title: The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.
abstract: BACKGROUND
  HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.
  
  
  RESULTS
  Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage.
  
  
  CONCLUSIONS
  The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
authors.affiliation: Department of Molecular Genetics & Microbiology, Stony Brook University, Life Sciences Bldg, Rm 248, Stony Brook, NY 11794-5222, USA. carol.carter@stonybrook.edu.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Watanabe
authors.forename: Min-Huei
authors.initials: MH
authors.lastname: Chen
authors.forename: Mahfuz
authors.initials: M
authors.lastname: Khan
authors.forename: Lorna
authors.initials: L
authors.lastname: Ehrlich
authors.forename: Kimdar Sherefa
authors.initials: KS
authors.lastname: Kemal
authors.forename: Barbara
authors.initials: B
authors.lastname: Weiser
authors.forename: Binshan
authors.initials: B
authors.lastname: Shi
authors.forename: Chaoping
authors.initials: C
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Powell
authors.forename: Kathryn
authors.initials: K
authors.lastname: Anastos
authors.forename: Harold
authors.initials: H
authors.lastname: Burger
authors.forename: Carol A
authors.initials: CA
authors.lastname: Carter
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D054884:Host-Pathogen Interactions; D008854:Microscopy, Electron; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D057074:Virus Release; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D000099177:Tsg101 Protein
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D050505:Mutant Proteins; D014157:Transcription Factors; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099177:Tsg101 Protein; C117020:PDCD6IP protein, human; C083002:p6 gag protein, Human immunodeficiency virus 1