ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding.
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.
Unit of Virus Host Cell Interactions, UMI 3265, Université Joseph Fourier-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9, France.
Grégory
G
Effantin
Aurélien
A
Dordor
Virginie
V
Sandrin
Nicolas
N
Martinelli
Wesley I
WI
Sundquist
Guy
G
Schoehn
Winfried
W
Weissenhorn
D020285:Cryoelectron Microscopy; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D015497:HIV-1 / Q000737:chemistry* / Q000502:physiology; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry; D058105:Polymerization; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D020349:Surface Plasmon Resonance; D057074:Virus Release / Q000502:physiology*
C507674:CHMP2A protein, human; C503812:CHMP2B protein, human; C498543:CHMP3 protein, human; C478397:CHMP4B protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D010447:Peptide Hydrolases