Host ABCE1 is at Plasma Membrane HIV Assembly Sites and Its Dissociation from Gag is Linked to Subsequent Events of Virus Production

Abstract

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next $2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.

The biological regulation of ABCE1

“…Interestingly, ABCE1 protein is specifically recruited into the discrete sites at plasma membrane, then associating with Gag, furthermore forming Gag-ABCE1 protein complexes throughout the assembly process. These intermediates do not resolve until the virus maturation company with dissociation ABCE1 protein from Gag, which is followed by virus release (25). How might Gag recruit ABCE1 protein?…”

A Temporospatial Map That Defines Specific Steps at Which Critical Surfaces in the Gag MA and CA Domains Act during Immature HIV-1 Capsid Assembly in Cells

“…All plasmids utilized in this study were generated via site-directed mutagenesis in HIV-1 provirus strain LAI with a deletion in env (HIV-1⌬Env) (48). Some mutant proviruses also encoded an inactive protease (HIV-1⌬Env, Pro Ϫ ), where indicated, as described previously (40). The sequences of the oligonucleotides used to generate each mutation are available upon request.…”

“…Formation of the ϳ80S intermediate, in which Gag first associates with ABCE1 and DDX6, occurs when Gag co-opts the cellular precursor complex that contains these and other host proteins (43). When assembly of the immature capsid is completed, ABCE1, DDX6, and other cellular proteins from the precursor complex dissociate from the immature capsid (40,43,46).…”

“…These complexes are true assembly intermediates, since they appear in a sequential progression in pulse-chase analyses, with newly synthesized Gag first forming the ϳ10S complex, followed by the ϳ80S intermediate, ϳ150S intermediate, and ϳ500S intermediate, before culminating in the ϳ750S completely assembled HIV-1 immature capsid (40,41). The order of intermediates in the assembly pathway initially indicated by pulse-chase experiments has been confirmed through the analysis of assembly-defective mutants (40,(42)(43)(44). In these studies, cells expressing an assembly-defective Gag mutant accumulate only those intermediates that precede the step at which assembly is blocked.…”

“…In these studies, cells expressing an assembly-defective Gag mutant accumulate only those intermediates that precede the step at which assembly is blocked. The ϳ80S, ϳ150S, and ϳ500S assembly intermediates (termed the high-molecular-mass intermediates) contain Gag as well as cellular proteins derived from a host precursor complex, at least two of which-the ATP-binding cassette protein E1 (ABCE1) and the DEAD box RNA helicase DDX6 -facilitate immature capsid assembly (40,42,43,45,46). Formation of the ϳ80S intermediate, in which Gag first associates with ABCE1 and DDX6, occurs when Gag co-opts the cellular precursor complex that contains these and other host proteins (43).…”