Search Results (12)

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines. Full article

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups. Full article

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology. Full article

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gα_i/o, Gα_Ss/L, Gα_12/13 and Gα_q/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor–acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for Gα_Ss/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity. Full article

SARS-CoV-2 is a positive-strand RNA virus in the Coronaviridae family that is responsible for morbidity and mortality worldwide. To better understand the molecular pathways leading to SARS-CoV-2 virus assembly, we examined a virus-like particle (VLP) system co-expressing all structural proteins together with an mRNA reporter encoding nanoLuciferase (herein nLuc). Surprisingly, the 19 kDa nLuc protein itself was encapsidated into VLPs, providing a better reporter than nLuc mRNA itself. Strikingly, infecting nLuc-expressing cells with the SARS-CoV-2, NL63 or OC43 coronaviruses yielded virions containing packaged nLuc that served to report viral production. In contrast, infection with the flaviviruses, dengue or Zika, did not lead to nLuc packaging and secretion. A panel of reporter protein variants revealed that the packaging is size-limited and requires cytoplasmic expression, indicating that the large virion of coronaviruses can encaspidate a small cytoplasmic reporter protein. Our findings open the way for powerful new approaches to measure coronavirus particle production, egress and viral entry mechanisms. Full article

Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase–nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3′,5,5′-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC₁₀) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples. Full article

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell–cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell–cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell–cell fusion in Vero cells. Full article

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5′ untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5′ UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5′ UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity. Full article

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV. Full article

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. Full article

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. Full article

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. Full article