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Nanoscopic visualization of HIV RNA trafficking in living cells

Connor Magura

Geisinger Commonwealth School of Medicine


Introduction A dynamic view of the process by which HIV recruits and utilizes a separate RNA export mechanism that is different from endogenous mRNA export has not been previously demonstrated. With our new high-speed super-resolution (SPEED) microscopy technique, we have successfully tracked individual HIV-RNA export complexes consisting of the REV transport protein, the Rev Response Element (RRE), and HIV viral RNA through the sub-micrometer nuclear pore complexes (NPCs) in living cells. Methods This study implements a real-time detection of HIV RNA in living human cells as a dynamic model for REV activity and HIV RNA trafficking. The implementation of the SPEED single molecule technique allows for a temporal resolution fast enough to capture transiting single-molecules as well as the spatial resolution necessary to localize the movements within a living cell and through a single NPC. Through this technique we were further able to understand the transport dynamics of these particles and elucidate the biophysical properties of REV and HIV RNA trafficking and interactions. Results Our high-resolution data reveals that the REV protein is constantly being transported both in and out of the nucleus. REV, together with the RRE and HIV RNA forms the RRE complex which utilizes the CRM-1 based transport differently than human cells to mediate nuclear export of HIV RNA. This results in a unique rate of success, which is approximately two-fold higher than endogenous mRNA export showing that HIV improves upon host export of RNA. Conclusions HIV has been shown in our study to not only take advantage of human cell RNA export methods but improve upon them. HIV accomplishes this by utilizing its REV and REV-Response-Element construction imprinted upon human CRM-1 mediated export machinery. By further understanding the molecular pathogenesis of HIV, alternative pathways can be explored and potentially lead to the next generation of anti-retroviral drug discovery that may aid in further prevention and curing of HIV.

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