The Human Endosomal Sorting Complex Required for Transport (ESCRT-I) and Its Role in HIV-1 Budding

Melissa D Stuchell,Jennifer E Garrus,Barbara Müller,Kirsten M Stray,Sanaz Ghaffarian,Rena Mckinnon,Hans-Georg Kräusslich,Scott G Morham,Wesley I Sundquist

doi:10.1074/jbc.m405226200

Abstract

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.

Highlights

Like other enveloped viruses, HIV-11 must bud from producer cells to spread infection
The PTAP late domain recruits TSG101, which in turn functions within a multiprotein complex (ESCRT-I) that links the virus to cellular machinery that normally functions in protein sorting and vesicle formation in the multivesicular body (MVB)
The N-terminal half of TSG101 appears to function primarily in recruiting the protein to the sites of virus budding, whereas the C-terminal half of the protein contains binding sites for other proteins that function in virus budding, including the other members of the ESCRT-I complex, VPS28 and VPS37

Summary

Introduction

HIV-11 must bud from producer cells to spread infection. Vps23p, the yeast ortholog of TSG101, binds two other proteins, Vps28p and Vps37p, to form a stable soluble ϳ350-kDa complex called ESCRT-I (endosomal sorting complex required for transport) [16]. Mammalian TSG101 and VPS28 function together as part of a soluble ϳ350-kDa ESCRT-I complex [22, 25]. TSG101/ESCRT-I Functions in HIV Release idues ϳ146 –215), a predicted central coiled-coil region (residues ϳ240 –311), and a conserved helical C-terminal domain (residues ϳ330 –390). 2) The C-terminal region of TSG101/Vps23p binds VPS28/Vps28p [14, 25, 29] This interaction is important for MVB formation and may control the steady-state levels of TSG101 [30]. This interaction is important for MVB formation and may control the steady-state levels of TSG101 [30]. 3) deletion of a predicted central coiledcoil region within Vps23p blocks incorporation of Vps37p into the ESCRT-I complex, suggesting that this region of Vps23p may form (or contain) the Vps37p-binding site [16]

Objectives

Methods

Results

Conclusion