Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay
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RESEARCH
Open Access
Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay Hongzhan Xu1, Tamera Franks1, Gregory Gibson2, Kelly Huber1, Nadia Rahm3, Caterina Strambio De Castillia3,4, Jeremy Luban3,4, Christopher Aiken5, Simon Watkins2, Nicolas Sluis-Cremer1 and Zandrea Ambrose1*
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.
Background The replication cycle of HIV-1 is complex; while many of the critical steps have been described in great detail, some, including uncoating of the viral core, remain poorly understood. After fusion with the host cell, HIV1 releases the core into the cytoplasm. The core contains the conical viral capsid, composed of a polymer of capsid protein (CA) subunits, encasing the viral RNA (vRNA) genome. The viral RNA undergoes reverse transcription, forming viral DNA (vDNA) in the cytoplasm. The vDNA together with the nucleocapsid (NC), reverse transcriptase (RT), Vpr, and integrase (IN) form the preintegration complex (PIC). The PIC is transported to the nucleus by way of microtubules and actin filaments in
* Correspondence: [email protected] 1 Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Full list of author information is available at the end of the article
the cytoplasm [1,2], and subsequently enters the nucleus by mechanisms which have only recently begun to be examined in detail [3]. Inside the nu
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