The Cyclosporin A Washout Assay to Detect HIV-1 Uncoating in Infected Cells
Uncoating is an early step of HIV-1 replication in which the viral capsid disassembles by p24 capsid (p24CA) protein dissociating from the viral complex. Although uncoating is required for HIV-1 replication, many questions remain about the mechanism of this process as well as its impact on other steps in viral replication. Here we describe a recently developed assay to study the process of uncoating in HIV-1-infected cells. The CsA washout assay is a cell-based assay that utilizes the HIV-1 restriction factor TRIM-CypA to detect and inhibit infection of coated viral complexes. Owl monkey kidney (OMK) cells are infected with a GFP reporter virus and TRIM-CypA restriction is switched on at various times postinfection allowing the kinetics of uncoating to be monitored in infected cells. This assay also can be used to examine the effect of different viral or cellular factors on the process of uncoating.
Key wordsHIV-1 Capsid Uncoating Retrovirus CsA washout assay TRIM-CypA
- 1.Bukrinsky MI, Sharova N, McDonald TL, Pushkarskaya T, Tarpley WG, Stevenson M (1993) Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A 90:6125–6129PubMedCentralCrossRefPubMedGoogle Scholar
- 12.Tang S, Murakami T, Agresta BE, Campbell S, Freed EO, Levin JG (2001) Human immunodeficiency virus type 1 N-terminal capsid mutants that exhibit aberrant core morphology and are blocked in initiation of reverse transcription in infected cells. J Virol 75:9357–9366PubMedCentralCrossRefPubMedGoogle Scholar
- 16.Stremlau M, Perron M, Lee M, Li Y, Song B, Javanbakht H, Diaz-Griffero F, Anderson DJ, Sundquist WI, Sodroski J (2006) Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci U S A 103: 5514–5519PubMedCentralCrossRefPubMedGoogle Scholar
- 17.Perez-Caballero D, Hatziioannou T, Zhang F, Cowan S, Bieniasz PD (2005) Restriction of human immunodeficiency virus type 1 by TRIM-CypA occurs with rapid kinetics and independently of cytoplasmic bodies, ubiquitin, and proteasome activity. J Virol 79: 15567–15572PubMedCentralCrossRefPubMedGoogle Scholar