Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells

• Type of Material: Article
• Authors: Ashwanth C. Francis, Emory University; Mariana Marin, Emory University; Jiong Shi, Vanderbilt University; Christopher Aiken, Vanderbilt University; Gregory Melikian, Emory University
• Language: English
• Date: 2016-06-20
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2016 Francis et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.ppat.1005709
• Title of Journal or Parent Work: PLoS Pathogens
• ISSN: 1553-7366
• Volume: 12
• Issue: 6
• Start Page: e1005709
• End Page: e1005709
• Grant/Funding Information: This work was supported by the NIH R01 grant GM054787 to GBM and Pittsburgh Center for HIV Protein Interactions (P50GM082251)grants to GBM and CA.; National Institute of General Medical Sciences GM054787 to Gregory B Melikyan.; National Institute of General Medical Sciences P50GM082251 (Collaboration Development Fund) to Gregory B Melikyan.
• Keywords: Microbiology; IMMUNODEFICIENCY-VIRUS TYPE-1; GAG POLYPROTEIN; CYCLOPHILIN-A; BINDING-SITE; Life Sciences & Biomedicine; Parasitology; Science & Technology; INFECTED-CELLS; HOST PROTEINS; Virology; TARGET-CELL; NUCLEAR ENTRY; REVERSE-TRANSCRIPTASE; RED FLUORESCENT PROTEIN
• Research Categories: Health Sciences, Pathology; Health Sciences, Immunology; Health Sciences, General

Abstract

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.