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Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells
摘要： Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.
关键词： HIV-1,Gag protein,virus assembly,CD4+ T cells,single-molecule localisation microscopy
更新于2025-09-23 15:21:01
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Co-ordinated Split Aptamer Assembly and Disassembly on Gold Nanoparticle for Functional Detection of HIV-1 Tat
摘要： Human immunodeficiency virus (HIV) is a life threatening, weakens the immune system upon infection, thus ultimately resulting in the fatal health issues. This situation necessitates the generation of different strategies for HIV detection. HIV-1 Tat, a transactivator of HIV gene expression, was chosen in this study as the target of a non-functional split aptamer. Implementation of split aptamer has been demonstrated in this work for colorimetric detection of HIV-1 Tat. An unmodified gold nanoparticle (GNP)-based colorimetric assay was used for the visible detection of the proof, displays color transitions from red to purple in relation to the dose-dependency of HIV-1 Tat against the split aptamer in ionic solutions. The visible color transition was characterized using UV-Vis spectrophotometer showing spectrum shift and supported by Scanning Electron Microscopy observation. With addition of sodium chloride, the color of the solution started to change to purple and spectrum started to shift to higher wavelength due to aggregation at HIV-1 Tat concentration as low as 10 nM. Specificity test was conducted with duplexed split aptamer and HIV-1 p24 has shown slight color changes. With HIV-1 Nef, GNP solution retains the color similar to the control, which indicated the specific split aptamer interaction to HIV-1 Tat.
关键词： Colorimetry,HIV-1 Tat,Gold nanoparticle,Split aptamer
更新于2025-09-23 15:19:57
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Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions
摘要： Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.
关键词： broadly neutralizing antibodies,STED microscopy,HIV-1,Env glycoprotein,MPER
更新于2025-09-11 14:15:04
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High-performance interactive analysis of split aptamer and HIV-1 Tat on multiwall carbon nanotube-modified field-effect transistor
摘要： Interaction between split RNA aptamer and the clinically important target, HIV-1 Tat was investigated on a biosensing surface transduced by functionally choreographed multiwall carbon nanotubes (MWCNTs). Acid oxidation was performed to functionalize MWCNTs with carboxyl functional groups. X-ray photoelectron spectroscopy analysis had profound ~2.91% increment in overall oxygen group and ~1% increment was noticed with a specific carboxyl content owing to C=O and O–C=O bonding. The interaction between split RNA aptamer and HIV-1 Tat protein was quantified by electrical measurements with the current signal (Ids) over a gate voltage (Vgs). Initially, 34.4 mV gate voltage shift was observed by the immobilization of aptamer on MWCNT. With aptamer and HIV-1 Tat interaction, the current flow was decreased with the concomitant gate voltage shift of 23.5 mV. The attainment of sensitivity with split aptamer and HIV-1 Tat interaction on the fabricated device was 600 pM. To ensure the genuine interaction of aptamer with HIV-1 Tat, other HIV-1 proteins, Nef and p24 were interacted with aptamer and they displayed the negligible interferences with gate voltage shift of 3.5 mV and 5.7 mV, which shows 4 and 2.5 folds lesser than HIV-1 Tat interaction, respectively.
关键词： Field effect transistor,Multiwall carbon nanotube,Split aptamer,HIV-1 Tat
更新于2025-09-09 09:28:46