RESUMO
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.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , HIV-1/química , Liberação de Vírus/fisiologia , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , HIV-1/fisiologia , Modelos Moleculares , Peptídeo Hidrolases/química , Polimerização , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteólise , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Ressonância de Plasmônio de SuperfícieRESUMO
Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Sítios de Ligação , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
HIV-1 employs its structural proteins to orchestrate assembly and budding at the plasma membrane of host cells. The Gag polyprotein is sufficient to form virus-like particles in the absence of other viral proteins and provides a platform to interact with numerous cellular factors that regulate Gag trafficking to the site of assembly and budding. Notably endosomal sorting complexes required for transport have attained much attention over the last decade because of their essential role in virion release. Here we review recent advances in understanding the role of host cell factors recruited by Gag during HIV-1 assembly and budding.
