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

Abstract

BackgroundUncoating 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.ResultsIn 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.ConclusionsTaken 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.

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

“…Interestingly, in the E45A capsid mutant the peak stiffness appeared sooner, after 4 h of transcription. This kinetic difference may be related to the observation that E45A cores appear to be more permeable to small molecules than WT cores (24), suggesting that reverse transcription may occur more rapidly owing to enhanced dNTP influx. Recent data suggest that nucleotides are transported into the core through electrostatic channels that were found in CA hexamers (25).…”

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

“…The use of GFP-Vpr for analysis of viral postentry events, pioneered by Hope and colleagues (33), has provided numerous valuable insights into postentry events during HIV-1 replication (22,24,(34)(35)(36). However, GFP-Vpr dissociates from the RTCs/PICs shortly after infection and has not been observed in the nuclei of infected cells (33,36,37). To date, only a small number of studies have detected PICs in the nuclei of infected cells by using virions labeled with fluorescently tagged IN (38)(39)(40)(41), and one recent study showed that there are nuclear IN complexes that are not associated with viral cDNA (42), suggesting that fluorescently tagged IN may not be a suitable marker for labeling PICs in the nuclei.…”

Cellular and viral determinants of retroviral nuclear entry

“…Interestingly, several imaging-based experiments have shown that a minor amount of viral capsid protein (CA) is associated with the RTC, indicating disassembly of some viral cores in the cytoplasm (Xu et al 2013;Campbell and Hope 2015). Uncoating may occur when the viral cores pass through the NPC (Lahaye et al 2013;Campbell and Hope 2015).…”