Cryo Electron Tomography of Native HIV-1 Budding Sites

Lars-Anders Carlson,Alex De Marco,Heike Oberwinkler,Hans-Georg Kräusslich,Kay Grünewald,John A G Briggs,Anja Habermann,Thomas J Hope

doi:10.1371/journal.ppat.1001173

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.

Highlights

HIV-1 particles are assembled at the cell membrane, as the 55 kDa viral polyprotein Gag multimerizes on its inner face [1]
The production of new HIV-1 particles is initiated at the plasma membrane where the viral polyprotein Gag assembles into a budding site, and proceeds through release of an immature virion which is subsequently transformed to the infectious virion by proteolytic cleavage of Gag
We show that some HIV-1 infected T-cells preferentially carry these budding sites, suggesting that they have lost a crucial control of the proteolytic maturation of the virus

Summary

Introduction

HIV-1 particles are assembled at the cell membrane, as the 55 kDa viral polyprotein Gag multimerizes on its inner face [1]. Gag recruits other viral components such as the RNA genome and the surface spike proteins, as well as cellular proteins of the ESCRT machinery required for virus release [2,3,4]. The viral protease (PR) is essential to convert the immature form of the virion into an infectious mature particle. Both forms of the virion are pleiomorphic structures, with the repetitive structural elements of the virus arranged non-symmetrically and variably from one particle to the other. C-terminally of CA, the nucleocapsid (NC) domain binds the RNA genome, and the p6 domain recruits the ESCRT machinery to facilitate particle release [6,7]. CA and NC, as well as NC and p6, are separated by short spacer peptides (SP1 and SP2, respectively) which are processed during maturation

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Conclusion