scholarly journals Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy

Author(s):  
Derrick Lau ◽  
James C. Walsh ◽  
Claire F. Dickson ◽  
Andrew Tuckwell ◽  
Jeffrey H. Stear ◽  
...  
2020 ◽  
Author(s):  
Derrick Lau ◽  
James C. Walsh ◽  
Claire F. Dickson ◽  
Andrew Tuckwell ◽  
Jeffrey H. Stear ◽  
...  

The HIV capsid is a multifunctional protein capsule for delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labelled capsid-binding analytes (‘prey’ molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using fluorescent capsid as bait further allows quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for discovery and characterization of molecules used by HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.


2002 ◽  
Vol 277 (41) ◽  
pp. 38045-38052 ◽  
Author(s):  
Jo Vercammen ◽  
Goedele Maertens ◽  
Melanie Gerard ◽  
Erik De Clercq ◽  
Zeger Debyser ◽  
...  

Author(s):  
James K. Koehler ◽  
Steven G. Reed ◽  
Joao S. Silva

As part of a larger study involving the co-infection of human monocyte cultures with HIV and protozoan parasites, electron microscopic observations were made on the course of HIV replication and infection in these cells. Although several ultrastructural studies of the cytopathology associated with HIV infection have appeared, few studies have shown the details of virus production in “normal,” human monocytes/macrophages, one of the natural targets of the virus, and suspected of being a locus of quiescent virus during its long latent period. In this report, we detail some of the interactions of developing virons with the membranes and organelles of the monocyte host.Peripheral blood monocytes were prepared from buffy coats (Portland Red Cross) by Percoll gradient centrifugation, followed by adherence to cover slips. 90-95% pure monocytes were cultured in RPMI with 5% non-activated human AB serum for four days and infected with 100 TCID50/ml of HIV-1 for four hours, washed and incubated in fresh medium for 14 days.


1997 ◽  
Vol 23 (3) ◽  
pp. 83-92 ◽  
Author(s):  
D. Seilhean ◽  
A. Dzia-Lepfoundzou ◽  
V. Sazdovitch ◽  
B. Cannella ◽  
C. S. Raine ◽  
...  

2000 ◽  
Vol 14 (2) ◽  
pp. 50-55
Author(s):  
FORTHEPEDIATRICPULMONARYANDCA ◽  
H COHEN ◽  
X CHEN ◽  
S SUNKLE ◽  
L DAVIS ◽  
...  

2020 ◽  
Author(s):  
Juan Ambrosioni ◽  
Elisa Petit ◽  
Geoffroy Liegeon ◽  
Montserrat Laguno ◽  
José M Miró
Keyword(s):  

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