Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion

Journal of Biological Chemistry

Chande

et al. 2017

Self Cite

118

189

Edited by Charles E. SamuelThe host proteins, SERINC3 and SERINC5, have been recently shown to incorporate into HIV-1 particles and compromise their ability to fuse with target cells, an effect that is antagonized by the viral Nef protein. Envelope (Env) glycoproteins from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC-mediated restriction, and the mechanism by which SERINCs interfere with HIV-1 fusion remains unclear. Here, we show that incorporation of SERINC5 into virions in the absence of Nef inhibits the formation of small fusion pores between viruses and cells. Strikingly, we found that SERINC5 promotes spontaneous functional inactivation of sensitive but not resistant Env glycoproteins. Although SERINC5-Env interaction was not detected by co-immunoprecipitation, incorporation of this protein enhanced the exposure of the conserved gp41 domains and sensitized the virus to neutralizing antibodies and gp41-derived inhibitory peptides. These results imply that SERINC5 restricts HIV-1 fusion at a step prior to small pore formation by selectively inactivating sensitive Env glycoproteins, likely through altering their conformation. The increased HIV-1 sensitivity to anti-gp41 antibodies and peptides suggests that SER5 also delays refolding of the remaining fusion-competent Env trimers.

Section: Cell Lines Reagents and Plasmidsmentioning

confidence: 99%

Section: Ser5 Inhibits Hiv-cell Fusion and Env-mediated Cell-cell Fusionmentioning

confidence: 99%

See 1 more Smart Citation

SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins

Sood

Journal of Biological Chemistry

Chande

et al. 2017

Self Cite

118

189

“…We also tested the effects of the above-described agents/inhibitors on HIV-1 Env-mediated cell-cell fusion, using a dual-splitprotein assay (38,39). In this assay, fusion is detected based upon de novo assembly of functional GFP and Renilla luciferase proteins from their respective complementary domains (designated DSP1 and DSP2) expressed separately in fusing partners.…”

Section: Resultsmentioning

confidence: 99%

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

Giroud

Hammonds

et al. 2015

J Virol

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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...

“…Cell fusion was measured using the dual-split luciferase assay, as described previously. 32 Robust luciferase signal resulting from fusion between HXB2 Env-expressing 293T cells and NP2-derived target cells was abrogated in the presence of C52L (Fig. 3C).…”

Section: Dphb Selectively Inhibits Fusion Of X4-tropic and Dual-tropimentioning

confidence: 99%

Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors

Giroud

ASSAY and Drug Development Technologies

et al. 2017

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HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus-cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound-4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid (DPHB)-that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions.