title: Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay.
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.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Hongzhan
authors.initials: H
authors.lastname: Xu
authors.forename: Tamera
authors.initials: T
authors.lastname: Franks
authors.forename: Gregory
authors.initials: G
authors.lastname: Gibson
authors.forename: Kelly
authors.initials: K
authors.lastname: Huber
authors.forename: Nadia
authors.initials: N
authors.lastname: Rahm
authors.forename: Caterina
authors.initials: C
authors.lastname: Strambio De Castillia
authors.forename: Jeremy
authors.initials: J
authors.lastname: Luban
authors.forename: Christopher
authors.initials: C
authors.lastname: Aiken
authors.forename: Simon
authors.initials: S
authors.lastname: Watkins
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
authors.forename: Zandrea
authors.initials: Z
authors.lastname: Ambrose
mesh_terms: D002460:Cell Line; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis; D014773:Virology / Q000379:methods*; D057646:Virus Uncoating
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D014529:Uridine