Virion Attachment and Entry: HIV gp120 Env Biotinylation, gp120 Env, or Integrin Ligand-Binding Assay

Abstract

The HIV-1 entry receptors are CD4 and a chemokine receptor (CCR5 or CXCR4). In addition it has recently been demonstrated that HIV-1 gp120 binds to and signals through integrin α₄β₇, the gut-homing receptor (Arthos et al., Nat Immunol 9(3):301–309, 2008). Integrin α₄β₇ is not an entry receptor for HIV-1, although it can facilitate virion attachment to target cells (Arthos et al., Nat Immunol 9(3):301–309, 2008; Cicala et al., Proc Natl Acad Sci U S A 106:20877–20882, 2009). Recombinant HIV-1 gp120s bind to integrin α₄β₇ in a manner similar to its natural ligands (MAdCAM-1, V-CAM-1, fibronectin) (Andrew et al., J Immunol 153:3847–3861, 1994). gp120-α₄β₇ interactions are detected in a manner similar to assays developed for the natural ligands of α₄β₇. In this chapter we describe a method for the analysis of integrin-gp120 binding via a cell-based binding assay. In vitro ligand-integrin affinity can be modified by the presence of divalent cations (Mn²⁺, Mg²⁺, Ca²⁺) (Leitinger et al., Leitinger Biochim Biophys Acta 1498:91–98, 2000). Here we describe a protocol to detect biotinylated recombinant HIV-1 gp120 binding to integrin α₄β₇ in both primary cells and cell lines expressing the gut-homing receptor.