Nef Does Not Affect the Efficiency of Human Immunodeficiency Virus Type 1 Fusion with Target Cells

Tobiume, Minoru; Lundquist, Christopher A.; Miller, Michael D.; Aiken, Christopher

doi:10.1128/jvi.77.19.10645-10650.2003

Cited by 89 publications

(96 citation statements)

References 54 publications

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“…HIV-1 pseudoviruses bearing HXB2, Bal26, and R3A envelope glycoproteins and the b-lactamase-Vpr chimera (BlaM-Vpr) were produced by cotransfecting HEK293T/17 cells with the HIV-based pR8DEnv packaging vector, pMM310 plasmid expressing BlaM-Vpr, 47 pcRev, 48 and the vector encoding HXB2 Env 49 or Bal26 (a gift from P. Clapham, University of Massachusetts, Worchester, MA) or R3A (a gift from Dr. J. Hoxie, University of Pennsylvania, Philadelphia, PA), using the jetPRIME Ò transfection reagent (VWR, Radnor, PA). The transfection medium was replaced with fresh DMEM/ 10% FBS after 12 h and cells were cultured for 36 h, after which time the virus-containing culture medium was collected, passed through a 0.45-mm filter, aliquoted, and stored at -80°C.…”

Section: Virus Productionmentioning

confidence: 99%

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Marin

Giroud

et al. 2015

ASSAY and Drug Development Technologies

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HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors. Here, we adapted and optimized an enzymatic HIV-cell fusion assay, which reports the transfer of virus-encapsulated b-lactamase into the cytoplasm, to high-throughput screening (HTS) with a 384-well format. The assay was robustly performed in HTS format and was validated by the pilot screen of a small library of pharmacologically active compounds. Several hits identified by screening included a prominent cluster of purinergic receptor antagonists. Functional studies demonstrated that P2X1 receptor antagonists selectively inhibited HIV-1 fusion without affecting the fusion activity of an unrelated virus that enters cells through an endocytic route. The inhibition of HIV-cell fusion by P2X1 antagonists was not through downmodulation of the cell surface expression of CD4 or coreceptors, thus implicating P2X1 receptor in the HIV-1 fusion step. The ability of these antagonists to inhibit viruses regardless of their coreceptor (CXCR4 or CCR5) preference indicates that fusion is blocked at a late step downstream of coreceptor binding. A future large-scale screening campaign for HIV-1 fusion inhibitors, using the above functional readout, will likely reveal novel classes of inhibitors and suggest potential targets for antiviral therapy.

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Section: Virus Productionmentioning

confidence: 99%

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Marin

Giroud

et al. 2015

ASSAY and Drug Development Technologies

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“…When release of viral contents alone is measured, levels of infection, rather than fusion itself, have traditionally been measured. Because a multitude of steps occur downstream of the fusion step, only a small fraction of the fused virus particles may cause infection, and thus infection levels need not correspond to fusion levels (Tobiume et al, 2003;Daecke et al, 2005). In the past few years, assays have been developed that directly monitor the release of virus contents into a host cell subsequent to viruscell fusion, and these provide a direct measure of fusion.…”

Section: Introductionmentioning

confidence: 99%

“…In the past few years, assays have been developed that directly monitor the release of virus contents into a host cell subsequent to viruscell fusion, and these provide a direct measure of fusion. For example, one can introduce ␤-lactamase into the viral core (Cavrois et al, 2002;Tobiume et al, 2003;Barnard et al, 2004;Wyma et al, 2004;Daecke et al, 2005) or associate luciferase with the viral envelope proteins (Kolokoltsov and Davey, 2004) and quantify fusion from measures of these enzymatic activities within cells. But these assays have limited temporal resolution, and the transfer of enzymes is not measured in real time.…”

Section: Introductionmentioning

confidence: 99%

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Markosyan

Cohen

Melikyan

2005

MBoC

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A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge. INTRODUCTIONIn the process of membrane fusion, two continuities are created: separate membranes join and distinct aqueous compartments become one. To identify the mechanisms of membrane fusion, both continuities should be followed and the temporal order in which they occur identified. In the case of the HIV-1 fusion protein Env, mechanistic studies have largely relied on fusion of cells expressing the protein to cells expressing CD4 and cognate chemokine receptors (e.g., Munoz-Barroso et al., 1998;Melikyan et al., 2000;Reeves et al., 2002;Abrahamyan et al., 2003;Gallo et al., 2003;Markosyan et al., 2003). But monitoring lipid dye spread in HIV-1 Env-mediated cell-cell fusion has not proved to be very useful in following membrane continuity, for several reasons. The dye does not reliably move between cells, and when it does move, the time course is slow, in large part because the dye segregates nonuniformly over the cell membrane. Also, the dye enters intracellular pools, making it difficult to follow transfer between fused membranes. Furthermore, in cell-cell fusion, the two bound cells are in contact over an appreciable area, so fusion could potentially occur at many sites (Frolov et al., 2000;Leikina and Chernomordik, 2000); the sites of origin for the observed lipid and aqueous dye spread could well be different.By studying fusion of viral particles to cells, these problems can be overcome. The small size of virus minimizes the area of contact and accurately reflects the biological situation. Also, membrane retrieval processes that internalize lipid dye are absent in virions, and the viral envelope is simpler than a cell membrane. But until recently, suitable assays to follow both lipid and contents mixing and to time-order the...

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“…The expression of Nef in the virus-producing cell is required for infectivity enhancement, indicating that Nef modifies the virion during particle assembly or maturation (2). Nef-defective HIV-1 particles fuse efficiently with cells (5,10,23), though one recent study reported a defect in the incorporation of envelope glycoprotein in Nefdefective HIV-1 particles (20). Nef-defective virions are impaired for reverse transcription in target cells despite containing normal levels of viral genomic RNA and a normal quantity of active reverse transcriptase enzyme (2,8,21).…”

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confidence: 99%

Selective Restriction of Nef-Defective Human Immunodeficiency Virus Type 1 by a Proteasome-Dependent Mechanism

Aiken

2007

J Virol

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The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nefdefective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells.

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Nef Does Not Affect the Efficiency of Human Immunodeficiency Virus Type 1 Fusion with Target Cells

Cited by 89 publications

References 54 publications

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Selective Restriction of Nef-Defective Human Immunodeficiency Virus Type 1 by a Proteasome-Dependent Mechanism

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