A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer

Hu, Kimberly; Clément, Jean‐François; Abrahamyan, Levon; Strebel, Klaus; Bouvier, Michel; Kleiman, Lawrence; Mouland, Andrew J.

doi:10.1016/j.jviromet.2005.04.012

Cited by 35 publications

(31 citation statements)

References 47 publications

(67 reference statements)

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“…BRET2 Assay-The BRET assay used for determination of the protein/protein interaction has been described previously in detail (15). Briefly, 293T cells were co-transfected with hGFP2-Vif.⌬C and hRLuc-hA3G and harvested 40 h after transfection.…”

Section: Methodsmentioning

confidence: 99%

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

Cen

Peng

et al. 2010

Journal of Biological Chemistry

100

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APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(؉) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD 50 of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.

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Section: Methodsmentioning

confidence: 99%

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

Cen

Peng

et al. 2010

Journal of Biological Chemistry

100

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“…The BRET 2 signal is much shorter than that of BRET 1 (Bacart et al, 2008). Previously, the BRET 2 sensor was used to detect thrombin activity in vitro, and HIV protease was expressed with a plasmid vector in the cells (Dacres et al, 2009;Hu et al, 2005). To our knowledge, this is the first report of the use of a BRET 2 -based probe for the detection of a viral protease during natural viral infection and growth.…”

Section: Discussionmentioning

confidence: 96%

Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

et al. 2011

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Feline calicivirus (FCV) is an important veterinary pathogen and causes respiratory disease in cats. Because it grows well in cell culture, FCV is often used as a model virus of non-culturable caliciviruses. In this study, a cell-free and two cell culture-based biosensor assay systems were established to detect FCV protease activity. The assays utilize luciferase sensor technology or second-generation bioluminescence resonance energy transfer (BRET2). A luciferase sensor was designed to contain an FCV protease cleavage motif within the permutated luciferase (GloSensor). The BRET2-based probe contained the same cleavage motif flanked by a renilla luciferase and a variant of green fluorescent protein. To confirm the specificity of these assay systems, GloSensor or a BRET2-based probe containing a mutation in the cleavage motif was also constructed. In a cell-free assay, GloSensor showed increased luminescence in proportion to the amount of FCV protease, while no signal change was observed when the construct harboring the mutant cleavage motif was used. A feline cell line stably expressing GloSensor or the BRET2-based probe was established. Increased levels of GloSensor luminescence, and decreased levels of BRET2 signals were observed according to input FCV titers. In contrast, no significant signal change was observed in the cells stably expressing the mutant cleavage motif. GloSensor and the BRET2-based probe were capable of detecting the inhibitory activity of ribavirin in FCV-infected cells. Our results demonstrate that these biosensors are useful to detect FCV protease activity induced in infected cells, and well worth consideration for screening of anti-FCV protease compounds in cell-free system as well as anti-FCV compounds in cultured cells.

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“…In a recent study, by developing BRET-based cellular RTK assays, it has been shown that discriminating pathway specific signals is important to facilitate the pathway specific drug candidate identification process [30], which will eventually require a HTS screen. Also, a HIV protease based BRET2 biosensor assay was developed [51], which can be adapted for high throughput screening of new HIV-1 protease inhibitors. Cells expressing the HIV-1 protease can be employed to screen for antiviral compounds that also target the proteases of other viruses.…”

Section: High-throughput Screeningmentioning

confidence: 99%

The New Era of Bioluminescence Resonance Energy Transfer Technology

2011

CPB

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Bioluminescence resonance energy transfer (BRET) assay is a comparatively new cell-based assay technology that is assuming more prominent roles in the field of studying protein-protein interactions, protein dimerization and signal transduction. In the last few years BRET related research has gained significant momentum in terms of adding versatility in the assay format as well as a variety of new applications where it has been suitably used. Beyond the scope of quantitative measurement of protein-protein interactions and protein dimerization, molecular imaging applications based on BRET assays have broaden its scope as a great tool for high-throughput screening (HTS) of pharmacologically important compounds. This article will highlight the landmarks in BRET research, with those which have significant contributions towards making it an attractive single format assay that shuttles between in vitro and in vivo measurements.

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A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer

Cited by 35 publications

References 47 publications

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

The New Era of Bioluminescence Resonance Energy Transfer Technology

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