scholarly journals Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1964
Author(s):  
Xinling Wang ◽  
Miao Cao ◽  
Yanling Wu ◽  
Wei Xu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Highly Active Antiretroviral Therapy ◽  
Binding Protein ◽  
Heptad Repeat ◽  
Target Cells ◽  
Single Chain ◽  
High Concentration ◽  
Gp120 Binding ◽  
Binding Antibody ◽  
Hiv 1

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.

scholarly journals High Concentration of Raltegravir in Semen of HIV-Infected Men: Results from a Substudy of the EASIER-ANRS 138 Trial

2009 ◽  
Vol 54 (2) ◽  
pp. 937-939 ◽  
Author(s):  
Caroline Barau ◽  
Constance Delaugerre ◽  
Joséphine Braun ◽  
Nathalie de Castro ◽  
Valérie Furlan ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Plasma Samples ◽  
Drug Penetration ◽  
High Concentration ◽  
Highly Active ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Rna Levels

ABSTRACT Raltegravir concentrations and human immunodeficiency virus type 1 (HIV-1) RNA levels in semen samples from 10 treatment-experienced HIV-1-infected patients were measured after 24 weeks of raltegravir-based highly active antiretroviral therapy (HAART). Semen and plasma HIV-1 RNA levels were below 100 copies/ml and 50 copies/ml, respectively, in all samples. The median raltegravir concentrations in semen samples (n = 10) and in plasma samples (n = 9) drawn simultaneously were 345 (range, 83 to 707) ng/ml and 206 (range, 106 to 986) ng/ml, respectively. The median semen-to-plasma ratio of raltegravir concentration was 1.42 (range, 0.52 to 6.66), indicating good although variable levels of drug penetration of raltegravir in the seminal compartment.

scholarly journals Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 566
Author(s):  
Mario Cano-Muñoz ◽  
Samuele Cesaro ◽  
Bertrand Morel ◽  
Julie Lucas ◽  
Christiane Moog ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Coiled Coil ◽  
Helical Structure ◽  
Heptad Repeat ◽  
Single Chain ◽  
Cell Infection ◽  
Polar Residues ◽  
Hiv 1

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell–activating polypeptide

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2342-2350 ◽  
Author(s):  
Marielle Maurice ◽  
Els Verhoeyen ◽  
Patrick Salmon ◽  
Didier Trono ◽  
Stephen J. Russell ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Genetic Material ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Blood Lymphocytes ◽  
Hiv 1

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1–derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G0 to G1 phases of the cell cycle. Compared to unmodified HIV-1–based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

scholarly journals Transactive response DNA-binding Protein (TDP-43) regulates early HIV-1 entry and infection.

2021 ◽  
Author(s):  
Romina Cabrera-Rodriguez ◽  
Silvia Perez-Yanes ◽  
Rafaela Gonzalez-Montelongo ◽  
Jose M Lorenzo-Salazar ◽  
Judith Estevez-Herrera ◽  
...  
Keyword(s):  
Dna Binding ◽  
Hiv Infection ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Viral Envelope ◽  
Target Cells ◽  
Histone Deacetylase 6 ◽  
Elite Controllers ◽  
Experimental Conditions ◽  
Hiv 1

The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

scholarly journals Targeting the Tumor Microenvironment with Fluorescence-Activatable Bispecific Endoglin/Fibroblast Activation Protein Targeting Liposomes

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 370 ◽  
Author(s):  
Felista L. Tansi ◽  
Ronny Rüger ◽  
Ansgar M. Kollmeier ◽  
Markus Rabenhold ◽  
Frank Steiniger ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Targeted Delivery ◽  
Near Infrared ◽  
Fibroblast Activation Protein ◽  
Target Cells ◽  
Single Chain ◽  
High Concentration ◽  
Single Chain Antibody ◽  
Stromal Fibroblasts ◽  
Fibroblast Activation

Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.

scholarly journals Recognition by Human Monoclonal Antibodies of Free and Complexed Peptides Representing the Prefusogenic and Fusogenic Forms of Human Immunodeficiency Virus Type 1 gp41

2000 ◽  
Vol 74 (13) ◽  
pp. 6186-6192 ◽  
Author(s):  
Miroslaw K. Gorny ◽  
Susan Zolla-Pazner
Keyword(s):  
Human Immunodeficiency Virus ◽  
Monoclonal Antibodies ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an α-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which participates in complex formation with N51.

scholarly journals Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors

2005 ◽  
Vol 79 (14) ◽  
pp. 9359-9362 ◽  
Author(s):  
Susann Lucke ◽  
Thomas Grunwald ◽  
Klaus Überla
Keyword(s):  
Human Immunodeficiency Virus ◽  
Lentiviral Vector ◽  
Binding Protein ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Novel Approach ◽  
Immunodeficiency Virus ◽  
Efficient Vector ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 103- to 104-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.

scholarly journals Mutations That Increase the Stability of the Postfusion gp41 Conformation of the HIV-1 Envelope Glycoprotein Are Selected by both an X4 and R5 HIV-1 Virus To Escape Fusion Inhibitors Corresponding to Heptad Repeat 1 of gp41, but the gp120 Adaptive Mutations Differ between the Two Viruses

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Min Zhuang ◽  
Russell Vassell ◽  
Chen Yuan ◽  
Paul W. Keller ◽  
Hong Ling ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Envelope Glycoprotein ◽  
Resistance Mutation ◽  
Heptad Repeat ◽  
Target Cells ◽  
Resistance Mutations ◽  
Adaptive Mutations ◽  
Helix Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

ABSTRACT Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes. IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.

scholarly journals Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 609 ◽  
Author(s):  
Xiuzhu Geng ◽  
Zixuan Liu ◽  
Danwei Yu ◽  
Bo Qin ◽  
Yuanmei Zhu ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mutational Analysis ◽  
Heptad Repeat ◽  
Titration Calorimetry ◽  
Target Cells ◽  
Viral Fusion ◽  
Fusion Inhibitors ◽  
Structural And Functional Properties ◽  
Relationship Of ◽  
Hiv 1

Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional properties of a single Asn-145 residue, which locates at the middle CHR site of gp41 and is extremely conserved among all the HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. By mutational analysis, we found that Asn-145 plays critical roles for Env-mediated cell-cell fusion and HIV-1 entry. As determined by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC), the substitution of Asn-145 with alanine (N145A) severely impaired the interactions between the NHR and CHR helices. Asn-145 was also verified to be important for the antiviral activity of CHR-derived peptide fusion inhibitors and served as a turn-point for the inhibitory potency. Intriguingly, Asn-145 could regulate the functionality of the M-T hook structure at the N-terminus of the inhibitors and displayed comparable activities with the C-terminal IDL anchor. Crystallographic studies further demonstrated the importance of Asn-145-mediated interhelical and intrahelical interactions in the 6-HB structure. Combined, the present results have provided valuable information for the structure-function relationship of HIV-1 gp41 and the structure-activity relationship of gp41-dependent fusion inhibitors.

scholarly journals Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

2014 ◽  
Vol 111 (51) ◽  
pp. 18207-18212 ◽  
Author(s):  
Sara Crespillo ◽  
Ana Cámara-Artigas ◽  
Salvador Casares ◽  
Bertrand Morel ◽  
Eva S. Cobos ◽  
...  
Keyword(s):  
Heptad Repeat ◽  
Repeat Region ◽  
Single Chain ◽  
Anti Hiv ◽  
Hiv 1
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