processed over 26,000 published HIV-1 gp120 sequences, from which we extracted 6,605 sequences for phylogenetic analysis. We reconstructed time-scaled phylogenies by maximum likelihood and fit a binomial-Poisson model to the observed distribution of indels between closely related pairs of sequences in each tree (cherries). By focusing on cherries in each tree, we obtained phylogenetically independent indel reconstructions, and the shorter time scales in cherries reduced the bias due to purifying selection. Rate estimates ranged from to indels/nt/year and varied significantly among variable regions and subtypes. Indel rates were significantly lower in V3 relative to V1, and were also lower in HIV-1 subtype B relative to the 01_AE reference. We also found that V1, V2, and V4 tended to accumulate significantly longer indels. Furthermore, we observed that the nucleotide composition of indels was distinct from the flanking sequence, with higher frequencies of G and lower frequencies of T. Indels affected N-linked glycosylation sites more often in V1 and V2 than expected by chance, consistent with positive selection on glycosylation patterns within these regions. These results represent the first comprehensive measures of indel rates in HIV-1 gp120 across multiple subtypes and CRFs, and identifies novel and unexpected patterns for further research in the molecular evolution of HIV-1.

Rational Design of Colchicine Derivatives as anti-HIV Agents via QSAR and Molecular Docking

Human immunodeficiency virus (HIV) is an infective agent that causes an acquired immunodeficiency syndrome (AIDS). Therefore, the rational design of inhibitors for preventing the progression of the disease is required. This study aims to construct quantitative structure-activity relationship (QSAR) models, molecular docking and newly rational design of colchicine and derivatives with anti-HIV activity. A data set of 24 colchicine and derivatives with anti-HIV activity were employed to develop the QSAR models using machine learning methods (e.g. multiple linear regression (MLR), artificial neural network (ANN) and support vector machine (SVM)), and to study a molecular docking. The significant descriptors relating to the anti-HIV activity included JGI2, Mor24u, Gm and R8p+ descriptors. The predictive performance of the models gave acceptable statistical qualities as observed by correlation coefficient (Q2) and root mean square error (RMSE) of leave-one out cross-validation (LOO-CV) and external sets. Particularly, the ANN method outperformed MLR and SVM methods that displayed LOO-CV 2 Q and RMSELOO-CV of 0.7548 and 0.5735 for LOOCV set, and Ext 2 Q of 0.8553 and RMSEExt of 0.6999 for external validation. In addition, the molecular docking of virus-entry molecule (gp120 envelope glycoprotein) revealed the key interacting residues of the protein (cellular receptor, CD4) and the site-moiety preferences of colchicine derivatives as HIV entry inhibitors for binding to HIV structure. Furthermore, newly rational design of colchicine derivatives using informative QSAR and molecular docking was proposed. These findings serve as a guideline for the rational drug design as well as potential development of novel anti-HIV agents.

Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle

The p6 domain of HIV-1 Gag contains highly conserved peptide motifs that recruit host machinery to sites of virus assembly, thereby promoting particle release from the infected cell. We previously reported that mutations in the YPXnL motif of p6, which binds the host protein Alix, severely impair HIV-1 replication. Propagation of the p6-Alix binding site mutants in the Jurkat T cell line led to the emergence of viral revertants containing compensatory mutations not in Gag but in Vpu and the envelope (Env) glycoprotein subunits gp120 and gp41. The Env compensatory mutants replicate in Jurkat T cells and primary human peripheral blood mononuclear cells, despite exhibiting severe defects in cell-free particle infectivity and Env-mediated fusogenicity. Remarkably, the Env compensatory mutants can also rescue a replication-delayed integrase (IN) mutant, and exhibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), demonstrating that they confer a global replication advantage. In addition, confirming the ability of Env mutants to confer escape from DTG, we performed de novo selection for DTG resistance and observed resistance mutations in Env. These results identify amino acid substitutions in Env that confer broad escape from defects in virus replication imposed by either mutations in the HIV-1 genome or by an antiretroviral inhibitor. We attribute this phenotype to the ability of the Env mutants to mediate highly efficient cell-to-cell transmission, resulting in an increase in the multiplicity of infection. These findings have broad implications for our understanding of Env function and the evolution of HIV-1 drug resistance.

Inhibition of the thioredoxin system by PX-12 (1-methylpropyl 2-imidazolyl disulfide) impedes HIV-1 infection in TZM-bl cells

Human immunodeficiency virus (HIV-1) entry is initiated by the binding between the viral envelope glycoprotein gp120 and the host receptor CD4, and followed by reduction of structural disulfides of gp120 and CD4. The host thioredoxin-1 (Trx1) efficiently reduces disulfides of gp120 and CD4 in vitro, and recently CD4-dependent HIV-1 entry was shown to be inhibited by anti-Trx1-antibodies, indicating a central role for Trx1. 1-methylpropyl-2-imidazolyl disulfide (PX-12) is a reversible inhibitor of the Trx1 system that may also cause a slow irreversible thioalkylation of Trx1. It was developed as an antitumor agent, however, the current study aimed to determine if it also has an anti-HIV-1 effect. We show that PX-12 has anti-HIV-1(IIIB) activity in TZM-bl cells, in fact, no virus was detected inside the cells in the presence of 10 µM PX-12. Moreover, PX-12 inhibited the enzymatic activity of Trx1 and the Trx1-dependent disulfide reduction of gp120. Microtubule polymerization and formation of acetylated microtubules were also inhibited, activities shown to be required for HIV-1 life cycle propagation. In conclusion, our data strengthens the notion that the early steps of the HIV-1 life cycle depends on the Trx1 system and indicate that the Trx1 system may be a rational drug target for HIV-1 treatment.

HIV-1 Envelope Recognition by Polyreactive and Cross-Reactive Intestinal B Cells

SummaryMucosal immune responses to HIV-1 involve the recognition of the viral envelope glycoprotein (gp)160 by tissue-resident B cells and subsequent secretion of antibodies. To characterize the B cells “sensing” HIV-1 in the gut of infected individuals, we probed monoclonal antibodies produced from single intestinal B cells binding to recombinant gp140 trimers. A large fraction of mucosal B cell antibodies were polyreactive and showed only low affinity to HIV-1 envelope glycoproteins, particularly the gp41 moiety. A few high-affinity gp140 antibodies were isolated but lacked neutralizing, potent ADCC, and transcytosis-blocking capacities. Instead, they displayed cross-reactivity with defined self-antigens. Specifically, intestinal HIV-1 gp41 antibodies targeting the heptad repeat 2 region (HR2) cluster II cross-reacted with the p38α mitogen-activated protein kinase 14 (MAPK14). Hence, physiologic polyreactivity of intestinal B cells and molecular mimicry-based self-reactivity of HIV-1 antibodies are two independent phenomena, possibly diverting and/or impairing mucosal humoral immunity to HIV-1.

High affinity CXCR4 inhibitors generated by linking low affinity peptides

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model of GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and binding free energy calculation to determine how the peptide interacts with CXCR4 and inhibits its downstream signaling. These results demonstrate that this combinational approach is effective for generating nanomolar active inhibitors of CXCR4 and may be applicable to other GPCRs.

HIV gp120 upregulates tonic inhibition through α5-containing GABAARs

HIV-Associated Neurocognitive disorder (HAND) affects nearly half of infected patients. The HIV envelope protein gp120 is shed by infected cells and is a potent neurotoxin in vitro that reproduces many aspects of HAND when expressed in vivo. Here, we show that HIV gp120 increases the amplitude of a tonic current mediated by γ-aminobutyric acid type-A receptors (GABAARs).Treating rat hippocampal cultures with 600 pM gp120IIIB for 4 h increased a tonic bicuculline-sensitive current, which remained elevated for 24 h. The increased current resulted from upregulation of extrasynaptic α5-containing GABAARs, as indicated by inhibition with the selective inverse agonist basmisanil. Treatment with gp120 increased α5-GABAAR immunoreactivity on the cell surface without new protein synthesis. The increase in tonic inhibition was prevented by a C-X-C chemokine receptor type 4 (CXCR4) antagonist or elimination of microglia from the culture. Treatment with interleukin-1β (IL-1β) increased the tonic current and an IL-1 receptor antagonist blocked the gp120-evoked response. Pharmacological or genetic inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the gp120-evoked increase in tonic current and direct activation of a mutant form of p38 MAPK expressed in neurons increased the current.Collectively, these data show that gp120 activates CXCR4 to stimulate microglia to release IL-1β. Subsequent stimulation of IL-1 receptors activates p38 MAPK in neurons leading to the upregulation of α5-containing GABAARs. Increased tonic inhibition impairs neuroplasticity and inhibition of α5-containing GABAARs improves cognitive function in disease models. Thus, gp120-induced upregulation of α5-containing GABAARs presents a novel therapeutic target for HAND.

Capturing the inherent structural dynamics of the HIV-1 envelope glycoprotein fusion peptide

The N-terminal fusion peptide (FP) of the human immunodeficiency virus (HIV)-1 envelope glycoprotein (Env) gp41 subunit plays a critical role in cell entry. However, capturing the structural flexibility in the unbound FP is challenging in the native Env trimer. Here, FP conformational isomerism is observed in two crystal structures of a soluble clade B transmitted/founder virus B41 SOSIP.664 Env with broadly neutralizing antibodies (bNAbs) PGT124 and 35O22 to aid in crystallization and that are not specific for binding to the FP. Large rearrangements in the FP and fusion peptide proximal region occur around M530, which remains anchored in the tryptophan clasp (gp41 W623, W628, W631) in the B41 Env prefusion state. Further, we redesigned the FP at position 518 to reinstate the bNAb VRC34.01 epitope. These findings provide further structural evidence for the dynamic nature of the FP and how a bNAb epitope can be restored during vaccine design.

Optimization of Formulation Conditions for Broadly Neutralizing Antibodies (bnAb): Utilizing Isothermal Chemical Denaturation as a High-throughput Screening Method

Broadly neutralizing antibodies (bnAb) were identified at the Vaccine Research Center (VRC) at NIH, towards the development of an anti-HIV vaccine. Examples of two such mAbs are CAP256 LS and N6 LS, targeting the V1V2 and CD4-binding sites, respectively, on the gp120 envelope glycoprotein. As part of mAb formulation development, it is critical to identify condition which can maintain the conformational and colloidal stability of the antibodies at 100mg/ml in frozen or liquid phase for two years. The factors which have the maximum impact on stability are formulation pH, ionic strength and stabilizers. High throughput methods to screen these three factors and identify optimal conditions are described here. The initial characterization of the mAbs using techniques including Differential Scanning Calorimetry (DSC), Dynamic Light Scattering (DLS), and Circular Dichroism (CD) indicated that they have very different physico-chemical properties. An assessment of intrinsic stability was conducted using Isothermal Chemical Denaturation (ICD) at a wide pH (4-8) and salt concentration range (0-200 mM). pH had the strongest effect on both CAP256LS and N6LS intrinsic stability. CAP256LS was most stable at a higher pH (>7) while N6LS stability improved above pH 6. Ionic strength had little effect on intrinsic stability for both mAbs. The stabilizing effects of various excipients on CAP256LS was screened using DLS, UV-Visible Spectroscopy, and Size Exclusion Chromatography (SEC). ICD was utilized to more rapidly screen the optimal concentration of these excipients. Balancing the results of the pH and ionic strength screens for both assays, optimal conditions were selected for both mAbs, and the high-throughput screening capacity of the ICD in conjunction with traditional stability-indicating assays helped identify optimal formulation conditions.

The Lupus Susceptibility Locus Sgp3 Encodes the Suppressor of Endogenous Retrovirus Expression SNERV

Elevated endogenous retrovirus (ERV) transcription and anti-ERV antibody reactivity are implicated in lupus pathogenesis. Overproduction of non-ecotropic ERV (NEERV) envelope glycoprotein gp70 and resultant nephritis occur in lupus-prone mice, but whether NEERV mis-expression contributes to lupus etiology is unclear. Here we identified suppressor of NEERV (Snerv) 1 and 2, Krüppel-associated box zinc-finger proteins (KRAB-ZFPs) that repressed NEERV by binding the NEERV long terminal repeat to recruit the transcriptional regulator KAP1. Germline Snerv1/Snerv2 deletion increased activating chromatin modifications, transcription, and gp70 expression from NEERV loci. F1 crosses of lupus-prone New Zealand Black (NZB) and 129 mice to Snerv1/Snerv2-/- mice failed to restore NEERV repression, demonstrating that loss of SNERV underlies the lupus autoantigen gp70 overproduction that promotes nephritis in susceptible mice and that SNERV encodes for Sgp3 (in NZB mice) and Gv-1 loci (in 129 mice). Increased ERV expression in lupus patients inversely correlated with three putative ERV-suppressing KRAB-ZFPs, suggesting that loss of KRAB-ZFP-mediated ERV control may contribute to human lupus pathogenesis.

Effects of CD4 Binding on Conformational Dynamics, Molecular Motions, and Thermodynamics of HIV-1 gp120.

Human immunodeficiency virus type-1 (HIV-1) infection is triggered by its envelope (Env) glycoprotein gp120 binding to the host-cell receptor CD4. Although structures of Env/gp120 in the liganded state are known, detailed information about dynamics of the liganded gp120 has remained elusive. Two structural models, the CD4-free gp120 and the gp120-CD4 complex, were subjected to µs-scale multiple-replica molecular dynamics (MD) simulations to probe the effects of CD4 binding on the conformational dynamics, molecular motions, and thermodynamics of gp120. Comparative analyses of MD trajectories in terms of structural deviation and conformational flexibility reveal that CD4 binding effectively suppresses the overall conformational fluctuations of gp120. Despite the largest fluctuation amplitude of the V1/V2 region in both forms of gp120, the presence of CD4 prevents it from approaching the gp120 core. Comparison of the constructed free energy landscapes (FELs) shows that CD4 binding reduces the conformational entropy and conformational diversity while enhancing the stability of gp120. Further comparison of the representative structures extracted from free energy basins/minima of FELs reveals that CD4 binding weakens the reorientation ability of V1/V2 and hence hinders gp120 from transitioning out of the liganded state to the unliganded state. Therefore, locking gp120 conformation via restraining V1/V2 reorientation with small molecules seems to be a promising strategy to control HIV-1 infection. Our computer simulation results support the conformational selection mechanism for CD4 binding to gp120 and facilitate the understanding of HIV-1 immune evasion mechanisms.

Broadly applicable PCR restriction fragment length polymorphism method for genotyping bovine leukemia virus.

Bovine leukemia virus (BLV) is a causative agent of enzootic bovine lymphoma (EBL). BLV is prevalent worldwide, and ten genotypes have been classified based on the sequence of the envelope glycoprotein (gp51) gene. In this study, we present a simple and generally applicable PCR restriction fragment length polymorphism (PCR-RFLP) method to identify BLV genotypes. While the genotyping results obtained by previously described PCR-RFLP methods matched only 78.96% to the results of phylogenetic analysis, we demonstrated that our PCR-RFLP method can identify 90.4% of the sequences available in the database in silico. The method was validated with 20 BLV sequences from EBL tumor tissues and 3 BLV sequences from blood of BLV infected cattle, and was found to show high specificity. We utilized this method to determine genotypes of blood samples from 18 BLV seropositive cattle in Kanagawa and Niigata, as well as 12 EBL cattle in Chiba, Japan. Our analysis with the modified PCR-RFLP detected two genotypes, Genotypes 1 and 3. Genotype 1 was detected as the main genotype, while Genotype 3 was sporadically observed. This technique can be used as a reliable system for screening a large number of epidemiological samples.

The Botanical Glycoside Oleandrin Inhibits Human T-cell Leukemia Virus Type-1 Infectivity and Env-Dependent Virological Synapse Formation.

At present, there are no antiretroviral drugs that inhibit incorporation of the envelope glycoprotein into newly-synthesized virus particles. The botanical glycoside, oleandrin, derived from extracts of Nerium oleander, has previously been shown to reduce the levels of the gp120 envelope glycoprotein on human immunodeficiency virus type-1 (HIV-1) particles and inhibit HIV-1 infectivity in vitro. We therefore tested whether oleandrin or an extract from N. oleander could also inhibit the infectivity of the human T-cell leukemia virus type-1 (HTLV-1): A related enveloped retrovirus and emerging tropical infectious agent. The treatment of HTLV-1+ lymphoma T-cells with either oleandrin or a N. oleander extract did not significantly inhibit viral replication or the release of p19Gag-containing particles into the culture supernatants. However, the collected virus particles from treated cells exhibited reduced infectivity on primary human peripheral blood mononuclear cells (huPBMCs). Unlike HIV-1, extracellular HTLV-1 particles are poorly infectious and viral transmission typically occurs via direct intercellular interactions across a virological synapse. We therefore investigated whether oleandrin or a N. oleander extract could inhibit virus transmission from a GFP-expressing HTLV-1+ lymphoma T-cell-line to huPBMCs in co-culture assays. These results demonstrated that both oleandrin and the crude phytoextract inhibited the formation of virological synapses and the transmission of HTLV-1 in vitro. Importantly, these findings suggest oleandrin may have broad antiviral activity against enveloped viruses by reducing the incorporation of the envelope glycoprotein into mature particles, a stage of the infection cycle not targeted by modern HAART.

HIV Proteins and Endothelial Dysfunction: Implications in Cardiovascular Disease.

With the success of antiretroviral therapy (ART), a dramatic decrease in viral burden and opportunistic infections and an increase in life expectancy has been observed in human immunodeficiency virus (HIV) infected individuals. However, it is now clear that HIV- infected individuals have enhanced susceptibility to non-AIDS (Acquired immunodeficiency syndrome)-related complications such as cardiovascular disease (CVD). CVDs such as atherosclerosis have become a significant cause of morbidity and mortality in individuals with HIV infection. Though studies indicate that ART itself may increase the risk to develop CVD, recent studies suggest a more important role for HIV infection in contributing to CVD independently of the traditional risk factors. Endothelial dysfunction triggered by HIV infection has been identified as a critical link between infection, inflammation/immune activation, and atherosclerosis. Considering the inability of HIV to actively replicate in endothelial cells, endothelial dysfunction depends on both HIV-encoded proteins as well as inflammatory mediators released in the microenvironment by HIV-infected cells. Indeed, the HIV proteins, gp120 (envelope glycoprotein) and Tat (transactivator of transcription), are actively secreted into the endothelial cell micro-environment during HIV infection, while Nef can be actively transferred onto endothelial cells during HIV infection. These proteins can have significant direct effects on the endothelium. These include a range of responses that contribute to endothelial dysfunction, including enhanced adhesiveness, permeability, cell proliferation, apoptosis, oxidative stress as well as activation of cytokine secretion. This review summarizes the current understanding of the interactions of HIV, specifically its proteins with endothelial cells and its implications in cardiovascular disease. We analyze recent in vitro and in vivo studies examining endothelial dysfunction in response to HIV proteins. Furthermore, we discuss the multiple mechanisms by which these viral proteins damage the vascular endothelium in HIV patients. A better understanding of the molecular mechanisms of HIV protein associated endothelial dysfunction leading to cardiovascular disease is likely to be pivotal in devising new strategies to treat and prevent cardiovascular disease in HIV-infected patients.

The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: implications for baculovirus phylogeny and host specificity

The Mythimna unipuncta nucleopolyhedrovirus isolate KY310 (MyunNPV-KY310) is an alphabaculovirus isolated from a true armyworm (Mythimna unipuncta) population in Kentucky, USA. Occlusion bodies of this virus were examined by electron microscopy and the genome sequence was determined by 454 pyrosequencing. MyunNPV-KY310 occlusion bodies consisted of irregular polyhedra measuring 0.8-1.8µm in diameter and containing multiple virions, with one to six nucleocapsids per virion. The genome sequence was determined to be 156,647bp with a nucleotide distribution of 43.9% G+C. 152 ORFs and six homologous repeat (hr) regions were annotated for the sequence, including the 38 core genes of family Baculoviridae and an additional group of 26 conserved alphabaculovirus genes. BLAST queries and phylogenetic inference confirmed that MyunNPV-KY310 is most closely related to the alphabaculovirus Leucania separata nucleopolyhedrovirus isolate AH1, which infects Mythimna separata. In contrast, MyunNPV-KY310 did not exhibit a close relationship with Mythimna unipuncta nucleopolyhedrovirus isolate #7, an alphabaculovirus from the same host species. MyunNPV-KY310 lacks the gp64 envelope glycoprotein, which is a characteristic of group II alphabaculoviruses. However, this virus and five other alphabaculoviruses lacking gp64 are placed outside the group I and group II clades in core gene phylogenies, further demonstrating that viruses of genus Alphabaculovirus do not occur in two monophyletic clades. Potential instances of MyunNPV-KY310 ORFs arising by horizontal transfer were detected. Although there are now genome sequences of four different baculoviruses from M. unipuncta, comparison of their genome sequences provides little insight into the genetic basis for their host specificity.

Defining specific residue‐to‐residue interactions between the gp120 bridging sheet and the N‐terminal segment of CCR5: applications of transferred NOE NMR

Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core YU 2 gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5.