A Quantitative Fusion Assay to Study Measles Virus Entry.

We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter system is comprised of two expression vectors, each encoding a segment of Renilla luciferase fused to a segment of GFP. To regain function, the two segments need to associate, which is dependent on cell-cell fusion between effector cells expressing the MeV fusion machinery and target cells expressing the corresponding MeV receptor. By measuring reconstituted luciferase activity, we can follow the kinetics of cell-cell fusion and quantify the extent of fusion. This assay lends itself to the study of the MeV fusion machinery comprised of the attachment and fusion glycoproteins, the matrix protein, and the MeV receptors. Moreover, entry inhibitors targeting attachment or fusion can be readily screened using this assay. Finally, this assay can be easily adopted to study the entry of other members of the Paramyxoviridae, as we have demonstrated for the henipaviruses.

Enhancing luciferase activity and stability through generative modeling of natural enzyme sequences.

The availability of natural protein sequences synergized with generative AI provides new paradigms to engineer enzymes. Although active enzyme variants with numerous mutations have been designed using generative models, their performance often falls short of their wild type counterparts. Additionally, in practical applications, choosing fewer mutations that can rival the efficacy of extensive sequence alterations is usually more advantageous. Pinpointing beneficial single mutations continues to be a formidable task. In this study, using the generative maximum entropy model to analyze Renilla luciferase (RLuc) homologs, and in conjunction with biochemistry experiments, we demonstrated that natural evolutionary information could be used to predictively improve enzyme activity and stability by engineering the active center and protein scaffold, respectively. The success rate to improve either luciferase activity or stability of designed single mutants is ~50%. This finding highlights nature's ingenious approach to evolving proficient enzymes, wherein diverse evolutionary pressures are preferentially applied to distinct regions of the enzyme, ultimately culminating in an overall high performance. We also reveal an evolutionary preference in RLuc toward emitting blue light that holds advantages in terms of water penetration compared to other light spectra. Taken together, our approach facilitates navigation through enzyme sequence space and offers effective strategies for computer-aided rational enzyme engineering.

Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer.

Bioluminescent resonance energy transfer (BRET) is a natural phenomenon resulting from a non-radiative energy transfer between a bioluminescent donor (Renilla luciferase) and a fluorescent protein acceptor. BRET signal is dependent on the distance and the orientation between the donor and the acceptor and could be used to study protein-protein interactions and conformational changes within proteins at real-time in living cells. This protocol describes the use of BRET technique to study NLRP3 oligomerization in living cells before and during NLRP3 inflammasome activation.

Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity.

The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.

A Split Renilla Luciferase Complementation Assay for the Evaluation of Hsp90/Aha1 Complex Disruptors and Their Activity at the Aha1 C-Terminal Domain.

Disruption of interactions between Hsp90 and the cochaperone protein, Aha1, has emerged as a therapeutic strategy to inhibit Aha1-driven cancer metastasis and tau aggregation in models of tauopathy. A combination of split Renilla luciferase assays was developed to screen and quantify the ability of small molecules to disrupt interactions between Hsp90 and both full length Aha1 protein (Aha1-FL) and the Aha1 C-terminal domain (Aha1-CTD). This luminescence-based approach was used to identify withaferin A and gedunin as disruptors of Hsp90/Aha1 interactions and provided insight into the binding regions for gambogic acid and gedunin on the Hsp90 homodimer. All compounds tested that disrupted Hsp90/Aha1-CTD interactions were found to disrupt interactions between Hsp90 and Aha1-FL, suggesting that interactions between Hsp90 and the Aha1-CTD play a key role in the stability of Hsp90/Aha1 complexes.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Establishment of Recombinant Trisegmented Mopeia Virus Expressing Two Reporter Genes for Screening of Mammarenavirus Inhibitors.

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions.

Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate.

Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the ß6- and ß7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light.

Visible Light Bioluminescence Imaging Platform for Animal Cell Imaging.

The present protocol introduces a visible light bioluminescence imaging (BLI) platform together with 12 novel coelenterazine (CTZ) analogues and luciferase sets. We exemplify to create diverse hues of bioluminescence (BL) ranging from blue to far red with the combination of marine luciferases and the three groups of CTZ analogues. We also show how to characterize the new CTZ analogues in detail such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to artificial luciferases and are completely dark with Renilla luciferase derivatives in contrast. The 3d is highly specific to only NanoLuc. This BL imaging system covering the visible region provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.

CD38 Enhances TLR9 Expression and Activates NLRP3 Inflammasome after Porcine Parvovirus Infection.

(1) Background: Porcine Parvovirus (PPV) is a single-stranded DNA virus without envelope which causes great harm in relation to porcine reproductive disorders in clinic. Cluster of Differentiation 38 (CD38) is a transmembrane protein widely existing in mammals. Its various functions make it a very popular research object, including in the viral infection field. (2) Methods: Western blotting and an EdU Cell Proliferation Kit were used to evaluate the effect of CD38-deficient cells. Relative quantitative real-time RT-PCR was used to detect the transcription levels of cytokines after PPV infection. The renilla luciferase reporter gene assay was used to verify the activation function of CD38 on downstream factors. The fluorescence probe method was used to detect the level of intracellular reactive oxygen species (ROS). (3) Results: This study found that the loss of CD38 function inhibited the up-regulated state of Toll-like Receptor 9 (TLR9), Interferon-α (IFN-α), and Myxovirus Resistance 1 (Mx1) after PPV infection. The luminescence of the group transfected with both CD38 expression plasmid and TLR9 promoter renilla luciferase reporter plasmid was significantly up-regulated compared with the control, suggesting that CD38 may activate the promoter of TLR9. In addition, CD38 deficiency not only activated the transcription of Sirtuin-1 (SIRT1), but also inhibited ROS level and the transcription of NLR Family Pyrin Domain Containing 3 (NLRP3). (4) Conclusion: (i) CD38 may participate in the TLR9/IFN-α/Mx1 pathway by activating the expression of TLR9 after PPV infected PK-15 cells; (ii) CD38 may activate the NLRP3/CASP1 pathway by increasing ROS level; (iii) CD38 deficiency activates the expression of SIRT1 and can prevent the normal proliferation of PPV.

Paramyxovirus-Like Particles as Protein Delivery Vehicles.

We have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using two different VLP production platforms, one based on parainfluenza virus 5 (PIV5) and the other based on Nipah virus, and in both cases efficient cargo packaging and delivery could be achieved. These findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. IMPORTANCE Therapeutic proteins including transcription factors and genome editors have enormous clinical potential but are currently limited in part due to the challenges of safely and efficiently delivering these proteins to the interiors of target cells. Here, we have developed a new strategy for protein delivery based on manipulation of paramyxovirus genome packaging interactions.

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids.

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

Monitoring the Mitochondrial Presequence Import Pathway In Living Mammalian Cells with a New Molecular Biosensor.

Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol, and imported into the organelle. Mitochondrial protein import is therefore vital for the maintenance of mitochondrial function and cell survival. Alterations in this process are suspected to contribute to various diseases, including neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Our understanding of the cytosolic signaling mechanisms and posttranslational modifications regulating the mitochondrial import process is still in its infancy and hampered by the lack of tools for its dynamic assessment in cells. We recently engineered an inducible molecular biosensor for monitoring one of the main mitochondrial import routes, the so-called presequence pathway, using a quantitative luminescence-based readout. Here, we provide basic guidelines for using this probe in common cell types of general use in the scientific community: HEK293T cells, human fibroblasts, and mouse primary neurons. These guidelines can serve as a starting point for the development of more elaborated protocols for the dynamic investigation of the presequence import pathway and its regulation in relevant physiological and pathological conditions.

A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant.

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.

Color-tunable bioluminescence imaging portfolio for cell imaging.

The present study describes a color-tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red-shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artificial Luciferase 16 (ALuc16) show 187 nm- and 105 nm-redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d. The optical properties of the new CTZ analogues were investigated such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to ALucs and are completely dark with RLuc derivatives, and 3d is highly specific to only NanoLuc. We further determined the theoretical background of the red-shifted BL maximum wavelengths (λBL) values according to the extended π conjugation of the CTZ backbone using Density Functional Theory (DFT) calculations. This color-tunable BL imaging system provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.

Effects of codon usage on gene expression are promoter context dependent.

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.

Nonviral DNA Delivery System with Supramolecular PEGylation Formed by Host-Guest Pseudo-Block Copolymers.

A cationic supramolecular system based on host-guest pseudoblock copolymers was developed for nonviral DNA delivery. In this system, the macromolecular host was a cationic star-shaped polymer composed of a ß-cyclodextrin (ß-CD) core and multiple poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) chains grafted on the core, while the macromolecular guest was a linear adamantyl-ended poly(ethylene glycol) (mPEG-Ad). Pseudoblock copolymers were self-assembled from the polymeric host-guest pairs (typically, 1:1 molar ratio) in aqueous media through the inclusion of an adamantyl group at the end of guest polymer into the ß-CD cavity of host polymers. Through such an approach, the resultant supramolecular system was integrated with not only a superior DNA condensing ability due to the host polymer but also an outstanding polyplex-stabilizing ability as well as biocompatibility due to the guest polymer. The cationic star-shaped host polymers alone were capable of condensing plasmid DNA efficiently into nanoparticles (70-100 nm) with positive surface charge. They showed obviously lower cytotoxicity than PEI 25K (commercial branched polyethylenimine with a molecular weight around 25 kDa) in cell lines of L929, MB231, and Hela under high dose. In serum-free or serum-containing culture conditions, these host polymers exhibited either higher or lower in vitro DNA transfection efficiency as compared with PEI 25K in the three cell lines under study, which was dependent on the N/P ratios and PDMAEMA arm length. Upon incorporation of the PEG block through host-guest complexation with mPEG-Ad (i.e., supramolecular PEGylation), the resulting host-guest supramolecular systems exhibited even lower cytotoxicity than the host polymers alone. The polyplexes between plasmid DNA (pDNA) and the host-guest systems showed significantly improved stability in BSA-PBS buffer solution (pH 7.4) and enhanced in vitro DNA transfection efficiency in the cases of higher N/P ratios or longer PDMAEMA arms in all tested cell lines under both serum-free and serum-containing culture conditions, as compared with the corresponding polyplexes without supramolecular PEGylation. Further, through forming pseudoblock copolymer, the DNA transfection ability of the supramolecular system can be easily modulated and optimized either by changing the ratio between the guest and host or by using different hosts with varied PDMAEMA arm lengths.

Renilla Luciferase Reporter Assay to Study 3'-UTR-Driven Posttranscriptional Regulation of OPRM1.

MOR expression levels at a specific cell type or tissue significantly contribute to its role in pain transmission and in other responses involving opioid receptors. Therefore, molecular processes regulating MOR levels have gained more and more interest. Recently, posttranscriptional regulation mechanisms have been shown to play a relevant role in influencing MOR expression levels, with polymorphisms and mutations within OPRM1 3'-UTR region impacting the differential opioid-mediated response observed within individuals. Here we report a Renilla luciferase reporter assay format suitable for dissecting the contribution of different and distinct OPRM1 3'-UTR elements to MOR expression levels in a model of glial cells, both under basal conditions and following specific treatments.

Monitoring Opioid Receptor Interaction in Living Cells by Bioluminescence Resonance Energy Transfer (BRET).

Bioluminescence resonance energy transfer (BRET ) is a natural phenomenon that has been successfully applied for the study of protein-protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the discovery of new functions and new way of signaling and trafficking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fluorescent protein (such as EYFP ) it is possible to study opioid receptor dimerization using BRET .