Cryo Electron Tomography of Native HIV-1 Budding Sites

Abstract

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

“…A morphological connection between F-actin and nascent HIV-1 assembly sites was also suggested by our previous cryoelectron tomography (cET) analysis of budding sites at the membrane of HIV-1 Gag-or GagPol-expressing glioblastoma cells (21). F-actin structures were observed in the close vicinity of most budding sites analyzed and were pointing into the nascent particle in ϳ50% of all cases.…”

“…F-actin structures were observed in the close vicinity of most budding sites analyzed and were pointing into the nascent particle in ϳ50% of all cases. Furthermore, tips of actin filaments appeared to be connected with the inner rims of the immature Gag lattice in some cases, suggesting a direct interaction (21). We have now extended this cET analysis to budding sites of HIV-1 Gag(LZ), which lacks the putative actin interaction domain in NC.…”

“…Adenoviral vectors for HIV-1 Gag (AdGag) and GagPol (AdGagPol) were constructed from Rev-independent expression plasmids (13), using BD Adeno-X Expression System 1 (BD Biosciences Clontech), as described previously (21). A Rev-independent Gag leucine zipper [Gag(LZ)] coding sequence was constructed by overlap PCR using an MA-CA-SP1-encoding fragment derived from pcDNA3.1synGagPol (22) and an LZ-p6-encoding fragment derived from a chimeric Gag variant containing codons 247 to 280 of the GCN4 leucine zipper in place of NC-SP2(Z wt p6) (16) (forward primer 5=-ACCAACACCGCCACCATCA TGCTGCAGCGTATGAAGCAGCTCGAGG-3= and reverse primer 5=-T ATGGTACCTTATTGTGACGAGGGGTCGTTGCCAAAGAG-3=).…”

“…Subtomogram averaging analyses were performed as described previously (21). Subvolumes of 57.73 nm 3 were extracted along the surface of the particles and preoriented normal to the surface.…”

Cellular structural biology as revealed by cryo-electron tomography

“…Cryo-ET has matured into a reliable method for obtaining a structural blueprint of the unperturbed macromolecular organization inside cells at a resolution of a few nanometers (Woodward et al, 2015;Stauffer et al, 2014;Elad et al, 2013;Fridman et al, 2012;Patla et al, 2010;Carlson et al, 2010). Here, we focus on the study of macromolecules, organelles, entire cells and multi-cell specimens, primarily from the Eukaryotic kingdom, and discuss some of the recent advances and applications of this technique that make it possible to obtain a realistic view of cellular landscapes in their physiological environment.…”