91 research outputs found
Effect of simultaneous ESCRT-I and ALIX disruption on ubiquitin-dependent and PPxY-dependent particle release.
<p>(A) Quantitative Western blot analysis of VLP production from 293T cells transfected with plasmids expressing Lck-Gag-Ub, Lck-Gag-PY or MLV Gag-HA and siRNAs targeting Tsg101, ALIX or both. Corresponding cell lysates were also probed with antibodies to PFV, HA, Tsg101 and/or ALIX, as appropriate, and as indicated. (B) Quantitation of VLP release following knockdown of Tsg101, ALIX or both. Values are plotted as the mean+SD of two or three independent experiments and represent the levels of particles released relative to those released from cells transfected with control luciferase siRNAs, which was assigned a value of 1.</p
Diversity of pathways that can be used to engage the ESCRT machinery.
<p>The ESCRT pathway can be engaged by Gag using a variety of natural and artificial mechanisms (i)–(v) that include ubiquitin ligation to trans-acting proteins, (perhaps including the ubiquitin ligase itself) or fusion to Gag. These studies suggest that ubiquitin functions like a transferable L-domain, recruiting class E VPS factors such as ESCRT-I and ALIX, independently of the identity if the protein to which it is attached.</p
Quantitative western blotting analysis of virions tethered by the C8Fac and N5Fac proteins.
<p>Quantitative western blotting analysis of virions tethered by the C8Fac and N5Fac proteins.</p
Axially oriented tetherin homodimers directly trap virions.
<p>(<b>A</b>) Schematic representation of the protease-induced virion release assay. Modified tetherin dimers programmed with a protease cleavage site (scissors) are indicated as blue helices, while the WT tetherin dimers are indicated as red helices. If trapped viruses are liberated upon protease treatment only when tetherin is programmed with a protease site, it confirms a role for tetherin as a direct tether in virion entrapment. The liberated virions are subjected to quantitative western blotting analyses to estimate the numbers of tetherin dimers associated with a single virion and their orientation. (<b>B</b>) Schematic representation of the polarities that would be adopted by the C8Fac and N5Fac proteins if HA-tagged proteolytic fragments are observed to partition with liberated virions. (<b>C</b>) Western blot analyses of virions, 293T cells stably expressing C8Fac and N5Fac tetherin proteins, and virions liberated upon Factor Xa treatment. The samples were probed using anti-HA and anti-CA antibodies. (<b>D</b>) Western blot analyses of PNGase-F-treated cells and liberated virions from the above panel. The samples were probed using an anti-HA antibody. Stars indicate non-specific bands. (<b>E</b>) Western blot analyses of 293T cells stably expressing C8Fac and N5Fac tetherin proteins and virions liberated upon Factor Xa or subtilisin A treatment. The samples were probed using an anti-CA antibody.</p
Stimulation of PPxY-dependent VLP production by chimeric HECT ubiquitin ligases.
<p>(A) Quantitative Western blot (LICOR) analysis of VLP release from 293T cells co-expressing Lck-Gag-PY and either YFP alone (None) or the indicated YFP-fused WWP1 C2/WW domains linked to the indicated HECT domains. Note that the unfused YFP is not visible in the “None” lane because it migrates to a different position on the blot. (B) Quantitation of Lck-Gag-PY protein in particles by quantitative Western blot analysis (LICOR). Values plotted are the levels of VLP associated Lck-Gag-PY protein generated in the presence of the indicated YFP-fused chimeric ubiquitin ligase, relative to that generated in the presence of YFP only (None). Data represent the mean and standard deviation of four independent experiments.</p
Antiviral activity of modified tetherin proteins in stable cell lines.
<p>(<b>A</b>) 293T cells stably expressing the modified tetherin proteins were analyzed by flow cytometry to determine the relative surface expression levels of tetherin, using a mouse anti-human tetherin antibody. The mean fluorescence intensity (MFI) for endogenous tetherin in HeLa cells was 5000, while the MFIs for WT, C8Fac, N5Fac and Flag N5Fac tetherin proteins were 6200, 15000, 8800, and 12000 respectively. (<b>B</b>) Western blot analyses of the stable 293T cell lysates and virions harvested from them, following infection with HIV-1 or its Vpu-deficient counterpart at a MOI of 1. All samples were probed with an anti-CA antibody. The three lanes for each tetherin protein are replicates of the experiment.</p
Class E VPS factors and associated proteins encoding ubiquitin binding domains.
<p>SPR: surface plasmon resonance; NMR: nuclear magnetic resonance; IP: immunoprecipitation or bead based ‘pull-down’ assays; Y2H: yeast 2-hybrid.</p
Antiviral activity of the panel of modified tetherin proteins.
<p>(<b>A</b>) Western blot analyses of the 293T cell lysates that were cotransfected with a Vpu-deficient (HIV-1 ΔVpu) proviral pNL4-3 plasmid along with varying amounts of the indicated modified tetherin proteins. All samples were probed with an anti-HA antibody. (<b>B</b>) 293T cells were cotransfected with WT (HIV-1 WT) or Vpu-deficient (HIV-1 ΔVpu) proviral pNL4-3 plasmids along with varying amounts of the indicated modified tetherin proteins. Infectious virion yield was measured by inoculating HeLa-TZM indicator cells with culture supernatant and is given as the logarithm to the base 10 of the relative light units (RLU). (<b>C</b>) Western blot analyses of transfected 293T cell lysates and virions corresponding to the above panel. All samples were probed with an anti-CA antibody. The numbers at the bottom represent measurement of CA protein levels in virion pellets (LI-COR).</p
Quantitative western blotting analysis of virions tethered by the Flag N5Fac protein.
<p>Quantitative western blotting analysis of virions tethered by the Flag N5Fac protein.</p
Preferential insertion of tetherin C-termini into virion envelopes.
<p>(<b>A</b>) Schematic representation of the polarities that would be adopted by the Flag N5Fac protein if HA- or FLAG-tagged proteolytic fragments are observed to partition with liberated virions. (<b>B</b>) Western blot analyses of 293T cells and liberated virions, obtained by the protease treatment of Flag N5Fac cells at various time points following infection. The samples were probed using anti-HA, anti-FLAG and anti-CA antibodies. (<b>C</b>) Western blot analyses of 293T cells expressing serial dilutions of the CA-HA-Flag protein and the PNGase-F-treated liberated virion lysates. The samples were probed with anti-CA, anti-HA and anti-FLAG antibodies. (<b>D</b>) The CA, HA and FLAG band intensities for the control protein were determined using a LiCOR Odyssey scanner and were plotted against the dilution (left panels). Regression analysis was performed over the linear range of signal intensities (right panels), and the unknown amounts of CA, HA and FLAG epitopes in the PNGase-F-treated, Factor Xa-liberated virion lysates were deduced by interpolation from the standard curves. (<b>E</b>) Western blot analyses of 293T cells stably expressing the Flag N5Fac tetherin protein and virions liberated upon Factor Xa or subtilisin A treatment. The samples were probed using an anti-CA antibody.</p
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