Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

PLoS Pathog. 2015 Aug 28;11(8):e1005141. doi: 10.1371/journal.ppat.1005141. eCollection 2015 Aug.

Abstract

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu's mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Antigens, CD / metabolism*
  • CD4-Positive T-Lymphocytes / virology
  • Clathrin / metabolism
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • GPI-Linked Proteins / metabolism
  • HEK293 Cells
  • HIV-1 / metabolism*
  • Human Immunodeficiency Virus Proteins / metabolism*
  • Humans
  • Immunoprecipitation
  • Phosphorylation
  • Protein Binding
  • Protein Transport / physiology
  • RNA, Small Interfering
  • Serine
  • Transfection
  • Viral Regulatory and Accessory Proteins / metabolism*

Substances

  • Adaptor Proteins, Vesicular Transport
  • Antigens, CD
  • BST2 protein, human
  • Clathrin
  • GPI-Linked Proteins
  • Human Immunodeficiency Virus Proteins
  • RNA, Small Interfering
  • Viral Regulatory and Accessory Proteins
  • vpu protein, Human immunodeficiency virus 1
  • Serine