title: In vitro uncoating of HIV-1 cores.
abstract: The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone(1, 2). Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form(3) in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells(4). This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability(4, 5, 6). It should also be useful for studying the role of cellular factors in HIV-1 uncoating.
authors: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine. vaibhav.b.shah@vanderbilt.edu
authors: Vaibhav B
authors: VB
authors: Shah
authors: Christopher
authors: C
authors: Aiken
mesh_terms: D002213:Capsid / Q000737:chemistry*; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods*; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D013395:Sucrose; D014461:Ultracentrifugation / Q000379:methods; D014771:Virion / Q000737:chemistry*; D014773:Virology / Q000379:methods*
chemical_list: D013395:Sucrose