Vaccinia‐Based Reporter Gene Cell‐Fusion Assays to Quantitate Functional Interactions of <scp>HIV</scp> Envelope Glycoprotein with Receptors

Dey, Barna; Berger, Edward A.

doi:10.1002/0471142735.im1210s54

Cited by 4 publications

(4 citation statements)

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“…To test the ability of fusion-inactive Env subunits to functionally complement one another in the context of mixed Env trimers, we first employed a vaccinia-based quantitative cell fusion assay system wherein fusion between effector cells expressing Env and target cells expressing the necessary receptors leads to reporter gene activation (β-galactosidase production) [ 36 , 37 ]. We examined complementation between variants in gp120 that were inactive due to inability to interact with CD4 (CD4 BS mutation) or coreceptor (mismatched specificity, or mutation in the V3 loop), as well as variants in gp41 with mutations at different points within the FP and HR1 regions, as well as modifications of the membrane proximal tryptophan-rich region (TRR) and the transmembrane (TM) domain (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer

Salzwedel

Berger

2009

Retrovirology

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Background: The HIV-1 Env glycoprotein mediates virus entry by catalyzing direct fusion between the virion membrane and the target cell plasma membrane. Env is composed of two subunits: gp120, which binds to CD4 and the coreceptor, and gp41, which is triggered upon coreceptor binding to promote the membrane fusion reaction. Env on the surface of infected cells is a trimer consisting of three gp120/gp41 homo-dimeric protomers. An emerging question concerns cooperative interactions between the protomers in the trimer, and possible implications for Env function.

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Section: Resultsmentioning

confidence: 99%

“…Env-mediated cell fusion activity was measured using a quantitative vaccinia-based reporter gene assay as described previously [ 36 , 37 ]. Each vaccinia virus was used at a multiplicity of infection of 10.…”

Section: Methodsmentioning

confidence: 99%

Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer

Salzwedel

Berger

2009

Retrovirology

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“…The HIV-1 fusion assay is based on the vaccinia virus T7 RNA polymerase expression system with recombinant derivatives of the vaccinia virus (v) strain WR obtained through the NIH, USA [17]. “Effector” or “Donor” cells are infected with vTF7-3 expressing the bacteriophage T7 RNA polymerase gene under the control of the v p7.5 promoter from Dr. T. Fuerst and Dr B. Moss.…”

Section: Methodsmentioning

confidence: 99%

Characterization of HIV-1 entry inhibitors with broad activity against R5 and X4 viral strains

et al. 2015

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BackgroundCombined antiretroviral therapy has drastically reduced mortality and morbidity of HIV-infected individuals. Nevertheless long-term toxicity and appearance of viral resistance hampers the prolonged effectiveness of combination therapy, requiring a continuous input of drugs to replace those utilized in combination regimens. We here investigated the anti-HIV activity of novel derivatives of the suradista chemical class.MethodsCompounds were tested on acute HIV-1 infection of activated peripheral blood mononuclear cells. HIV production was monitored by enzyme-linked immunosorbent assay measuring the protein p24 released in culture supernatants. Fusion assays were carried out to study the mechanism of action of these compounds. A modified version of a previously established recombinant vaccinia virus-based assay was used measuring activation of a reporter gene upon fusion of two distinct cell populations. Flow cytometry was performed in competition assays for the binding of several antibodies targeting different sites of the viral envelope glycoprotein gp120, or the receptor CD4, or the coreceptors CXCR4 and CCR5.ResultsFour compounds inhibited replication of a prototypic R5 (BaL) and X4 (IIIB) laboratory-adapted HIV-1 strain at low micromolar concentrations, in the absence of cytotoxicity. Approximately a ten fold greater activity was achieved against the X4 as compared to the R5 strain.The compounds blocked X4 and R5 HIV-1 fusion, a step of viral entry. This activity appeared specific for HIV-1, as entry of human herpesvirus 6 (HHV-6) and influenza virus was not substantially affected. Further investigation of the inhibitory mechanism revealed that these new molecules target the viral envelope, rather than the coreceptors, as previously shown for a congener of the same class characterized by a long plasmatic half-life. Indeed ND-4043, the most active compound, specifically competed with binding of monoclonal antibodies against the CD4-binding site (CD4-BS) and coreceptor-binding site (CoR-BS) of gp120. These compounds displayed broad anti-HIV activity, as they inhibited various primary R5, X4 and, importantly, dualtropic R5X4 HIV-1 isolates. Of the four derivatives tested, the dimeric compounds were consistently more potent than the monomeric ones.ConclusionsGiven their unique features, these molecules represent promising candidates for further development and exploitation as anti-HIV therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0461-9) contains supplementary material, which is available to authorized users.

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“…The concept of using HIV-1 reporter viruses to monitor HIV-1 replication was first introduced using a replication competent HIV-1 reporter virus containing the chloramphenicol acetyltransferase gene in place of HIV-1 Nef sequences. Cells are then infected with the recombinant reporter virus and virus replication is quantified by measuring the expression of the virally encoded reporter gene (Adelson et al, 2003;Dey & Berger, 2003). For reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection.…”

Section: Hiv-1 Replication Screensmentioning

confidence: 99%

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors

Abad¹,

Bedoya²,

Bermejo³

2011

Recent Translational Research in HIV/AIDS

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Vaccinia‐Based Reporter Gene Cell‐Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors

Cited by 4 publications

References 49 publications

Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer

Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer

Characterization of HIV-1 entry inhibitors with broad activity against R5 and X4 viral strains

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors

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