Dynamics in HIV Gag Lattice Detected by Time-Lapse iPALM ^†

Abstract

Immature HIV virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. This lattice has significant void spaces that were previously characterized by fluorescence correlation spectroscopy, cryoelectron tomography, and iPALM imaging. In the current study, we demonstrate that HIV virus-like particles (VLPs,) assembled by the viral protein Gag tagged at its C terminus with the photoactivable fluorescent protein Dendra, and are of the same size as virus-like particles assembled using only HIV Gag (140 ± 15 nm). We show that the Gag-Dendra lattice observed within these VLPs has similar gaps as those observed in Gag-only VLPs. We further used time-lapse iPALM microscopy to image the Gag–Dendra lattice within the lumen of VLPs at two timepoints. The reconstruction of these time-lapse images shows significant lattice dynamics within the lumen of purified VLPs. The addition of disuccinimidyl suberate (DSS) to the purified VLPs completely abrogated these dynamics. A method to quantify the dynamics of the Gag–Dendra lattice using correlation function analysis is further presented. The HIV Gag lattice, along with the structural lattices of many other viruses, have been mostly considered static. Our study provides an important tool to investigate the dynamics within these lattices and also highlights the effects of fluorescent tags on virion structures.