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    Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

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    Authors
    Xu, Hongzhan
    Franks, Tamera
    Gibson, Gregory
    Huber, Kelly
    Rahm, Nadia
    De Castillia, Caterina Strambio
    Luban, Jeremy
    Aiken, Christopher
    Watkins, Simon
    Sluis-Cremer, Nicolas
    Ambrose, Zandrea
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    UMass Chan Affiliations
    Program in Molecular Medicine
    Document Type
    Journal Article
    Publication Date
    2013-07-09
    Keywords
    Cell Line
    HIV-1
    Humans
    Microscopy, Confocal
    RNA, Viral
    Staining and Labeling
    Uridine
    Viral Proteins
    Virology
    *Virus Uncoating
    Virology
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    Abstract
    BACKGROUND: Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. RESULTS: In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. CONCLUSIONS: Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process.
    Source

    Xu H, Franks T, Gibson G, Huber K, Rahm N, De Castillia CS, Luban J, Aiken C, Watkins S, Sluis-Cremer N, Ambrose Z. Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay. Retrovirology. 2013 Jul 9;10:70. doi: 10.1186/1742-4690-10-70. Link to article on publisher's site

    DOI
    10.1186/1742-4690-10-70
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/30097
    PubMed ID
    23835323
    Related Resources

    Link to Article in PubMed

    Rights
    Copyright 2013 Xu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    ae974a485f413a2113503eed53cd6c53
    10.1186/1742-4690-10-70
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