Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.
Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process.
Department of Pediatric, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Ashwanth C
AC
Francis
Department of Pediatric, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Mariana
M
Marin
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center. Nashville, Tennessee, United States of America.
Jiong
J
Shi
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center. Nashville, Tennessee, United States of America.
Christopher
C
Aiken
Department of Pediatric, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Children's Healthcare of Atlanta, Atlanta, Georgia, United States of America.
Gregory B
GB
Melikyan
D015153:Blotting, Western; D021984:Cyclophilin A; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D015658:HIV Infections; D015497:HIV-1; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D061848:Optical Imaging / Q000379:methods; D059008:Time-Lapse Imaging / Q000379:methods*; D057646:Virus Uncoating / Q000502:physiology*
D005456:Fluorescent Dyes; D021984:Cyclophilin A