The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.
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Issue Date
11/21/2013
Authors
Watanabe, Susan M
Chen, Min-Huei
Khan, Mahfuz
Ehrlich, Lorna
Kemal, Kimdar Sherefa
Weiser, Barbara
Shi, Binshan
Chen, Chaoping
Powell, Michael
Anastos, Kathryn
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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.
Citation
Watanabe SM, Chen MH, Khan M, Ehrlich L, Kemal KS, Weiser B, Shi B, Chen C, Powell M, Anastos K, Burger H, Carter CA. The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities. Retrovirology. 2013 Nov 21;10:143. doi: 10.1186/1742-4690-10-143. PMID: 24257210; PMCID: PMC3907034.
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R01 GM111028/GM/NIGMS NIH HHS/United States , U01 AI035004/AI/NIAID NIH HHS/United States , 8G12MD007602/MD/NIMHD NIH HHS/United States , NIH U3U 01AI35004/AI/NIAID NIH HHS/United States , GM111028/GM/NIGMS NIH HHS/United States , AI068463/AI/NIAID NIH HHS/United States , AI152015/AI/NIAID NIH HHS/United States , NIH R21 AI095150/AI/NIAID NIH HHS/United States , NIH R21A1080351/PHS HHS/United States