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Journal articles on the topic 'NanoLuciferase'

Author: Grafiati

Published: 28 June 2021

Last updated: 7 February 2022

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1

Sfarcic, Ivana, Theresa Bui, Erin C. Daniels, and Emily R. Troemel. "Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans." Genetics 213, no. 4 (October 4, 2019): 1197–207. http://dx.doi.org/10.1534/genetics.119.302655.

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Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

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Pulkina, A. A., E. A. Romanovskaya-Romanko, A. S. Mustafaeva, A. Yu Egorov, and M. A. Stukova. "Rapid Neutralizing Antibody Assessment Using Influenza Viruses with Luciferase Reporter." Biotekhnologiya 37, no. 2 (2021): 81–91. http://dx.doi.org/10.21519/0234-2758-2021-37-2-81-91.

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Influenza viruses cause an acute respiratory infection, especially in the autumn-winter period. They are characterized by a high mutation frequency and cause annual seasonal epidemics. The detection of virus-neutralizing antibodies is an important criterion in the assessment of population immunity and the influenza vaccine effectiveness. In this study, a method for determining the titer of virus-neutralizing antibodies in blood serum has been developed. A new test, called the luciferase neutralization assay, uses measurement of a bioluminescent signal as a detection method. The influenza A reporter viruses of various subtypes were constructed that encode the nanoluciferase protein in the non-structural NS1 protein reading frame. The developed method was used to compare paired sera of volunteers before and after their immunization with a seasonal influenza vaccine. The proposed method was also compared with certified antibody assay methods: neutralization reaction and hemagglutination inhibition reaction. The tests showed a high correlation, while the luciferase neutralization assay reduced the time and simplified the detection procedure. microneutralization, reporter virus, influenza virus, bioluminescence, nanoluciferase The study was supported by the grant of the President of the Russian Federation for young PhDs (no. 075-15-2019-226); and also by the grant of the Government of Saint-Petersburg for undergraduate and graduate students (September 25, 2018, no 124).

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Sahihi, Mehdi, Juan Sanz García, and Isabelle Navizet. "Bioluminescent Nanoluciferase–Furimamide Complex: A Theoretical Study on Different Protonation States." Journal of Physical Chemistry B 124, no. 13 (March 10, 2020): 2539–48. http://dx.doi.org/10.1021/acs.jpcb.9b11597.

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4

Wires, Emily S., Doug Howard, Mark J. Henderson, Xiaokang Yan, Kathleen A. Trychta, Emily J. Heathward, Yajun Zhang, Molly Lutrey, Christopher Richie, and Brandon K. Harvey. "218. Monitoring ER Stress Activation of the ATF6 Pathway Using Nanoluciferase." Molecular Therapy 24 (May 2016): S85. http://dx.doi.org/10.1016/s1525-0016(16)33027-1.

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5

Degrelle, Séverine A., Hussein Shoaito, and Thierry Fournier. "New Transcriptional Reporters to Quantify and Monitor PPARγ Activity." PPAR Research 2017 (2017): 1–7. http://dx.doi.org/10.1155/2017/6139107.

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The peroxisome-proliferator-activated-receptor-γ (PPARγ) is a member of the nuclear receptor superfamily that plays a critical role in diverse biological processes, including adipogenesis, lipid metabolism, and placental development. To study the activity of PPARγ, we constructed two new reporter genes: a fluorescent GFP-tagged histone-2B (PPRE-H2B-eGFP) and a secreted nanoluciferase (PPRE-pNL1.3[secNluc]). This study demonstrates their usage to monitor PPARγ activity in different cell types and screen for PPARγ’s potential ligands.

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6

Calverley, Ben C., Karl E. Kadler, and Adam Pickard. "Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging." Cells 9, no. 9 (September 10, 2020): 2070. http://dx.doi.org/10.3390/cells9092070.

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The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

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7

Abe, Taisho, Riku Nagai, Hiroaki Imataka, and Nono Takeuchi-Tomita. "Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA." Journal of Biochemistry 167, no. 5 (March 16, 2020): 441–50. http://dx.doi.org/10.1093/jb/mvaa021.

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Abstract We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter protein, nanoLuciferase, with a molecular weight of 19 kDa. The protein synthesis by the system is appropriately regulated by controlling its composition, including translation factors, amino acids and antibiotics. We found that a high eEF1A concentration relative to the ribosome concentration is critically required for efficient IRES-mediated translation initiation, to ensure its dominance over IRES-independent random internal translation initiation.

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8

Kim, Jiho, and Regis Grailhe. "Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy." Cytometry Part A 89, no. 8 (May 3, 2016): 742–46. http://dx.doi.org/10.1002/cyto.a.22870.

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9

Zhang, Lei, Ge Song, Ting Xu, Qing-Ping Wu, Xiao-Xia Shao, Ya-Li Liu, Zeng-Guang Xu, and Zhan-Yun Guo. "A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase." Biochimie 95, no. 12 (December 2013): 2454–59. http://dx.doi.org/10.1016/j.biochi.2013.09.008.

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10

Ji, Ben-Jun, Ge Song, Zhou Zhang, and Zhan-Yun Guo. "Efficient overexpression of human interleukin-6 in Escherichia coli using nanoluciferase as a fusion partner." Process Biochemistry 50, no. 10 (October 2015): 1618–22. http://dx.doi.org/10.1016/j.procbio.2015.06.008.

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11

Silberstein, Erica, Carylinda Serna, Stenio Perdigão Fragoso, Rana Nagarkatti, and Alain Debrabant. "A novel nanoluciferase-based system to monitor Trypanosoma cruzi infection in mice by bioluminescence imaging." PLOS ONE 13, no. 4 (April 19, 2018): e0195879. http://dx.doi.org/10.1371/journal.pone.0195879.

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12

White, Carl W., Birgit Caspar, Hannah K. Vanyai, Kevin D. G. Pfleger, and Stephen J. Hill. "CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes." Cell Chemical Biology 27, no. 5 (May 2020): 499–510. http://dx.doi.org/10.1016/j.chembiol.2020.01.010.

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13

Laschet, Céline, Nadine Dupuis, and Julien Hanson. "A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR–G protein interactions." Journal of Biological Chemistry 294, no. 11 (December 28, 2018): 4079–90. http://dx.doi.org/10.1074/jbc.ra118.006231.

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14

Duyên, Trần Thị Mỹ, and Trần Thị Tuyết Hoa. "Nghiên cứu tạo protein tín hiệu nanoluciferase: Ứng dụng tạo cảm biến sinh học nhận diện kháng sinh." Can Tho University Journal of Science 56(2) (2020): 146. http://dx.doi.org/10.22144/ctu.jvn.2020.041.

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15

Ren, Wenjie, Zhenfeng Li, Yang Xu, Debin Wan, Bogdan Barnych, Yanping Li, Zhui Tu, Qinghua He, Jinheng Fu, and Bruce D. Hammock. "One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B1 in Cereal." Journal of Agricultural and Food Chemistry 67, no. 18 (March 18, 2019): 5221–29. http://dx.doi.org/10.1021/acs.jafc.9b00688.

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16

Wang, Feng, Zhen-Feng Li, De-Bin Wan, Natalia Vasylieva, Yu-Dong Shen, Zhen-Lin Xu, Jin-Yi Yang, et al. "Enhanced Non-Toxic Immunodetection of Alternaria Mycotoxin Tenuazonic Acid Based on Ferritin-Displayed Anti-Idiotypic Nanobody-Nanoluciferase Multimers." Journal of Agricultural and Food Chemistry 69, no. 16 (April 18, 2021): 4911–17. http://dx.doi.org/10.1021/acs.jafc.1c01128.

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17

Azad, Taha, Ragunath Singaravelu, Emily E. F. Brown, Zaid Taha, Reza Rezaei, Rozanne Arulanandam, Stephen Boulton, Jean-Simon Diallo, Carolina S. Ilkow, and John C. Bell. "SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition." Biosensors and Bioelectronics 180 (May 2021): 113122. http://dx.doi.org/10.1016/j.bios.2021.113122.

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18

Li, Wan, Mengjia Zhang, Huijun Zheng, Peng Zhou, Zheng Liu, Anan Jongkaewwattana, Rui Luo, and Qigai He. "Construction of a Recombinant Porcine Epidemic Diarrhea Virus Encoding Nanoluciferase for High-Throughput Screening of Natural Antiviral Products." Viruses 13, no. 9 (September 18, 2021): 1866. http://dx.doi.org/10.3390/v13091866.

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Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

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19

Huang, Yikun, André O’Reilly Beringhs, Qi Chen, Donghui Song, Wilfred Chen, Xiuling Lu, Tai-Hsi Fan, Mu-Ping Nieh, and Yu Lei. "Genetically Engineered Bacterial Outer Membrane Vesicles with Expressed Nanoluciferase Reporter for in Vivo Bioluminescence Kinetic Modeling through Noninvasive Imaging." ACS Applied Bio Materials 2, no. 12 (November 26, 2019): 5608–15. http://dx.doi.org/10.1021/acsabm.9b00690.

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20

Li, Zhenfeng, Yi Wang, Natalia Vasylieva, Debin Wan, Zihan Yin, Jiexian Dong, and Bruce D. Hammock. "An Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Heptamer Fusion for the Detection of Tetrabromobisphenol A in Sediment." Analytical Chemistry 92, no. 14 (June 19, 2020): 10083–90. http://dx.doi.org/10.1021/acs.analchem.0c01908.

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21

Wang, Feng, Zhen-Feng Li, Yuan-Yuan Yang, De-Bin Wan, Natalia Vasylieva, Yu-Qi Zhang, Jun Cai, et al. "Chemiluminescent Enzyme Immunoassay and Bioluminescent Enzyme Immunoassay for Tenuazonic Acid Mycotoxin by Exploitation of Nanobody and Nanobody–Nanoluciferase Fusion." Analytical Chemistry 92, no. 17 (July 24, 2020): 11935–42. http://dx.doi.org/10.1021/acs.analchem.0c02338.

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22

Vasudevan, Lakshmi, and Christophe P. Stove. "A novel nanobody-based bio-assay using functional complementation of a split nanoluciferase to monitor Mu- opioid receptor activation." Analytical and Bioanalytical Chemistry 412, no. 29 (September 14, 2020): 8015–22. http://dx.doi.org/10.1007/s00216-020-02945-6.

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23

Azad, Taha, Ragunath Singaravelu, Zaid Taha, Taylor R. Jamieson, Stephen Boulton, Mathieu J. F. Crupi, Nikolas T. Martin, et al. "Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry." Molecular Therapy 29, no. 6 (June 2021): 1984–2000. http://dx.doi.org/10.1016/j.ymthe.2021.02.007.

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24

Song, Ge, Qing-Ping Wu, Ting Xu, Ya-Li Liu, Zeng-Guang Xu, Shi-Fu Zhang, and Zhan-Yun Guo. "Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model." Journal of Photochemistry and Photobiology B: Biology 153 (December 2015): 311–16. http://dx.doi.org/10.1016/j.jphotobiol.2015.10.014.

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25

Yu, Sheng, Zhenfeng Li, Jingzhang Li, Shimei Zhao, Shanguang Wu, Hongjing Liu, Xiongjie Bi, et al. "Generation of dual functional nanobody-nanoluciferase fusion and its potential in bioluminescence enzyme immunoassay for trace glypican-3 in serum." Sensors and Actuators B: Chemical 336 (June 2021): 129717. http://dx.doi.org/10.1016/j.snb.2021.129717.

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26

He, Sheng-Xiang, Ge Song, Jia-Ping Shi, Yu-Qi Guo, and Zhan-Yun Guo. "Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor." Biochimie 106 (November 2014): 140–48. http://dx.doi.org/10.1016/j.biochi.2014.08.012.

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27

Liu, Yu, Ge Song, Xiao-Xia Shao, Ya-Li Liu, and Zhan-Yun Guo. "Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model." Biochimica et Biophysica Acta (BBA) - Biomembranes 1848, no. 2 (February 2015): 688–94. http://dx.doi.org/10.1016/j.bbamem.2014.11.026.

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28

Kim, Dae Gyu, Chul Min Park, Srigouri Huddar, Semi Lim, Sunghoon Kim, and Sunkyung Lee. "Anticancer Activity of Pyrimethamine via Ubiquitin Mediated Degradation of AIMP2-DX2." Molecules 25, no. 12 (June 15, 2020): 2763. http://dx.doi.org/10.3390/molecules25122763.

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While aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a tumor suppressor, its exon 2-depleted splice variant (AIMP2-DX2 or shortly DX2) is highly expressed in human lung cancer, and the ratio of DX2 to AIMP2 increases according to the progression of lung cancer. In this study, pyrimethamine inhibited the level of DX2 (IC50 = 0.73 µM) in A549 cells expressing nanoluciferase-tagged DX2. In a panel of 5 lung cancer cell lines with various DX2 levels, pyrimethamine most potently suppressed the growth of H460 cells, which express high levels of DX2 (GI50 = 0.01 µM). An immunoblot assay in H460 cells showed that pyrimethamine decreased the DX2 level dose-dependently but did not affect the AIMP2 level. Further experiments confirmed that pyrimethamine resulted in ubiquitination-mediated DX2 degradation. In an in vivo mouse xenograft assay using H460 cells, intraperitoneal administration of pyrimethamine significantly reduced the tumor size and weight, comparable with the effects of taxol, without affecting body weight. Analysis of tumor tissue showed a considerably high concentration of pyrimethamine with a decreased levels of DX2. These results suggest that pyrimethamine, currently used as anti-parasite drug, could be repurposed to treat lung cancer patients expressing high level of DX2.

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29

Wouters, Elise, Adrián Marín, James Dalton, Jesús Giraldo, and Christophe Stove. "Distinct Dopamine D2 Receptor Antagonists Differentially Impact D2 Receptor Oligomerization." International Journal of Molecular Sciences 20, no. 7 (April 4, 2019): 1686. http://dx.doi.org/10.3390/ijms20071686.

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Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40–60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a–D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D2R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D2R homodimerization.

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30

Ali, Amanat, Elizabeth K. M. Johnstone, Bincy Baby, Heng B. See, Angela Song, K. Johan Rosengren, Kevin D. G. Pfleger, Mohammed Akli Ayoub, and Ranjit Vijayan. "Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor." International Journal of Molecular Sciences 22, no. 1 (December 28, 2020): 209. http://dx.doi.org/10.3390/ijms22010209.

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Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

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Spillmann, Martin, Larissa Thurner, Nina Romantini, Mirjam Zimmermann, Benoit Meger, Martin Behe, Maria Waldhoer, Gebhard F. X. Schertler, and Philipp Berger. "New Insights into Arrestin Recruitment to GPCRs." International Journal of Molecular Sciences 21, no. 14 (July 13, 2020): 4949. http://dx.doi.org/10.3390/ijms21144949.

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G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed us to probe the relevance of each of the two arrestin binding sites in the different GPCRs for arrestin binding. We observed remarkable differences between the roles of each arresting binding site in the tested GPCRs and propose that the distinct advantages of our system for probing receptor interaction with effector proteins will help elucidate the molecular basis of GPCR signaling efficacy and specificity in different cell types.

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Nagai, Riku, Yichen Xu, Chang Liu, Ayaka Shimabukuro, and Nono Takeuchi-Tomita. "In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling." Methods and Protocols 4, no. 3 (July 4, 2021): 45. http://dx.doi.org/10.3390/mps4030045.

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The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

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33

Saxena, Gauri, James M. Moore, Meleri Jones, Gareth Pryce, Liaqat Ali, Georgia R. Leisegang, Vivek Vijay, et al. "Detecting and predicting neutralization of alemtuzumab responses in MS." Neurology - Neuroimmunology Neuroinflammation 7, no. 4 (June 4, 2020): e767. http://dx.doi.org/10.1212/nxi.0000000000000767.

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ObjectiveTo test the hypothesis that antidrug antibodies (ADAs) against alemtuzumab could become relevant after repeated treatments for some individuals, possibly explaining occasional treatment resistance.MethodsRecombinant alemtuzumab single-chain variable fragment antibody with a dual tandem nanoluciferase reporter linker was made and used to detect binding ADAs. Alemtuzumab immunoglobulin G Alexa Fluor 488 conjugate was used in a competitive binding cell-based assay to detect neutralizing ADAs. The assays were used to retrospectively screen, blinded, banked serum samples from people with MS (n = 32) who had received 3 or more cycles of alemtuzumab. Lymphocyte depletion was measured between baseline and about 1 month postinfusion.ResultsThe number of individuals showing limited depletion of lymphocytes increased with the number of treatment cycles. Lack of depletion was also a poor prognostic feature for future disease activity. ADA responses were detected in 29/32 (90.6%) individuals. Neutralizing antibodies occurred before the development of limited depletion in 6/7 individuals (18.8% of the whole sample). Preinfusion, ADA levels predicted limited, postinfusion lymphocyte depletion.ConclusionsAlthough ADAs to alemtuzumab have been portrayed as being of no clinical significance, alemtuzumab-specific antibodies appear to be clinically relevant for some individuals, although causation remains to be established. Monitoring of lymphocyte depletion and the antidrug response may be of practical value in patients requiring additional cycles of alemtuzumab. ADA detection may help to inform on retreatment or switching to another treatment.

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Zhang, Wei, Terunao Takahara, Takuya Achiha, Hideki Shibata, and Masatoshi Maki. "Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization." International Journal of Molecular Sciences 19, no. 2 (February 18, 2018): 605. http://dx.doi.org/10.3390/ijms19020605.

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35

Sherwood, Laura J., and Andrew Hayhurst. "Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus." Viruses 11, no. 4 (April 20, 2019): 364. http://dx.doi.org/10.3390/v11040364.

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We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb–APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb–APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕnglà virus genome. Antibody–antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕnglà NP C-terminal domain. The sdAb–APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb–APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.

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Henderson, Mark J., Marc A. Holbert, Anton Simeonov, and Lorena A. Kallal. "High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 2 (September 30, 2019): 137–47. http://dx.doi.org/10.1177/2472555219877183.

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Thermal shift assays (TSAs) can reveal changes in protein structure, due to a resultant change in protein thermal stability. Since proteins are often stabilized upon binding of ligand molecules, these assays can provide a readout for protein target engagement. TSA has traditionally been applied using purified proteins and more recently has been extended to study target engagement in cellular environments with the emergence of cellular thermal shift assays (CETSAs). The utility of CETSA in confirming molecular interaction with targets in a more native context, and the desire to apply this technique more broadly, has fueled the emergence of higher-throughput techniques for CETSA (HT-CETSA). Recent studies have demonstrated that HT-CETSA can be performed in standard 96-, 384-, and 1536-well microtiter plate formats using methods such as beta-galactosidase and NanoLuciferase reporters and AlphaLISA assays. HT-CETSA methods can be used to select and characterize compounds from high-throughput screens and to prioritize compounds in lead optimization by facilitating dose–response experiments. In conjunction with cellular and biochemical activity assays for targets, HT-CETSA can be a valuable addition to the suite of assays available to characterize molecules of interest. Despite the successes in implementing HT-CETSA for a diverse set of targets, caveats and challenges must also be recognized to avoid overinterpretation of results. Here, we review the current landscape of HT-CETSA and discuss the methodologies, practical considerations, challenges, and applications of this approach in research and drug discovery. Additionally, a perspective on potential future directions for the technology is presented.

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Kumar, Binod, Grant M. Hawkins, Tom Kicmal, Enya Qing, Emily Timm, and Tom Gallagher. "Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles." Cells 10, no. 4 (April 9, 2021): 853. http://dx.doi.org/10.3390/cells10040853.

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Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

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38

Gornalusse, Germán Gustavo, Lucia N. Vojtech, Claire N. Levy, Sean M. Hughes, Yeseul Kim, Rogelio Valdez, Urvashi Pandey, et al. "Buprenorphine Increases HIV-1 Infection In Vitro but Does Not Reactivate HIV-1 from Latency." Viruses 13, no. 8 (July 27, 2021): 1472. http://dx.doi.org/10.3390/v13081472.

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Background: medication-assisted treatment (MAT) with buprenorphine is now widely prescribed to treat addiction to heroin and other illicit opioids. There is some evidence that illicit opioids enhance HIV-1 replication and accelerate AIDS pathogenesis, but the effect of buprenorphine is unknown. Methods: we obtained peripheral blood mononuclear cells (PBMCs) from healthy volunteers and cultured them in the presence of morphine, buprenorphine, or methadone. We infected the cells with a replication-competent CCR5-tropic HIV-1 reporter virus encoding a secreted nanoluciferase gene, and measured infection by luciferase activity in the supernatants over time. We also surveyed opioid receptor expression in PBMC, genital epithelial cells and other leukocytes by qPCR and western blotting. Reactivation from latency was assessed in J-Lat 11.1 and U1 cell lines. Results: we did not detect expression of classical opioid receptors in leukocytes, but did find nociception/orphanin FQ receptor (NOP) expression in blood and vaginal lymphocytes as well as genital epithelial cells. In PBMCs, we found that at physiological doses, morphine, and methadone had a variable or no effect on HIV infection, but buprenorphine treatment significantly increased HIV-1 infectivity (median: 8.797-fold increase with 20 nM buprenorphine, eight experiments, range: 3.570–691.9, p = 0.0078). Using latently infected cell lines, we did not detect reactivation of latent HIV following treatment with any of the opioid drugs. Conclusions: our results suggest that buprenorphine, in contrast to morphine or methadone, increases the in vitro susceptibility of leukocytes to HIV-1 infection but has no effect on in vitro HIV reactivation. These findings contribute to our understanding how opioids, including those used for MAT, affect HIV infection and reactivation, and can help to inform the choice of MAT for people living with HIV or who are at risk of HIV infection.

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Vickers, Timothy A., Meghdad Rahdar, Thazha P. Prakash, and Stanley T. Crooke. "Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1." Nucleic Acids Research 47, no. 20 (September 9, 2019): 10865–80. http://dx.doi.org/10.1093/nar/gkz771.

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Abstract The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein–protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO–protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.

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Li, Jie, Xiaoxu Wang, Gaopan Dong, Chongzheng Yan, Yuanyuan Cui, Zheng Zhang, Lupei Du, and Minyong Li. "Novel furimazine derivatives for nanoluciferase bioluminescence with various C-6 and C-8 substituents." Organic & Biomolecular Chemistry, 2021. http://dx.doi.org/10.1039/d1ob01098k.

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41

Lee, Muhoon, Noriko Matsunaga, Shiori Akabane, Ippei Yasuda, Takuya Ueda, and Nono Takeuchi-Tomita. "Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA." Nucleic Acids Research, December 9, 2020. http://dx.doi.org/10.1093/nar/gkaa1165.

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Abstract Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

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42

White, Carl W., Laura E. Kilpatrick, Kevin D. G. Pfleger, and Stephen J. Hill. "A Nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding." iScience, December 2020, 102011. http://dx.doi.org/10.1016/j.isci.2020.102011.

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43

Lin, Jing-Yi, Kuo-Feng Weng, Chih-Kuang Chang, Yu-Nong Gong, Guo-Jen Huang, Hui-Lan Lee, Yen-Cheng Chen, et al. "Enterovirus A71 Induces Neurological Diseases and Dynamic Variants in Oral Infection of Human SCARB2-Transgenic Weaned Mice." Journal of Virology, August 11, 2021. http://dx.doi.org/10.1128/jvi.00897-21.

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Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally-inoculated virus can spread to the central nervous system and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. Importance Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae , poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and non-immunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real-time. Such a model offers several advantages over existing animal models, and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

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44

Jain, Paras, Spencer Garing, Deepshikha Verma, Rajagopalan Saranathan, Nicholas Clute-Reinig, Jacob Gadwa, Chelsea Peterson, et al. "Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility." Journal of Bacteriology 202, no. 22 (September 8, 2020). http://dx.doi.org/10.1128/jb.00411-20.

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ABSTRACT Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

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"Using the newly developed nanoluciferase as an ultrasensitive bioluminescent probe for ligand-receptor interaction studies." Receptors & Clinical Investigation, April 15, 2014. http://dx.doi.org/10.14800/rci.116.

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46

Li, Hong, Caiyun Wu, Manman Du, Yache Chen, Xin Hou, Yinong Yang, and Kabin Xie. "A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants." Molecular Breeding 41, no. 2 (February 2021). http://dx.doi.org/10.1007/s11032-021-01210-7.

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47

Oliveira, Débora Moraes de, Igor de Andrade Santos, Daniel Oliveira Silva Martins, Yasmim Garcia Gonçalves, Léia Cardoso-Sousa, Robinson Sabino-Silva, Gustavo Von Poelhsitz, et al. "Organometallic Complex Strongly Impairs Chikungunya Virus Entry to the Host Cells." Frontiers in Microbiology 11 (December 15, 2020). http://dx.doi.org/10.3389/fmicb.2020.608924.

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Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV) that is transmitted by the bite of the female of Aedes sp. mosquito. The symptoms include fever, muscle aches, skin rash, and severe joint pains. The disease may develop into a chronic condition and joint pain for months or years. Currently, there is no effective antiviral treatment against CHIKV infection. Treatments based on natural compounds have been widely studied, as many drugs were produced by using natural molecules and their derivatives. Alpha-phellandrene (α-Phe) is a naturally occurring organic compound that is a ligand for ruthenium, forming the organometallic complex [Ru2Cl4(p-cymene)2] (RcP). Organometallic complexes have shown promising as candidate molecules to a new generation of compounds that presented relevant biological properties, however, there is a lack of knowledge concerning the anti-CHIKV activity of these complexes. The present work evaluated the effects of the RcP and its precursors, the hydrate ruthenium(III) chloride salt (RuCl3⋅xH2O) (Ru) and α-Phe, on CHIKV infection in vitro. To this, BHK-21 cells were infected with CHIKV-nanoluciferase (CHIKV-nanoluc), a viral construct harboring the nanoluciferase reporter gene, at the presence or absence of the compounds for 16 h. Cytotoxicity and impact on infectivity were analyzed. The results demonstrated that RcP exhibited a strong therapeutic potential judged by the selective index > 40. Antiviral effects of RcP on different stages of the CHIKV replicative cycle were investigated; the results showed that it affected early stages of virus infection reducing virus replication by 77% at non-cytotoxic concentrations. Further assays demonstrated the virucidal activity of the compound that completely blocked virus infectivity. In silico molecular docking calculations suggested different binding interactions between aromatic rings of RcP and the loop of amino acids of the E2 envelope CHIKV glycoprotein mainly through hydrophobic interactions. Additionally, infrared spectroscopy spectral analysis indicated interactions of RcP with CHIKV glycoproteins. These data suggest that RcP may act on CHIKV particles, disrupting virus entry to the host cells. Therefore, RcP may represent a strong candidate for the development of anti-CHIKV drugs.

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48

Oliveira, Débora Moraes de, Igor de Andrade Santos, Daniel Oliveira Silva Martins, Yasmim Garcia Gonçalves, Léia Cardoso-Sousa, Robinson Sabino-Silva, Gustavo Von Poelhsitz, et al. "Organometallic Complex Strongly Impairs Chikungunya Virus Entry to the Host Cells." Frontiers in Microbiology 11 (December 15, 2020). http://dx.doi.org/10.3389/fmicb.2020.608924.

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Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV) that is transmitted by the bite of the female of Aedes sp. mosquito. The symptoms include fever, muscle aches, skin rash, and severe joint pains. The disease may develop into a chronic condition and joint pain for months or years. Currently, there is no effective antiviral treatment against CHIKV infection. Treatments based on natural compounds have been widely studied, as many drugs were produced by using natural molecules and their derivatives. Alpha-phellandrene (α-Phe) is a naturally occurring organic compound that is a ligand for ruthenium, forming the organometallic complex [Ru2Cl4(p-cymene)2] (RcP). Organometallic complexes have shown promising as candidate molecules to a new generation of compounds that presented relevant biological properties, however, there is a lack of knowledge concerning the anti-CHIKV activity of these complexes. The present work evaluated the effects of the RcP and its precursors, the hydrate ruthenium(III) chloride salt (RuCl3⋅xH2O) (Ru) and α-Phe, on CHIKV infection in vitro. To this, BHK-21 cells were infected with CHIKV-nanoluciferase (CHIKV-nanoluc), a viral construct harboring the nanoluciferase reporter gene, at the presence or absence of the compounds for 16 h. Cytotoxicity and impact on infectivity were analyzed. The results demonstrated that RcP exhibited a strong therapeutic potential judged by the selective index > 40. Antiviral effects of RcP on different stages of the CHIKV replicative cycle were investigated; the results showed that it affected early stages of virus infection reducing virus replication by 77% at non-cytotoxic concentrations. Further assays demonstrated the virucidal activity of the compound that completely blocked virus infectivity. In silico molecular docking calculations suggested different binding interactions between aromatic rings of RcP and the loop of amino acids of the E2 envelope CHIKV glycoprotein mainly through hydrophobic interactions. Additionally, infrared spectroscopy spectral analysis indicated interactions of RcP with CHIKV glycoproteins. These data suggest that RcP may act on CHIKV particles, disrupting virus entry to the host cells. Therefore, RcP may represent a strong candidate for the development of anti-CHIKV drugs.

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49

Yao, Zhong, Luka Drecun, Farzaneh Aboualizadeh, Sun Jin Kim, Zhijie Li, Heidi Wood, Emelissa J. Valcourt, et al. "A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies." Nature Communications 12, no. 1 (March 22, 2021). http://dx.doi.org/10.1038/s41467-021-22102-6.

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AbstractBetter diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.

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50

Xie, Xuping, Antonio E. Muruato, Xianwen Zhang, Kumari G. Lokugamage, Camila R. Fontes-Garfias, Jing Zou, Jianying Liu, et al. "A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19." Nature Communications 11, no. 1 (October 15, 2020). http://dx.doi.org/10.1038/s41467-020-19055-7.

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Abstract A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.

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