Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

• Type of Material: Article
• Authors: Tanay M. Desai, Emory University; Mariana Marin, Emory University; Chetan Sood, Emory University; Jiong Shi, Vanderbilt University; Fatima Nawaz, Vanderbilt University; Christopher Aiken, Vanderbilt University; Gregory Melikian, Emory University
• Language: English
• Date: 2015-10-29
• Publisher: BioMed Central
• Publication Version: Final Published Version
• Copyright Statement: © Desai et al. 2015
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1186/s12977-015-0215-z
• Title of Journal or Parent Work: Retrovirology
• ISSN: 1742-4690
• Volume: 12
• Issue: 1
• Start Page: 88
• End Page: 88
• Grant/Funding Information: Work in the Aiken lab was also supported by NIH grant P50GM082251.; The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 gp120 monoclonal antibody (902) from Dr. Bruce Chesebro; the psPAX2 vector was from Didier Trono.; This work was supported by the NIH R01 Grant GM054787 and Pittsburgh Center for HIV Protein Interactions (P50GM082251) Collaboration Development Fund to G.B.M.
• Keywords: Viral fusion; FRAP; Confocal imaging; Single particle tracking; Beta-lactamase assay; HIV capsid mutant; Fluorescence correlation spectroscopy; Vpr; Nuclear transport
• Research Categories: Health Sciences, Immunology; Health Sciences, Obstetrics and Gynecology; Health Sciences, General

Abstract

BACKGROUND: HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. RESULTS: Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. CONCLUSIONS: The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.