The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures.
Retroviral late domains (L domains) are short amino acid sequences in the Gag protein that facilitate the process of budding. L domains act by recruiting the ESCRT complexes, which normally function in the formation of multivesicular bodies. The PTAP late domain of human immunodeficiency virus (HIV) is believed to specifically recruit this machinery by binding the ESCRT protein TSG101. It was recently demonstrated that expression of a C-terminal fragment of TSG101 (TSG-3') blocked the budding of both PTAP-dependent and PPPY-dependent retroviruses. We show here that TSG-3' expression leads to the formation of large spherical entities that we call TICS (TSG-3'-induced cellular structures) in the cytoplasm. Rous sarcoma virus (RSV) and murine leukemia virus (MLV) Gag proteins are selectively recruited to these structures, but HIV type 1 Gag is completely excluded. Experiments with various HIV and RSV vector constructs as well as HIV and RSV chimeras suggest that recruitment to the TICS is late domain independent and does not involve recognition of any single amino acid sequence. TICS appear to have no limiting membrane and do not colocalize with markers for any membranous cellular compartment. Wild-type TSG101 is also recruited to TICS, but most other ESCRT proteins are excluded. These structures are similar in nature to aggresomes, colocalize with the aggresome marker GFP-250, and are highly enriched in ubiquitin but in other ways do not fully meet the description of aggresomes. We conclude that the block to retroviral budding by TSG-3' may be the result of its sequestration of Gag, depletion of free TSG101, or depletion of free ubiquitin.
Johnson
Marc C
MC
Cornell University, Ithaca, N.Y, USA. mcj7@cornell.edu
Spidel
Jared L
JL
Ako-Adjei
Danso
D
Wills
John W
JW
Vogt
Volker M
VM
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D022083:Cytoplasmic Structures / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics; D046911:Macromolecular Substances / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D025801:Ubiquitin / Q000032:analysis; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D049452:Green Fluorescent Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D000099177:Tsg101 Protein; C468293:Gag protein p12, moloney murine leukemia virus; C113029:gag protein p19, Rous sarcoma virus
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