High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Abstract

HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors. Here, we adapted and optimized an enzymatic HIV-cell fusion assay, which reports the transfer of virus-encapsulated b-lactamase into the cytoplasm, to high-throughput screening (HTS) with a 384-well format. The assay was robustly performed in HTS format and was validated by the pilot screen of a small library of pharmacologically active compounds. Several hits identified by screening included a prominent cluster of purinergic receptor antagonists. Functional studies demonstrated that P2X1 receptor antagonists selectively inhibited HIV-1 fusion without affecting the fusion activity of an unrelated virus that enters cells through an endocytic route. The inhibition of HIV-cell fusion by P2X1 antagonists was not through downmodulation of the cell surface expression of CD4 or coreceptors, thus implicating P2X1 receptor in the HIV-1 fusion step. The ability of these antagonists to inhibit viruses regardless of their coreceptor (CXCR4 or CCR5) preference indicates that fusion is blocked at a late step downstream of coreceptor binding. A future large-scale screening campaign for HIV-1 fusion inhibitors, using the above functional readout, will likely reveal novel classes of inhibitors and suggest potential targets for antiviral therapy.

P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

“…In agreement with our previously reported findings (24), the P2X1R antagonists NF279 and NF449 suppressed fusion between particles pseudotyped with CXCR4-tropic (HXB2), CCR5-tropic (BaL26), and dual-tropic (R3A) HIV-1 Env glycoproteins and TZM-bl cells in a dose-dependent manner ( Fig. 1A to C).…”

“…We first confirmed and expanded previously reported findings that P2X1R inhibitors selectively block HIV-1 fusion (13)(14)(15)24). The effects of antagonists of P2X1 (NF279 and NF449), P2Y11 (NF340), and P2X7 (A740003) receptors on HIV-1 fusion with HeLa-derived and CD4…”

“…Both compounds were nearly equally potent and were more effective against fusion mediated by laboratory-adapted HXB2 Env than against fusion mediated by BaL26 or R3A Env. We have previously shown that this inhibition was not due to downregulation of CD4 or coreceptor expression (24). Both NF279 and NF449 inhibited HXB2 pseudovirus (HXB2pp) fusion with lymphoid CEM-A cells (Fig.…”

“…This effect was HIV specific, since fusion of viruses pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) was insensitive to these inhibitors. In contrast, P2Y or P2X7 receptor antagonists did not suppress HIV-1 fusion (24). These findings further implicate P2X1R in HIV-1 fusion, but the mechanism by which these receptors regulate viral fusion remains unclear.…”

“…We previously conducted a pilot screen for HIV-1 fusion inhibitors and found that the P2X1R antagonists NF449 and NF279 inhibited the fusion of both R5-and X4-tropic HIV-1 strains without affecting the expression levels of CD4 and coreceptors (24). This effect was HIV specific, since fusion of viruses pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) was insensitive to these inhibitors.…”