Rab6A is required for the post-entry nuclear localization of HIV-1

Isaac James Zentner

doi:10.17918/00009691

While some aspects of the HIV-1 replication cycle have been thoroughly investigated, the proteins involved and the mechanism by which the viral contents are trafficked to the nucleus remains poorly understood. The small Rab GTPase, Rab6A, has been identified as a HIV-1 dependency factor by a number of siRNA screens. Rab6A, ubiquitously expressed in two closely related isoforms, Rab6A and Rab6A', is primarily involved in the retrograde membrane traffic to the ER via the Golgi and contrary to initial studies, likely functions in the cytosolic transport of the virus to the nucleus. To test this, we utilized siRNA knockdowns in cell lines and primary human monocyte-derived macrophages (MDMs), coupled with a HIV-1 single-round infection assay to accurately define the role of Rab6A in HIV-1 infection. Additional characterization of the function of Rab6A/A' in infection was achieved by silencing Rab6A-specific guanine nucleotide exchange factor, Ric1, and Rab6A/A' effector proteins DENND5A and BICD2. We found that depletion of Rab6A/A' in cell lines and MDMs confirmed its role in the early stage of infection. Contrary to previous reports, the role of Rab6A was not dependent on the HIV-1 envelope suggesting a post-entry pre-integration function. Diminishing Ric1 or overexpression of a dominant-negative mutant of Rab6A decreased infectivity similar to silencing, revealing that Rab6A activation is required for efficient viral replication. Using real-time PCR, Rab6A or Rab6A' depletion displayed minimal effects on early/late viral reverse transcription products, but resulted in undetectable (or extremely low) levels of 2-LTR circles and integrated pro-viruses. Moreover, nuclear localization of HIV-1 pre-integration complex was diminished when Rab6A/A' were absent. Consistent with a role of Rab6A in nuclear localization, silencing of BICD2, a protein involved in retrograde transport and known to interact with nuclear pore complexes, and not DENND5A, displayed a similar effect on the incoming virus as Rab6A/A' silencing. Lastly, a direct role of Rab6A on the HIV-1 nuclear entry pathways was ruled out by using HIV-1 capsid mutants in concert with Rab6A/A' depletion. Taken together, this study reveals a novel retrograde trafficking pathway regulated by activated Rab6A and Rab6A' that is required for efficient HIV-1 nuclear localization.

Rab6A is required for the post-entry nuclear localization of HIV-1

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Rab6A is required for the post-entry nuclear localization of HIV-1

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