A novel dual-luciferase assay for anti-HIV drug screening based on the CCR5/CXCR4 promoters

Lin, Feng; Lu, Wuhao; Ma, Yunyun; Guo, Wentao; Wang, Yuanyuan; Sun, Qianqian; Wu, Jianbo; Zhang, Xiaoli

doi:10.1016/j.jviromet.2018.02.016

Cited by 4 publications

(4 citation statements)

References 35 publications

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“…Our laboratory constructed pGL4.10-F2 based on pGL4.10, which contains firefly luciferase and Renilla luciferase genes and can be used for dual fluorescence reporter gene detection. 15 Pseudovirus-SARS-CoV-2 (B.1.617.2) was purchased from General Biology (Anhui, China), which encapsulates RNA sequences of green fluorescent protein (GFP) and luciferase. Infection efficiency can be determined by observing GFP expression and detecting luciferase activity.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The digested fragment was then ligated with pGL4.10-F2 plasmid that had been cut with XhoI and HindIII to generate a recombinant vector pGL4.10-F2-ACE2, in which the expression of the firefly luciferase reporter gene was driven by the ACE2 promoter. 15 Luciferase reporter assays HEK293T cells were inoculated in 6-well plates at 3.6 × 10 5 cells/well using cell culture medium containing 10% (v/v%) fetal bovine serum at 5% CO 2 and 37°C. When the cells covered 80% area of the well, pGL4.10-F2 and pGL4.10-F2-ACE2 were wrapped with liposomes and transfected into HEK293T cells.…”

Section: Materials and Reagentsmentioning

confidence: 99%

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Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study

Li,

Lu,

Ruan

et al. 2024

Antiviral Therapy

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Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Materials and Reagentsmentioning

confidence: 99%

See 1 more Smart Citation

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study

Li,

Lu,

Ruan

et al. 2024

Antiviral Therapy

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“…The sensitivity of BRET assays has recently been improved by introducing new BRET components: RLuc2 and RLuc8 with improved quantum yields, stability, and brightness [ 143 ] as well as a great variety of acceptors (GFP2, YFP, Venus, mOrange, TagRFP, TurboFP, semiconductor quantum dots or carbon-dots) [ 144 , 145 , 146 ]. However, the main application of RLuc is luciferase genetic reporter assay that has become an invaluable and routine tool for molecular biology research, including identification and characterization of protein functional variants [ 147 , 148 ], investigations of gene expression [ 148 , 149 , 150 ], transcription factors [ 151 , 152 , 153 , 154 , 155 ], receptor activity [ 156 ], miRNA expression [ 157 , 158 , 159 ], monitoring mRNA splicing events in cells [ 160 , 161 ], virus investigations [ 162 , 163 , 164 ], drug discovery [ 165 , 166 ], and identification and evaluation of virus inhibitors [ 167 , 168 ]. Moreover, RLuc is widely used for bioluminescence imaging of different processes in vivo [ 169 , 170 , 171 ] and guiding drug delivery to cells [ 172 , 173 ].…”

Section: Ctz-dependent Luciferase Analytical Applicationmentioning

confidence: 99%

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Krasitskaya

Bashmakova

Frank

2020

IJMS

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: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

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Curcuma Longa Induces the Transcription Factor FOXP3 to Downregulate Human Chemokine CCR5 Expression and Inhibit HIV-1 Infection

Feng

et al. 2023

Am. J. Chin. Med.

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HIV mutations occur frequently despite the substantial success of combination antiretroviral therapy, which significantly impairs HIV progression. Failure to develop specific vaccines, the occurrence of drug-resistant strains, and the high incidence of adverse effects due to combination antiviral therapy regimens call for novel and safer antivirals. Natural products are an important source of new anti-infective agents. For instance, curcumin inhibits HIV and inflammation in cell culture assays. Curcumin, the principal constituent of the dried rhizomes of Curcuma longa L. (turmeric), is known as a strong anti-oxidant and anti-inflammatory agent with different pharmacological effects. This work aims to assess curcumin’s inhibitory effects on HIV in vitro and to explore the underpinning mechanism, focusing on CCR5 and the transcription factor forkhead box protein P3 (FOXP3). First, curcumin and the RT inhibitor zidovudine (AZT) were evaluated for their inhibitory properties. HIV-1 pseudovirus infectivity was determined by green fluorescence and luciferase activity measurements in HEK293T cells. AZT was used as a positive control that inhibited HIV-1 pseudoviruses dose-dependently, with IC50 values in the nanomolar range. Then, a molecular docking analysis was carried out to assess the binding affinities of curcumin for CCR5 and HIV-1 RNase H/RT. The anti-HIV activity assay showed that curcumin inhibited HIV-1 infection, and the molecular docking analysis revealed equilibrium dissociation constants of [Formula: see text]9.8[Formula: see text]kcal/mol and [Formula: see text]9.3[Formula: see text]kcal/mol between curcumin and CCR5 and HIV-1 RNase H/RT, respectively. To examine curcumin’s anti-HIV effect and its mechanism in vitro, cell cytotoxicity, transcriptome sequencing, and CCR5 and FOXP3 amounts were assessed at different concentrations of curcumin. In addition, human CCR5 promoter deletion constructs and the FOXP3 expression plasmid pRP–FOXP3 (with an EGFP tag) were generated. Whether FOXP3 DNA binding to the CCR5 promoter was blunted by curcumin was examined using transfection assays employing truncated CCR5 gene promoter constructs, a luciferase reporter assay, and a chromatin immunoprecipitation (ChIP) assay. Furthermore, micromolar concentrations of curcumin inactivated the nuclear transcription factor FOXP3, which resulted in decreased expression of CCR5 in Jurkat cells. Moreover, curcumin inhibited PI3K-AKT activation and its downstream target FOXP3. These findings provide mechanistic evidence encouraging further assessment of curcumin as a dietary agent used to reduce the virulence of CCR5-tropic HIV-1. Curcumin-mediated FOXP3 degradation was also reflected in its functions, namely, CCR5 promoter transactivation and HIV-1 virion production. Furthermore, curcumin inhibition of CCR5 and HIV-1 might constitute a potential therapeutic strategy for reducing HIV progression.

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A novel dual-luciferase assay for anti-HIV drug screening based on the CCR5/CXCR4 promoters

Cited by 4 publications

References 35 publications

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Curcuma Longa Induces the Transcription Factor FOXP3 to Downregulate Human Chemokine CCR5 Expression and Inhibit HIV-1 Infection

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