High-throughput assay to identify inhibitors of Vpu-mediated down-regulation of cell surface BST-2

Antiviral Res. 2011 Sep;91(3):321-9. doi: 10.1016/j.antiviral.2011.07.007. Epub 2011 Jul 14.

Abstract

Bone marrow stromal cell antigen 2 (BST-2, also known as Tetherin) inhibits HIV-1 release and thereby severely impairs viral replication. HIV-1 accessory protein Vpu induces the down-regulation of cell surface BST-2, and counteracts the antiviral function of BST-2. Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. In this study, we have developed a high-throughput cell-based ELISA to identify small molecules that antagonize HIV-1 Vpu function and consequently inhibit HIV-1 replication through rescuing the antiviral activity of host BST-2. This cell-ELISA shows an excellent correlation with results obtained by flow cytometry (FACS). Under optimal conditions, a Z' factor of 0.605 was achieved in a 96-well format. Together, these results demonstrate that this assay can be used to quantify the cell surface level of BST-2 and be adapted to a high-throughput screening for novel anti-HIV compounds.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-HIV Agents / pharmacology*
  • Antigens, CD / metabolism*
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Membrane / virology
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • GPI-Linked Proteins / agonists
  • GPI-Linked Proteins / metabolism
  • Gene Silencing / drug effects
  • HIV Infections / drug therapy
  • HIV Infections / metabolism
  • HIV Infections / pathology
  • HIV Infections / virology
  • HIV-1 / drug effects*
  • HIV-1 / genetics
  • HIV-1 / metabolism
  • HeLa Cells
  • High-Throughput Screening Assays*
  • Human Immunodeficiency Virus Proteins* / antagonists & inhibitors
  • Human Immunodeficiency Virus Proteins* / genetics
  • Human Immunodeficiency Virus Proteins* / metabolism
  • Humans
  • Plasmids
  • Protein Binding / drug effects*
  • RNA, Small Interfering / pharmacology*
  • Real-Time Polymerase Chain Reaction
  • Stromal Cells / cytology
  • Stromal Cells / drug effects*
  • Stromal Cells / metabolism
  • Transfection
  • Viral Regulatory and Accessory Proteins* / antagonists & inhibitors
  • Viral Regulatory and Accessory Proteins* / genetics
  • Viral Regulatory and Accessory Proteins* / metabolism
  • Virus Attachment / drug effects
  • Virus Replication / drug effects

Substances

  • Anti-HIV Agents
  • Antigens, CD
  • BST2 protein, human
  • GPI-Linked Proteins
  • Human Immunodeficiency Virus Proteins
  • RNA, Small Interfering
  • Viral Regulatory and Accessory Proteins
  • vpu protein, Human immunodeficiency virus 1