Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
[Abstract] The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence ...
Mismatched Primer Extension Assays
Authors: Vasudevan Achuthan and
Jeffrey J. DeStefano,
date: 06/20/2015,
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[Abstract] Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used ...
HIV-1 Fusion Assay
[Abstract] The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a ...
HIV-1 Virus-like Particle Budding Assay
Authors: Nathan H Vande Burgt,
Luis J Cocka and
Paul Bates,
date: 07/20/2013,
view: 9752,
Q&A: 0
[Abstract] Viral replication culminates with the egress of the mature virion from the host cell. This step of the viral life cycle has recently garnered increased attention with the discovery of the cellular restriction factor, Tetherin, which tethers budded virions to the surface of infected cells and inhibits viral spread. The importance of this block in ...