Capsid Stability Assay

Diagram of the stabilization assay. In vitro-assembled HIV-1 CA-NC complexes, shown here as tubular structures, are formed by monomeric recombinant HIV-1 CA-NC fusion proteins under highly ionic conditions and recapitulate the surface of the HIV-1 core. When HIV-1 CA-NC complexes are incubated in destabilization buffer, they disassemble spontaneously. Disassembled capsids are layered on top of a 70 % sucrose cushion; however, the disassembled capsid does not cross the cushion after spinning at 100,000 × g for 1 h. In contrast, incubation of HIV-1 CA-NC in stabilization buffer preserves the assembled structures, which pellet when layered onto a 70 % sucrose cushion with spinning at 100,000 × g for 1 h. Input, a fraction of the sample layered onto the sucrose cushion before the centrifugation step. Pellet, a fraction of the capsid pelleted after the sample has been centrifuged at 100,000 × g for 1 h. Input and pellet samples were analyzed by Western b lotting using anti-p24 antibodies.

Over the years, sensitive assays to biochemically measure core stability have been developed, such as the “fate of the capsid” assay that measures core stability during infection of cells over time [ Stremlau et al. 2006, Diaz-Griffero et al. 2007a, Diaz-Griffero et al. 2007b ]. Even though this assay is widely used [ Diaz-Griffero et al. 2007a, Roa et al. 2012, Yang et al. 2013, Diaz-Griffero et al. 2007b, Shi et al. 2011, De laco et al. 2013, Perron et al. 2007, Diaz-Griffero et al. 2008, Ohkura et al. 2011, Berube et al. 2011], it is intensive and laborious and a more rapid assay to measure core stability is desirable.

My lab developed a rapid and simple assay to measure capsid stability in vitro using in vitro-assembled HIV-1 CA-NC complexes as a surrogate for the HIV-1 core [ Fricke et al. 2013 ]. This assay will assist the evaluation of drugs or proteins that change the stability of capsid. Furthermore, this assay could be used to identify novel cellular factors that bind to the HIV-1 core.

Fricke T, Brandariz-Nuñez A, Wang X, Smith AB, Diaz-Griffero F. Human cytosolic extract stabilize the HIV-1 core. 2013; 87(19): 10587-10597.DOI:10.1128/JVI.01705-13

Fricke T, Diaz-Griffero F. HIV-1 capsid stabilization assay. 2016; 1354:39-47.DOI:10.1007/978-1-4939-3046-3_3