ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding

Cell Microbiol. 2013 Feb;15(2):213-26. doi: 10.1111/cmi.12041. Epub 2012 Nov 6.

Abstract

The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome and for the abscission step of cytokinesis. ESCRT-III proteins form polymers that constrict membrane tubes, leading to fission. We have used electron cryomicroscopy to determine the molecular organization of pleiomorphic ESCRT-III CHMP2A-CHMP3 polymers. The three-dimensional reconstruction at 22 Å resolution reveals a helical organization of filaments of CHMP molecules organized in a head-to-tail fashion. Protease susceptibility experiments indicate that polymerization is achieved via conformational changes that increase the protomer stability. Combinatorial siRNA knockdown experiments indicate that CHMP3 contributes synergistically to HIV-1 budding, and the CHMP3 contribution is ~ 10-fold more pronounced in concert with CHMP2A than with CHMP2B. This is consistent with surface plasmon resonance affinity measurements that suggest sequential CHMP4B-CHMP3-CHMP2A recruitment while showing that both CHMP2A and CHMP2B interact with CHMP4B, in agreement with their redundant functions in HIV-1 budding. Our data thus indicate that the CHMP2A-CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy
  • Endosomal Sorting Complexes Required for Transport / chemistry*
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • Endosomal Sorting Complexes Required for Transport / ultrastructure
  • HIV-1 / chemistry*
  • HIV-1 / physiology
  • Models, Molecular
  • Peptide Hydrolases / chemistry
  • Polymerization
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Secondary
  • Proteolysis
  • RNA, Small Interfering / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Surface Plasmon Resonance
  • Virus Release / physiology*

Substances

  • CHMP2A protein, human
  • CHMP2B protein, human
  • CHMP3 protein, human
  • CHMP4B protein, human
  • Endosomal Sorting Complexes Required for Transport
  • RNA, Small Interfering
  • Recombinant Proteins
  • Peptide Hydrolases