scholarly journals Fluorescent Reporter Signals, EGFP, and DsRed, Encoded in HIV-1 Facilitate the Detection of Productively Infected Cells and Cell-Associated Viral Replication Levels

2012 ◽  
Vol 2 ◽  
Author(s):  
Kazutaka Terahara ◽  
Takuya Yamamoto ◽  
Yu-ya Mitsuki ◽  
Kentaro Shibusawa ◽  
Masayuki Ishige ◽  
...  
Keyword(s):  
Viral Replication ◽  
Fluorescent Reporter ◽  
Infected Cells ◽  
Hiv 1

scholarly journals Notwithstanding Circumstantial Alibis, Cytotoxic T Cells Can Be Major Killers of HIV-1-Infected Cells

2016 ◽  
Vol 90 (16) ◽  
pp. 7066-7083 ◽  
Author(s):  
Saikrishna Gadhamsetty ◽  
Tim Coorens ◽  
Rob J. de Boer
Keyword(s):  
T Cells ◽  
Viral Replication ◽  
Cytotoxic T Cells ◽  
Chronic Phase ◽  
Replication Rate ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Eclipse Phase ◽  
Hiv 1

ABSTRACTSeveral experiments suggest that in the chronic phase of human immunodeficiency virus type 1 (HIV-1) infection, CD8+cytotoxic T lymphocytes (CTL) contribute very little to the death of productively infected cells. First, the expected life span of productively infected cells is fairly long, i.e., about 1 day. Second, this life span is hardly affected by the depletion of CD8+T cells. Third, the rate at which mutants escaping a CTL response take over the viral population tends to be slow. Our main result is that all these observations are perfectly compatible with killing rates that are much faster than one per day once we invoke the fact that infected cells proceed through an eclipse phase of about 1 day before they start producing virus. Assuming that the major protective effect of CTL is cytolytic, we demonstrate that mathematical models with an eclipse phase account for the data when the killing is fast and when it varies over the life cycle of infected cells. Considering the steady state corresponding to the chronic phase of the infection, we find that the rate of immune escape and the rate at which the viral load increases following CD8+T cell depletion should reflect the viral replication rate, ρ. A meta-analysis of previous data shows that viral replication rates during chronic infection vary between 0.5 ≤ ρ ≤ 1 day−1. Balancing such fast viral replication requires killing rates that are several times larger than ρ, implying that most productively infected cells would die by cytolytic effects.IMPORTANCEMost current data suggest that cytotoxic T cells (CTL) mediate their control of human immunodeficiency virus type 1 (HIV-1) infection by nonlytic mechanisms; i.e., the data suggest that CTL hardly kill. This interpretation of these data has been based upon the general mathematical model for HIV infection. Because this model ignores the eclipse phase between the infection of a target cell and the start of viral production by that cell, we reanalyze the same data sets with novel models that do account for the eclipse phase. We find that the data are perfectly consistent with lytic control by CTL and predict that most productively infected cells are killed by CTL. Because the killing rate should balance the viral replication rate, we estimate both parameters from a large set of published experiments in which CD8+T cells were depleted in simian immunodeficiency virus (SIV)-infected monkeys. This confirms that the killing rate can be much faster than is currently appreciated.

scholarly journals Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

2015 ◽  
Vol 89 (18) ◽  
pp. 9639-9652 ◽  
Author(s):  
Mako Toyoda ◽  
Yoko Ogata ◽  
Macdonald Mahiti ◽  
Yosuke Maeda ◽  
Xiaomei T. Kuang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Viral Entry ◽  
Immune Escape ◽  
Elite Controllers ◽  
Functional Impairments ◽  
Disease Phenotypes ◽  
Infected Cells ◽  
Entry Receptor ◽  
Hiv 1

ABSTRACTHIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified viain vitromutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here,nefclones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linkedin vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.IMPORTANCEHIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressurein vivoremains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein functionin vivo.

scholarly journals Cellular minichromosome maintenance complex component 5 (MCM5) is incorporated into HIV-1 virions and modulates viral replication in the newly infected cells

Virology ◽  
2016 ◽  
Vol 497 ◽  
pp. 11-22 ◽  
Author(s):  
Steven Santos ◽  
Yuri Obukhov ◽  
Sergei Nekhai ◽  
Tatiana Pushkarsky ◽  
Beda Brichacek ◽  
...  
Keyword(s):  
Viral Replication ◽  
Minichromosome Maintenance ◽  
Complex Component ◽  
Infected Cells ◽  
Hiv 1

RANTES upregulation by HIV-1-infected cells: lack of correlation with viral replication and induction of interferon

AIDS ◽  
1999 ◽  
Vol 13 (2) ◽  
pp. 288 ◽  
Author(s):  
I. Abbate ◽  
F. Carletti ◽  
F. Dianzani ◽  
M.R. Capobianchi
Keyword(s):  
Viral Replication ◽  
Infected Cells ◽  
Hiv 1

scholarly journals Infection with a newly-designed dual fluorescent reporter HIV-1 effectively identifies latently infected CD4+ T cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jinfeng Cai ◽  
Hongbo Gao ◽  
Jiacong Zhao ◽  
Shujing Hu ◽  
Xinyu Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Stable Cell Line ◽  
Fluorescent Reporter ◽  
Major Barrier ◽  
Latently Infected ◽  
Integration Sites ◽  
Compound Screening ◽  
Infected Cells ◽  
Hiv 1

The major barrier to curing HIV-1 infection is a small pool of latently infected cells that harbor replication-competent viruses, which are widely considered the origin of viral rebound when ART is interrupted. The difficulty of distinguishing latently infected cells from the vast majority of uninfected cells has represented a significant bottleneck precluding comprehensive understandings of HIV-1 latency. Here we reported and validated a newly-designed dual fluorescent reporter virus, DFV-B, infection with which in primary CD4+ T cells can directly label latently infected cells and generate a latency model that was highly physiological relevant. Applying DFV-B infection in Jurkat T cells, we generated a stable cell line model of HIV-1 latency with diverse viral integration sites. High-throughput compound screening with this model identified ACY-1215 as a potent latency reversing agent, which could be verified in other cell models and in primary CD4+ T cells from ART-suppressed individuals ex vivo. In summary, we have generated a meaningful and feasible model to directly study latently infected cells, which could open up new avenues to explore the critical events of HIV-1 latency and become a valuable tool for the research of AIDS functional cure.

scholarly journals CD4 Incorporation into HIV-1 Viral Particles Exposes Envelope Epitopes Recognized by CD4-Induced Antibodies

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Shilei Ding ◽  
Romain Gasser ◽  
Gabrielle Gendron-Lepage ◽  
Halima Medjahed ◽  
William D. Tolbert ◽  
...  
Keyword(s):  
Viral Replication ◽  
Accessory Protein ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Profound Impact ◽  
Functional Consequence ◽  
Viral Particles ◽  
Primate Lentiviruses ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT CD4 downregulation on infected cells is a highly conserved function of primate lentiviruses. It has been shown to positively impact viral replication by a variety of mechanisms, including enhanced viral release and infectivity, decrease of cell reinfection, and protection from antibody-dependent cellular cytotoxicity (ADCC), which is often mediated by antibodies that require CD4 to change envelope (Env) conformation. Here, we report that incorporation of CD4 into HIV-1 viral particles affects Env conformation resulting in the exposure of occluded epitopes recognized by CD4-induced antibodies. This translates into enhanced neutralization susceptibility by these otherwise nonneutralizing antibodies but is prevented by the HIV-1 Nef accessory protein. Altogether, these findings suggest that another functional consequence of Nef-mediated CD4 downregulation is the protection of viral particles from neutralization by commonly elicited CD4-induced antibodies. IMPORTANCE It has been well established that Env-CD4 complexes expose epitopes recognized by commonly elicited CD4-induced antibodies at the surface of HIV-1-infected cells, rendering them vulnerable to ADCC responses. Here, we show that CD4 incorporation has a profound impact on Env conformation at the surface of viral particles. Incorporated CD4 exposes CD4-induced epitopes on Env, rendering HIV-1 susceptible to neutralization by otherwise nonneutralizing antibodies.

scholarly journals The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Yvonne Affram ◽  
Juan C. Zapata ◽  
Zahra Gholizadeh ◽  
William D. Tolbert ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Cell Surface ◽  
Viral Replication ◽  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Integral Protein ◽  
Viral Particles ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target. IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

scholarly journals Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture

2009 ◽  
Vol 54 (3) ◽  
pp. 1047-1054 ◽  
Author(s):  
Daniel A. Donahue ◽  
Richard D. Sloan ◽  
Björn D. Kuhl ◽  
Tamara Bar-Magen ◽  
Susan M. Schader ◽  
...  
Keyword(s):  
Viral Load ◽  
Viral Replication ◽  
Reverse Transcription ◽  
Antiretroviral Drugs ◽  
Proteolytic Processing ◽  
Viral Inhibition ◽  
Dependent Inhibition ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.

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