The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities

Susan M. Watanabe, Min Huei Chen, Mahfuz Khan, Lorna Ehrlich, Kimdar S. Kemal, Barbara Weiser, Binshan Shi, Chaoping Chen, Michael Powell, Kathryn Anastos, Harold Burger, Carol A. Carter

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Article number143
JournalRetrovirology
Volume10
Issue number1
DOIs
StatePublished - Nov 21 2013

Fingerprint

Capsid
HIV-1
Endosomal Sorting Complexes Required for Transport
Reading Frames
Mutation
Pseudopodia
Membranes
Peptide Hydrolases
Yeasts
Electrons
Amino Acids

Keywords

  • Alix
  • CA NTD
  • HIV-1
  • Nedd4
  • Protease
  • Tsg101
  • Viral budding
  • Viral particle maturation

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities. / Watanabe, Susan M.; Chen, Min Huei; Khan, Mahfuz; Ehrlich, Lorna; Kemal, Kimdar S.; Weiser, Barbara; Shi, Binshan; Chen, Chaoping; Powell, Michael; Anastos, Kathryn; Burger, Harold; Carter, Carol A.

In: Retrovirology, Vol. 10, No. 1, 143, 21.11.2013.

Research output: Contribution to journalArticle

Watanabe, SM, Chen, MH, Khan, M, Ehrlich, L, Kemal, KS, Weiser, B, Shi, B, Chen, C, Powell, M, Anastos, K, Burger, H & Carter, CA 2013, 'The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities', Retrovirology, vol. 10, no. 1, 143. https://doi.org/10.1186/1742-4690-10-143
Watanabe, Susan M. ; Chen, Min Huei ; Khan, Mahfuz ; Ehrlich, Lorna ; Kemal, Kimdar S. ; Weiser, Barbara ; Shi, Binshan ; Chen, Chaoping ; Powell, Michael ; Anastos, Kathryn ; Burger, Harold ; Carter, Carol A. / The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities. In: Retrovirology. 2013 ; Vol. 10, No. 1.
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AU - Watanabe, Susan M.

AU - Chen, Min Huei

AU - Khan, Mahfuz

AU - Ehrlich, Lorna

AU - Kemal, Kimdar S.

AU - Weiser, Barbara

AU - Shi, Binshan

AU - Chen, Chaoping

AU - Powell, Michael

AU - Anastos, Kathryn

AU - Burger, Harold

AU - Carter, Carol A.

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N2 - 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.

AB - 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.

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KW - Viral budding

KW - Viral particle maturation

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