scholarly journals 2549 Characterizing the expression kinetics of HIV-1 envelope protein

2018 ◽  
Vol 2 (S1) ◽  
pp. 7-7
Author(s):  
Djin-Ye Oh ◽  
Lihong Liu ◽  
Benjamin Trinité ◽  
En-Wei Hu-Van Wright ◽  
Vincent Sahi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Virus Production ◽  
Cellular Level ◽  
Hiv Treatment ◽  
Cell Surface Expression ◽  
Substantial Impact ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1 ◽  
Expression Kinetics

OBJECTIVES/SPECIFIC AIMS: Characterize the expression kinetics of HIV-1 Envelope and their relationship to virus production at the cellular level. METHODS/STUDY POPULATION: In vitro and ex vivo laboratory analyses. RESULTS/ANTICIPATED RESULTS: Initial studies addressing the kinetics of cell surface. Envelope (Env) expression reveal that Env expression to peaks on day 2 post infection. Next steps include a series of experiments to compare the kinetics of Env cell surface expression with broadly neutralizing antibody (bNAb)-mediated ADCC and the characterization of virus production kinetics in this same context. To be maximally effective, ADCC elimination of infected cells should occur before peak Env expression. DISCUSSION/SIGNIFICANCE OF IMPACT: Potent bNAbs to HIV-1 recognize vulnerable sites on the HIV-1 Envelope (Env) protein and are of great clinical interest due to their potential use in the prevention and treatment of HIV-1 infection. Their effectiveness depends not only on the neutralization of viral infectivity, but also on the elimination of productively infected cells via antibody-dependent cellular cytotoxicity (ADCC). On a cellular level, ADCC dynamics are determined by the timing and level of Env expression on the surface of HIV-infected cells. This study aims to delineate the expression kinetics of HIV-1 Envelope and their relationship to virus production. We expect that it will provide new insights into the utility of bNAb-mediated ADCC in treating and possibly curing HIV-1 infection; therefore results might have substantial impact on future HIV treatment strategies.

scholarly journals HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell–mediated killing

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1354-1363 ◽  
Author(s):  
Jonathan Richard ◽  
Sardar Sindhu ◽  
Tram N. Q. Pham ◽  
Jean-Philippe Belzile ◽  
Éric A. Cohen
Keyword(s):  
Dna Damage ◽  
Cell Surface ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Infected Cells ◽  
Nkg2d Receptor ◽  
Hiv 1

AbstractHIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

scholarly journals The clonal repopulation of HSPC gene modified with anti–HIV-1 RNAi is not affected by preexisting HIV-1 infection

2020 ◽  
Vol 6 (30) ◽  
pp. eaay9206
Author(s):  
Gajendra W. Suryawanshi ◽  
Wannisa Khamaikawin ◽  
Jing Wen ◽  
Saki Shimizu ◽  
Hubert Arokium ◽  
...  
Keyword(s):  
Integration Site ◽  
Hiv Treatment ◽  
Hematopoietic Stem ◽  
Substantial Impact ◽  
Infected Cells ◽  
And Control ◽  
The Impact ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Selection Of

Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1–infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both anti–HIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1–infected cells—all within the same animal. In our HIV-1–preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1–mediated selection of anti–HIV-1 RNAi-modified clones was evident in HIV-1–infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1–infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.

scholarly journals Kinetics of Antiviral Activity by Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes (CTL) and Rapid Selection of CTL Escape Virus In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6851-6857 ◽  
Author(s):  
C. A. Van Baalen ◽  
M. Schutten ◽  
R. C. Huisman ◽  
P. H. M. Boers ◽  
R. A. Gruters ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Virus Production ◽  
Virus Release ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1 ◽  
Selection Of

ABSTRACT The antiviral activity of a CD8+ cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8+ reverse transcriptase-specific CTLs to HLA-matched CD4+ T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E→K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.

scholarly journals Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

2016 ◽  
Vol 90 (20) ◽  
pp. 9018-9028 ◽  
Author(s):  
G. Martrus ◽  
A. Niehrs ◽  
R. Cornelis ◽  
A. Rechtien ◽  
W. García-Beltran ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Gag Protein ◽  
Novel Approach ◽  
Latently Infected ◽  
Infected Cells ◽  
First Time ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACTHIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection ofgagpolmRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4+T cells, four cell populations were detected according to their expression levels of viral mRNA and protein.gagpolmRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4+T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle.IMPORTANCEEarly after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation orde novoinfection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected andde novo-infected cells dependent on the presence of viral RNA or protein.

scholarly journals Poxvirus Complement Control Proteins Are Expressed on the Cell Surface through an Intermolecular Disulfide Bridge with the Viral A56 Protein

2010 ◽  
Vol 84 (21) ◽  
pp. 11245-11254 ◽  
Author(s):  
Brian C. DeHaven ◽  
Natasha M. Girgis ◽  
Yuhong Xiao ◽  
Paul N. Hudson ◽  
Victoria A. Olson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Disulfide Bridge ◽  
Eukaryotic Cell ◽  
Cell Surface Expression ◽  
Complement Control Proteins ◽  
Express Cell ◽  
Infected Cells

ABSTRACT The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.

Down-Regulation of HLA-G1 Cell Surface Expression in Human Cytomegalovirus Infected Cells

2003 ◽  
Vol 50 (4) ◽  
pp. 328-333 ◽  
Author(s):  
Nathalie Pizzato ◽  
Barbara Garmy-Susini ◽  
Philippe Le Bouteiller ◽  
Francoise Lenfant
Keyword(s):  
Cell Surface ◽  
Human Cytomegalovirus ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Down Regulation ◽  
Infected Cells

scholarly journals Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

1994 ◽  
Vol 5 (7) ◽  
pp. 819-828 ◽  
Author(s):  
Y Wang ◽  
G M Fuller
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
De Novo ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Synthesis Inhibitors ◽  
Different Cell Types ◽  
Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

scholarly journals Human Immunodeficiency Virus Type 1 Vpr Protein Does Not Modulate Surface Expression of the CD4 Receptor

2002 ◽  
Vol 76 (8) ◽  
pp. 4125-4130 ◽  
Author(s):  
Enrique Argañaraz ◽  
María José Cortés ◽  
Sydney Leibel ◽  
Juan Lama
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Surface ◽  
Target Cells ◽  
Viral Receptor ◽  
Cd4 Receptor ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The CD4 receptor is required for the entry of human immunodeficiency virus (HIV) into target cells. It has long been known that Nef, Env, and Vpu participate in the removal of the viral receptor from the cell surface. Recently, it has been proposed that the HIV type 1 (HIV-1) Vpr protein may also play a role in the downmodulation of CD4 from the surfaces of infected cells (L. Conti, B. Varano, M. C. Gauzzi, P. Matarrese, M. Federico, W. Malorani, F. Belardelli, and S. Gessani, J. Virol. 74:10207-10211, 2000). To investigate the possible role of Vpr in the downregulation of the viral receptor Vpr alleles from HIV-1 and simian immunodeficiency virus were transiently expressed in transformed T cells and in 293T fibroblasts, and their ability to modulate surface CD4 was evaluated. All Vpr alleles efficiently arrested cells in the G2 stage of the cell cycle. However, none of the tested Vpr proteins altered the expression of CD4 on the cell surface. In comparison, HIV-1 Nef efficiently downmodulated surface CD4 in all the experimental settings. Transformed T cells and primary lymphocytes were challenged with wild-type, Nef-defective, and Vpr-defective viruses. A significant reduction in the HIV-induced downmodulation of surface CD4 was observed in viruses lacking Nef. However, Vpr-deletion-containing viruses showed no defect in their ability to remove CD4 from the surfaces of infected cells. Our results indicate that Vpr does not play a role in the HIV-induced downmodulation of the CD4 receptor.

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