Real-Time Imaging Reveals that HIV-1 Vpr Dissociates from the Core and Accumulates in the Nucleus after Viral Fusion

Abstract

Viral protein R (Vpr) is an HIV-1 accessory protein that associates with capsids during viral assembly and is important for infections in non-dividing cells. Vpr functions in host cells include induction of G2 cell-cycle arrest, and regulation of cellular proliferation and apoptosis. Vpr has two nuclear localization sequences that direct its transport to the nucleus. Fluorescently labeled Vpr (YFP-Vpr) is widely used to visualize HIV-1 cores in the cytoplasm during entry. Here we report on the dissociation of YFP-Vpr from HIV-1 cores shortly after viral fusion and its subsequent accumulation in the nuclei. Real-time live cell imaging showed that, under conditions of productive entry and infection, Vpr dissociated from cores post-fusion and accumulated in nuclei over time-scales that correlated with the kinetics of viral fusion (t1/2∼15 min). Nuclear accumulation of Vpr scaled with the number of cell-bound virions and could be blocked by lysosomotropic agents or a fusion-inhibitory peptide. These effects were observed in two cell lines and were independent of the fusion proteins incorporated into viral particles. Fluorescence recovery after photobleaching of YFP-Vpr within the nucleus revealed quick (t1/2∼3 min) recovery, indicating that Vpr dissociation from capsids is a rate-limiting step in Vpr post-fusion transport. Fluorescence correlation spectroscopy measurements on post-fusion nuclear YFP-Vpr, yielded fast and slow diffusive components (D∼10 μm2/s and 0.8 μm2/s, respectively) similar to those measured for YFP-Vpr over-expressed in cells. These diffusion coefficients reflect that nuclear Vpr exists in two forms - as a monomer, and in large complexes with host proteins or perhaps even chromatin structures. Current efforts are underway to explore the determinants of the stability of Vpr-capsid complexes. This work was supported by the NIH R01 GM054787 grant.