scholarly journals Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

2015 ◽  
Vol 210 (4) ◽  
pp. 629-646 ◽  
Author(s):  
Jelle Hendrix ◽  
Viola Baumgärtel ◽  
Waldemar Schrimpf ◽  
Sergey Ivanchenko ◽  
Michelle A. Digman ◽  
...  
Keyword(s):  
Live Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gag Polyprotein ◽  
Viral Particles ◽  
Infected Cells ◽  
Retroviral Replication ◽  
Gag Assembly ◽  
Reduced Mobility ◽  
Hiv 1

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.

scholarly journals Determination of Number of Infected Cells and Concentration of Viral Particles in Plasma during HIV-1 Infections Using Shehu Transformation

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
M. Higazy ◽  
Sudhanshu Aggarwal ◽  
Y. S. Hamed
Keyword(s):  
Initial Conditions ◽  
Integral Transformation ◽  
Mathematical Representation ◽  
Infection Model ◽  
Viral Particles ◽  
Infected Cells ◽  
Linear Differential ◽  
The Mathematical Model ◽  
Ordinary Linear Differential Equations ◽  
Hiv 1

In this paper, the authors determine the number of infected cells and concentration of infected (viral) particles in plasma during HIV-1 (human immunodeficiency virus type one) infections using Shehu transformation. For this, the authors first defined some useful properties of Shehu transformation with proof and then applied Shehu transformation on the mathematical representation of the HIV-1 infection model. The mathematical model of HIV-1 infections contains a system of two simultaneous ordinary linear differential equations with initial conditions. Results depict that Shehu transformation is very effective integral transformation for determining the number of infected cells and concentration of viral particles in plasma during HIV-1 infections.

scholarly journals Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment

2016 ◽  
Vol 32 (7) ◽  
pp. 705-717 ◽  
Author(s):  
Zahra F. Parker ◽  
Ann H. Rux ◽  
Amber M. Riblett ◽  
Fang-Hua Lee ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Viral Attachment ◽  
Hiv 1

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 Nef Enhances Human Immunodeficiency Virus Replication and Responsiveness to Interleukin-2 in Human Lymphoid Tissue Ex Vivo

1999 ◽  
Vol 73 (5) ◽  
pp. 3968-3974 ◽  
Author(s):  
Svetlana Glushakova ◽  
Jean-Charles Grivel ◽  
Kalachar Suryanarayana ◽  
Pascal Meylan ◽  
Jeffrey D. Lifson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Dependent Manner ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Human Lymphoid Tissue ◽  
Hiv 1

ABSTRACT The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with anef mutant (ΔnefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than ΔnefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4+ T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nefgene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, ΔnefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.

scholarly journals An ultrasensitive planar array p24 Gag ELISA to detect individual HIV-1 viral particles and infected cells

2021 ◽  
Author(s):  
Alberto Bosque ◽  
Callie Levinger ◽  
J Natalie Howard ◽  
Pingtao Tang ◽  
Amit Joshi
Keyword(s):  
Biological Fluids ◽  
Low Frequency ◽  
Viral Reservoirs ◽  
Hiv Persistence ◽  
Viral Particles ◽  
Planar Array ◽  
Infected Cells ◽  
Major Loss ◽  
Hiv 1

Abstract Human Immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the SimoaTM planar array technology that can detect as low as a single HIV-1 particle and a single HIV-1 infected cell. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the ‘homebrew' planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids.

scholarly journals Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1109 ◽  
Author(s):  
Assia Mouhand ◽  
Marco Pasi ◽  
Marjorie Catala ◽  
Loussiné Zargarian ◽  
Anissa Belfetmi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleocapsid Protein ◽  
Nucleic Acid Binding ◽  
Binding Properties ◽  
Gag Polyprotein ◽  
C Terminus ◽  
Viral Particles ◽  
Hiv 1 ◽  
Unstructured Regions

HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with nucleic acids, and p1 and p6, two unstructured regions, p6 containing the motifs to bind ALIX, the cellular ESCRT factor TSG101 and the viral protein Vpr. The processing of Gag by the viral protease subsequently liberates NCp15 (NC-p1-p6), NCp9 (NC-p1) and NCp7, NCp7 displaying the optimal chaperone activity of nucleic acids. This review focuses on the nucleic acid binding properties of the NC domain in the different maturation states during the HIV-1 viral cycle.

Comparative study of ultrastructure of HIV and SIV

1990 ◽  
Vol 48 (3) ◽  
pp. 342-343
Author(s):  
Takashi Nakano ◽  
Shunro Imura ◽  
Masuyo Nakai
Keyword(s):  
Osmium Tetroxide ◽  
Staining Method ◽  
Culture Fluid ◽  
Ultrastructural Feature ◽  
Uranyl Acetate ◽  
Negative Staining ◽  
Thin Sections ◽  
Viral Particles ◽  
Infected Cells ◽  
Hiv 1

The ultrastructural feature of AIDS viruses (HIV and SIV) was observed by ultrathin-sectioning and negative staining method, and the surface structures of these viruses were compared.HIV-1 group including, HTLV-III and LAV-1 were used. And in HIV-2 group, LAV-2, which was isolated from a Senegalese AIDS patient, and GH-1, which was isolated from a Ghanan AIDS patient, were used. SIV group was used AGM-1, which was isolated from an African green monkey. The virions were used which was produced from Molt-4 cells after the infection of each viruses of these strains. The infected cells and viral particles, which were collected by centrifugation were fixed with 2% glutaraldehyde (GA), washed with PBS, and postfixed with 1% osmium tetroxide. The specimens were dehydrated in graded ethanol, and embedded in Epon 812. The thin sections were stained with uranyl acetate and lead citrate. On the other hand, for negative staining, 25% GA was added to the supernatant culture fluid and finally concentrated on 2% GA. Then virions were collected by ultracentrifugation.

scholarly journals Type I Phosphatidylinositol-4-Phosphate 5-Kinases α and γ Play a Key Role in Targeting HIV-1 Pr55Gag to the Plasma Membrane

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Baptiste Gonzales ◽  
Hugues de Rocquigny ◽  
Anne Beziau ◽  
Stephanie Durand ◽  
Julien Burlaud-Gaillard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Family Type ◽  
Type I ◽  
Gag Polyprotein ◽  
The Matrix ◽  
Intracellular Compartments ◽  
Infected Cells ◽  
Phosphatidylinositol 4 ◽  
Hiv 1

ABSTRACT HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1β, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1β is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process. IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag. However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.

scholarly journals Constitutive Siglec-1 expression confers susceptibility to HIV-1 infection of human dendritic cell precursors

2019 ◽  
Vol 116 (43) ◽  
pp. 21685-21693 ◽  
Author(s):  
Nicolas Ruffin ◽  
Ester Gea-Mallorquí ◽  
Flavien Brouiller ◽  
Mabel Jouve ◽  
Aymeric Silvin ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Constitutive Expression ◽  
Dependent Manner ◽  
Human Dendritic Cell ◽  
Restriction Factors ◽  
Functional Feature ◽  
Viral Particles ◽  
Intracellular Compartments ◽  
Functional Features ◽  
Hiv 1

The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population of blood circulating DC precursors (pre-DCs). Whether this new DC population possesses specific functional features as compared to the other blood DC subset upon pathogen encounter remained to be evaluated. A unique feature of pre-DCs among blood DCs is their constitutive expression of the viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not other blood DC subsets, are susceptible to infection by HIV-1 in a Siglec-1–dependent manner. Siglec-1 mediates pre-DC infection of CCR5- and CXCR4-tropic strains. Infection of pre-DCs is further enhanced in the presence of HIV-2/SIVmac Vpx, indicating that Siglec-1 does not counteract restriction factors such as SAMHD1. Instead, Siglec-1 promotes attachment and fusion of viral particles. HIV-1–infected pre-DCs produce new infectious viral particles that accumulate in intracellular compartments reminiscent of the virus-containing compartment of macrophages. Pre-DC activation by toll-like receptor (TLR) ligands induces an antiviral state that inhibits HIV-1 fusion and infection, but Siglec-1 remains functional and mediates replication-independent transfer of HIV-1 to activated primary T lymphocytes. Altogether, Siglec-1–mediated susceptibility to HIV-1 infection of pre-DCs constitutes a unique functional feature that might represent a preferential relationship of this emerging cell type with viruses.

HIV-1 Tat promotes monocyte chemoattractant protein-1 secretion followed by transmigration of monocytes

Blood ◽  
2001 ◽  
Vol 97 (2) ◽  
pp. 352-358 ◽  
Author(s):  
In-Woo Park ◽  
Jian-Feng Wang ◽  
Jerome E. Groopman
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Dependent Manner ◽  
Endothelial Monolayer ◽  
Monocyte Chemoattractant Protein 1 ◽  
Pkc Inhibitor ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Infected Cells ◽  
Hiv 1

Abstract The mechanism whereby HIV-infected cells transit from the bloodstream into tissues is not well defined. This phenomenon was addressed by studying the effects of HIV-1 Tat, a protein secreted by infected cells, on human lung microvascular endothelial cells (HMVEC-Ls). It was found that monocyte chemoattractant protein-1 (MCP-1) was released from HMVEC-Ls in a dose- and time-dependent manner after Tat treatment. MCP-1 is a potent β-chemokine that recruits monocytes and T cells and promotes cell adhesion and transmigration across an endothelial monolayer. It was also observed that MCP-1 and the culture medium from Tat-treated HMVEC-Ls were chemotactic for CD14+ monocytes from human peripheral blood and for THP-1, a promonocytic cell line used as a model system. To characterize the signaling pathways underlying the observed induction of MCP-1, HMVEC-Ls were treated with 2 different protein kinase inhibitors: PD98059, a MAP kinase inhibitor, and GF109203X, a protein kinase C (PKC) inhibitor. MCP-1 release was significantly reduced when PKC was inhibited, and slightly decreased when PI3 kinase was blocked; no effect on MCP-1 release was observed on MAP kinase inhibition. Similarly, transmigration of THP-1 cells was significantly impaired by the PKC inhibitor, but not by the other tested inhibitors. These data indicate that the HIV-1 Tat protein may act as a protocytokine by causing the release of MCP-1 from the endothelial monolayer, and thereby facilitating monocyte transmigration into tissues via a PKC signaling pathway.

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