Immature human immunodeficiency virus (HIV) virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. Using electron microscopy, we demonstrate that HIV virus-like particles (VLPs) assembled by the viral protein Gag and tagged at its C-terminus with the fluorescent protein Dendra2 have the same morphology and size as the VLPs assembled using only HIV Gag. We characterize the photophysical properties of Dendra2 and demonstrate that 60% of Dendra2 molecules can be photoswitched and reliably counted in our interferometric photoactivated localization microscopy (iPALM) setup. We further perform iPALM imaging on immobilized HIV Gag-Dendra2 VLPs and demonstrate that we can localize and count 900–1600 Dendra2 molecules within each immobilized VLP with a single-molecule localization precision better than (10 nm) 3 . Our molecular counts correspond to 1400–2400 Gag-Dendra2 proteins incorporated within each VLP. We further calculate temporal correlation functions of localization data, which we present as localization correlation analysis, and show dynamics within the lattice of immobilized VLPs in the timescale of 10–100 s. We further use our localization data to reconstruct time-lapse iPALM images of the Gag-Dendra2 lattice within the lumen of immobilized VLPs. The iPALM time-lapse images show significant lattice dynamics within the lumen of VLPs. Addition of disuccinimidyl suberate to the VLPs completely abrogated these dynamics as observed in both localization correlation analysis and time-lapse iPALM. In a complementary approach, we utilized HaXS8 cross-linking reactions between Halo and SNAP proteins and verified lattice dynamics in purified VLPs incorporating 10% Gag-SNAP, 10% Gag-Halo, and 80% Gag proteins. The HIV Gag lattice, along with the structural lattice of other enveloped viruses, has been mostly considered static. Our study provides an important tool to investigate the dynamics within these enveloped viruses.

Interferometric Photo-Activation-Localization-Microscopy (iPALM) localizes single fluorescent molecules with 20 nm lateral and 10 nm axial resolution. We present a method utilizing glass coverslip lithography for correlative imaging between iPALM and scanning electron microscopy (SEM). Using iPALM on HIV Gag-Dendra virus-like particles (VLPs) we localized the position of HIV Gag proteins. Based on these localizations we reconstructed the central cavity of the VLPs along with imperfections within the HIV Gag lattice. The SEM images and iPALM images overlap and show imaging from single VLPs immobilized on glass coverslips. The localization of many HIV proteins including accessory proteins and Gag-Pol remains unknown, we discuss how the specificity of iPALM coupled with SEM has the potential for resolving more of HIV proteins.

Immature HIV virions harbor a lattice of Gag molecules with significant ordering in CA-NTD, CA-CTD and SP1 regions. This ordering plays a major role during HIV maturation. To test the condition in which the Gag lattice forms in vivo, we assembled virus like particles (VLPs) by expressing only HIV Gag in mammalian cells. Here we show that these VLPs incorporate a similar number of Gag molecules compared to immature HIV virions. However, within these VLPs, Gag molecules diffuse with a pseudo-diffusion rate of 10 nm2/sec, this pseudo-diffusion is abrogated in the presence of melittin and is sensitive to mutations within the SP1 region. Using cryotomography, we show that unlike immature HIV virions, in the Gag lattice of VLPs the CA-CTD and SP1 regions are significantly less ordered. Our observations suggest that within immature HIV virions, other viral factors in addition to Gag, contribute to ordering in the CA-CTD and SP1 regions.