scholarly journals Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

2015 ◽  
Vol 90 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Eden P. Go ◽  
Albert Cupo ◽  
Rajesh Ringe ◽  
Pavel Pugach ◽  
John P. Moore ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Protein A ◽  
Native Conformation ◽  
Purification Method ◽  
Immunodeficiency Virus ◽  
Monomeric Gp120 ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACTWe investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505envgene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.IMPORTANCEIt is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

scholarly journals Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Isabelle Staropoli ◽  
Jérémy Dufloo ◽  
Anaïs Ducher ◽  
Pierre-Henri Commere ◽  
Anna Sartori-Rupp ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cellular Proteins ◽  
Viral Infectivity ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACT The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

scholarly journals Upregulation of human immunodeficiency virus-1 in chronically infected monocytic cell line by both contact with endothelial cells and cytokines

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1567-1572 ◽  
Author(s):  
ST Fan ◽  
K Hsia ◽  
TS Edgington
Keyword(s):  
Human Immunodeficiency Virus ◽  
Endothelial Cells ◽  
Viral Gene ◽  
Target Cells ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

Abstract Cells of monocytic lineage (Mo) persistently infected with human immunodeficiency virus (HIV) have been suspected to be a major reservoir for in vivo transmission of virus to susceptible target cells. Cellular events and mechanisms that upregulate viral gene expression in such cells are important issues. Because the traffic of such cells is central to biodistribution of HIV, we have explored the impact of interaction of endothelium with HIV-1-infected U1 promonocytic cells. Coculturing of U1 with human umbilical endothelial cells (HUVEC) for 24 to 72 hours in the absence of stimulation induced HIV-1 p24 biosynthesis significantly. Antibody-blocking experiments indicated that CD11/CD18 integrins play a role in upregulation of HIV expression elicited by interaction with HUVEC. Engagement of CD11b/CD18 by adherence of U1 to surfaces coated with either the cognate ligand fibrinogen or monoclonal antibody specific for CD11b/CD18 also enhanced p24 biosynthesis. Furthermore, endothelial cells were found to constitutively synthesize and secrete soluble factors that enhanced HIV- 1 synthesis. The enhancing factors, of estimated size 10 to 45 kD, were induced in HUVEC to high levels by monokines or by lipopolysaccharide, resulting in markedly enhanced HIV-1 expression by U1. These endothelial cell-derived HIV-1-enhancing factors consist of, among others, interleukin-6 (IL-6), IL-1 beta, and granulocyte-macrophage CSF (GM-CSF). Our results suggest that activation of HIV biosynthesis in infected Mo via interaction with endothelium may impact significantly on the tissue distribution and pathogenesis of HIV infections.

scholarly journals Baseline Resistance of Primary Human Immunodeficiency Virus Type 1 Strains to the CXCR4 Inhibitor AMD3100

2008 ◽  
Vol 82 (23) ◽  
pp. 11695-11704 ◽  
Author(s):  
Jessamina E. Harrison ◽  
Jonathan B. Lynch ◽  
Luz-Jeannette Sierra ◽  
Leslie A. Blackburn ◽  
Neelanjana Ray ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antiviral Agents ◽  
Viral Envelope ◽  
Cxcr4 Antagonist ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1

ABSTRACT We screened a panel of R5X4 and X4 human immunodeficiency virus type 1 (HIV-1) strains for their sensitivities to AMD3100, a small-molecule CXCR4 antagonist that blocks HIV-1 infection via this coreceptor. While no longer under clinical development, AMD3100 is a useful tool with which to probe interactions between the viral envelope (Env) protein and CXCR4 and to identify pathways by which HIV-1 may become resistant to this class of antiviral agents. While infection by most virus strains was completely blocked by AMD3100, we identified several R5X4 and X4 isolates that exhibited plateau effects: as the AMD3100 concentration was increased, virus infection and membrane fusion diminished to variable degrees. Once saturating concentrations of AMD3100 were achieved, further inhibition was not observed, indicating a noncompetitive mode of viral resistance to the drug. The magnitude of the plateau varied depending on the virus isolate, as well as the cell type used, with considerable variation observed when primary human T cells from different human donors were used. Structure-function studies indicated that the V1/V2 region of the R5X4 HIV-1 isolate DH12 was necessary for AMD3100 resistance and could confer this property on two heterologous Env proteins. We conclude that some R5X4 and X4 HIV-1 isolates can utilize the AMD3100-bound conformation of CXCR4, with the efficiency being influenced by both viral and host factors. Baseline resistance to this CXCR4 antagonist could influence the clinical use of such compounds.

scholarly journals Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Hanh T. Nguyen ◽  
Nirmin Alsahafi ◽  
Andrés Finzi ◽  
Joseph G. Sodroski
Keyword(s):  
Envelope Glycoprotein ◽  
Disulfide Bonds ◽  
Virus Entry ◽  
Envelope Proteins ◽  
Immunodeficiency Virus ◽  
Membrane Envelope ◽  
And Function ◽  
State 1 ◽  
Hiv 1

ABSTRACTMost broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.IMPORTANCEThe human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.

scholarly journals Inhibitory role of CXCR4 glycan in CD4-independent X4-tropic human immunodeficiency virus type 1 infection and its abrogation in CD4-dependent infection

2007 ◽  
Vol 88 (11) ◽  
pp. 3139-3144 ◽  
Author(s):  
Yoshinao Kubo ◽  
Masaru Yokoyama ◽  
Hiroaki Yoshii ◽  
Chiho Mitani ◽  
Chika Tominaga ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Virus Infection ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Envelope ◽  
Extracellular Region ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1

CXCR4 functions as an infection receptor of X4 human immunodeficiency virus type 1 (HIV-1) . CXCR4 is glycosylated at the N-terminal extracellular region, which is important for viral envelope (Env) protein binding. We compared the effects of CXCR4 glycan on the CD4-dependent and –independent infections in human cells by X4 viruses. We found that transduction mediated by Env proteins of CD4-independent HIV-1 strains increased up to 5.5-fold in cells expressing unglycosylated CXCR4, suggesting that the CXCR4 glycan inhibits CD4-independent X4 virus infection. Co-expression of CD4 on the target cell surface or pre-incubation of virus particles with soluble CD4 abrogates the glycan-mediated inhibition of X4 virus infection, suggesting that interaction of Env protein with CD4 counteracts the inhibition. These findings indicate that it will be advantageous for X4 HIV-1 to remain CD4-dependent. A structural model that explains the glycan-mediated inhibition is discussed.

scholarly journals Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness

2012 ◽  
Vol 93 (12) ◽  
pp. 2625-2634 ◽  
Author(s):  
Elena Capel ◽  
Glòria Martrus ◽  
Mariona Parera ◽  
Bonaventura Clotet ◽  
Miguel Angel Martínez
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Replication Capacity ◽  
Recombinant Viruses ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

scholarly journals Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer.

1995 ◽  
Vol 182 (1) ◽  
pp. 185-196 ◽  
Author(s):  
Q J Sattentau ◽  
J P Moore
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Neutralization ◽  
V3 Loop ◽  
Immunodeficiency Virus ◽  
Monomeric Gp120 ◽  
Hiv 1

The major target of the neutralizing antibody response to infection by the human immunodeficiency virus type 1 (HIV-1) is the outer envelope glycoprotein, gp120. The spectrum of HIV-1 neutralization specificity is currently represented by monoclonal antibodies (mAbs) that can be divided broadly into five groups. We have studied the binding of these mAbs to functional oligomeric and soluble monomeric gp120 derived from the molecular clone of a cell line-adapted isolate of HIV-1, and compared these binding properties with virus neutralization. Binding of all mAbs except those reactive with the V3 loop was much weaker to oligomeric than to monomeric gp120. This reduction in binding to oligomeric gp120 was determined mostly by a slower relative rate of association, although the dissociation rate also had some influence on relative variation in mAb affinity. Virus neutralization correlated broadly with mAb binding to the oligomeric rather than to the monomeric form of gp120, and neutralization potency was related to the estimated association rate. Thus, with the exception of the hypervariable V3 loop, regions of HIV-1 gp120 with the potential to induce a neutralization response are likely to be poorly presented for antibody recognition on the surface of cell line-adapted virions.

scholarly journals A Dual Infection/Competition Assay Shows a Correlation between Ex Vivo Human Immunodeficiency Virus Type 1 Fitness and Disease Progression

2000 ◽  
Vol 74 (19) ◽  
pp. 9222-9233 ◽  
Author(s):  
Miguel E. Quiñones-Mateu ◽  
Sarah C. Ball ◽  
Andre J. Marozsan ◽  
Vincent S. Torre ◽  
Jamie L. Albright ◽  
...  
Keyword(s):  
Immune Response ◽  
Human Immunodeficiency Virus ◽  
Disease Progression ◽  
Ex Vivo ◽  
Relative Fitness ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

ABSTRACT This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4+ T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

scholarly journals Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

2007 ◽  
Vol 82 (5) ◽  
pp. 2405-2417 ◽  
Author(s):  
Vineela Chukkapalli ◽  
Ian B. Hogue ◽  
Vitaly Boyko ◽  
Wei-Shau Hu ◽  
Akira Ono
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Molecular Mechanisms ◽  
Membrane Binding ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P2-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P2. To examine a putative Gag interaction with PI(4,5)P2, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P2. Using this assay, we observed that PI(4,5)P2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P2-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P2 on the membrane and that the MA basic domain mediates this interaction.

scholarly journals A Single Injection of Recombinant Measles Virus Vaccines Expressing Human Immunodeficiency Virus (HIV) Type 1 Clade B Envelope Glycoproteins Induces Neutralizing Antibodies and Cellular Immune Responses to HIV

2004 ◽  
Vol 78 (1) ◽  
pp. 146-157 ◽  
Author(s):  
Clarisse Lorin ◽  
Lucile Mollet ◽  
Frédéric Delebecque ◽  
Chantal Combredet ◽  
Bruno Hurtrel ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Responses ◽  
Neutralizing Antibodies ◽  
Measles Virus ◽  
Single Injection ◽  
Protein A ◽  
V3 Loop ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.

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