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

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

doi:10.1186/1742-4690-10-143

Abstract

BackgroundHIV-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.ResultsWhereas 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.ConclusionsThe 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.

Highlights

Human Immunodeficiency Virus type 1 (HIV-1) budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit Endosomal sorting complex required for transport (ESCRT) machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain
HIV-1 Gag does not have a PY motif, (i), it is known to be Ub-modified [21,22]; (ii), viral budding is impacted by translational fusion of Ub to Gag [23,24,25,26]; (iii), viral budding and infectivity have been shown to be affected by changes in the levels of free cellular Ub [23]; and (iv), Tsg101 has been reported to associate with Nedd4 [16,27]
Mutation of S40 to Phe (S40F), which does not alter the amino acid sequence in the overlapping pol reading frame, inhibits CA maturation, alters budding, and reduces viral infectivity To confirm the previously observed processing defect of S40F, we engineered the mutation into pNL4-3-ΔEnv and examined Gag processing products by Western analysis

Summary

Introduction

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. A apparent aspect of Human Immunodeficiency Virus type 1 (HIV-1) replication is the existence of multiple independent pathways for exit of progeny virus particles from the host cell. These but not absolutely, conserved amino acids [11,12,13]. This second budding pathway requires Alix interaction with Gag through both p6 and determinants in nucleocapsid (NC) [14]. It is likely that Nedd plays a role in HIV-1 egress

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