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

Abstract

HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCEInhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists. HIV-1 currently remains a major public health concern worldwide. In the face of virus's ability to develop resistance to antiviral agents, new therapeutic approaches focusing on host factors required for HIV-1 replication hold promise. HIV-1 enters target cells via a multistep process that is initiated upon binding of the gp120 subunit of the Env glycoprotein to its receptor and coreceptor, CD4 and CXCR4/CCR5, respectively (reviewed in reference 1). Coreceptor engagement promotes critical conformational changes in gp41 leading to fusion of the viral and cellular membranes (reviewed in refere...

Purinergic signaling in epilepsy

“…First, the antagonists used in these studies are specific for P2X7 receptors compared with other P2X receptors or other receptors and channels but may show off‐target activity. Such a case was reported recently for a P2X1 antagonist that also inhibits the activity of CCR5 and CXCR4 (Giroud et al, ). Second, the tissue and cellular distribution of P2X7 is still unclear; antibodies and mouse reporter gene lack specificity or can reasonably be questioned.…”

Purinergic Receptors: Elucidating the Role of these Immune Mediators in HIV-1 Fusion

“…There is a growing body of evidence that implicates purinergic receptors and both selective and non-selective antagonists of these receptors in HIV-1 infection [99][100][101][102][103][104][105]. Our laboratory and others have explored the role of purinergic receptors in HIV-1 infection and have reported that purinergic receptor antagonists can block HIV-1 infection and reduce inflammatory cytokine production associated with infection [96,106,107]. More specific probing appears to implicate drugs that target purinergic receptors in the inhibition of HIV-1 viral membrane fusion.…”

“…Hazleton, et al, demonstrated that purinergic receptors play a key role in HIV-1 replication in macrophages through selective pharmacologic inhibition of P2RX1, P2RX7, and P2RY1 [93]. Giroud, et al described a role for P2RX1 in blocking HIV-1 binding to the chemokine receptors CCR5 and CXCR4 [106]. Swartz, et al, demonstrated that non-selective antagonists of P2X receptors can inhibit HIV-1 infection of CD4 + lymphocytes through both cell-to-cell and cell-free mechanisms [96].…”

“…A screen of 1280 compounds identified the P2 × 1-antagonizing compounds NF279 and NF449 as inhibitors of virus-cell fusion in the TZM-bl cell line. NF449 and NF279 were further reported as fusion inhibitors in primary monocyte-derived macrophage (MDM), CEM-A, Jurkat, and NP2 cells as measured by the BlaM-Vpr assay [93,106]. Our laboratory developed a novel HIV-1 Cre-lox assay to measure cell-to-cell mediated virus-membrane fusion [96,116,117].…”

“…The P2Y11R antagonist NF340, an inhibitor of HIV-1 infection, was assessed as a fusion inhibitor via BlaM-Vpr assays. The compound demonstrated minimal activity as an inhibitor of HXB2, BaL26, and R3A Env-expressing HIV-1 entry in CEM-A or TZM-bl cells, and this minimal effect was seen only at high concentrations [106,115]. The BlaM-Vpr assay was further used to study the P2RY1 antagonist MRS2500 and the P2RY2 antagonist PSB 1114; neither MRS2500 nor PSB 1114 inhibited virus-cell fusion in TZM-bl cells [115].…”