RESUMEN
Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by â¼3.79-fold and â¼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.
Asunto(s)
COVID-19 , Luciferasas de Luciérnaga , Animales , Evaluación de Medicamentos , Luciferasas/genética , Luciferasas de Luciérnaga/metabolismo , Ratones , Proteínas de la Nucleocápside , SARS-CoV-2RESUMEN
Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.
Asunto(s)
Puntos Cuánticos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Manganeso , Puntos Cuánticos/toxicidad , Sulfuros , Compuestos de ZincRESUMEN
BACKGROUND: Much research has focused on quantifying the bony characteristics of patients with developmental dysplasia of the hip (DDH). Far less attention, however, has been paid to muscle abnormalities around the hip such as those in the gluteus medius (GM). METHODS: We retrospectively examined clinical and imaging data, such as the age of onset and computed tomography (CT) findings, in 108 consecutive hips. Subjects for the control group were selected from our radiology database. Two readers independently evaluated the length (LGM), cross-sectional area (CSA), width (WGM), and thickness (TGM) of the GM and arm of GM (AGM) and angle of the GM activation (AOA) and bony parameters including the acetabulum-head index (AHI), lateral central edge angle (LCEA), acetabular index (AI), femoral offset (FO), and height of the rotation centre of femoral head (HCFH) among all cases using the imaging data. RESULTS: The patient group included 108 hips. The AGM, LGM, CSA, and TGM were lower in the DDH patients, while AOA was higher. However, there was no significant difference in the WGM between the two groups. Multiple linear regression analysis showed that AGM and AOA were independent factors affecting LCEA. The following regression equation was used: Y(LCEA) = 5.377 * X1 (AGM) - 0.310 * X2 (AOA) - 11.331. The mechanical characteristics of the GM and many bony parameters were significantly correlated (the AGM and AHI, LCEA, AI, FO, but not HCFH; AOA and AHI, LCEA, AI, but not FO or HCFH). The CSA was positively correlated with only HCFH. The rest were not statistical significance linear correlation. The multivariate regression results showed that the age of onset was positively correlated with AGM (r = 0.467). The regression equation used was Y = 9.0 * X (age of onset) - 11.4. CONCLUSION: We found difference in the morphological and mechanical characteristics of the GM between hips with DDH and hips of normal morphology. Of note, the mechanical characteristics of the GM were influenced by bony parameters in patients with DDH.
Asunto(s)
Luxación Congénita de la Cadera , Luxación de la Cadera , Acetábulo , Luxación Congénita de la Cadera/complicaciones , Luxación Congénita de la Cadera/diagnóstico por imagen , Articulación de la Cadera/diagnóstico por imagen , Humanos , Persona de Mediana Edad , Osteotomía/métodos , Estudios RetrospectivosRESUMEN
PURPOSE: Our research developed a novel approach to quantitatively evaluate the boundary of necrotic lesions in osteonecrosis of the femoral head (ONFH) and to explore its diagnostic value in predicting bone collapse of the femoral head. METHODS: A retrospective cross-sectional study was conducted in our institution, and 146 hips (121 cases) identified as ONFH were recruited. The anterior and lateral boundaries of each enrolled subject were measured in standard anteroposterior (AP) view and frog-leg (FL) view of plain radiographic images, the intact rate of which was then calculated and presented as the anteroposterior view intact ratio (APIR) and frog-leg view intact ratio (FLIR), respectively. Univariate and multivariate logistic regression analyses were performed to identify risk factors for collapse. A receiver operating characteristic (ROC) curve analysis was performed to determine the sensitivity, specificity and cutoff value of the APIR and FLIR. A Kaplan-Meier (K-M) analysis was applied to calculate the survival rate of the femoral head, and bone collapse of the femoral head was regarded as the endpoint. RESULTS: Femoral head collapse was observed in 61 hips during the follow-up period. Patients with or without femoral head collapse were categorized into the collapse group and non-collapse group, respectively. The mean follow-up time was 3.7 years (2-9) for the collapse group and 7.7 years (5-20) for the non-collapse group. Univariate and multivariate logistic regression analysis and ROC analysis showed that APIR (< 25.61%) and FLIR (< 24.43%) were significantly associated with femoral head collapse. The K-M survival curves indicated that the overall survival rate of APIR (≥ 25.61%) was 94.8% at 7.5 years and 76.6% at 10 years, while that of FLIR (≥ 24.43%) was 87.3% at 7.5 years and ten years. CONCLUSION: The present study demonstrates that APIR and FLIR are of high diagnostic value in the early and middle stages of ONFH. APIR and FLIR can be used to predict the occurrence of femoral head collapse in patients with JIC classification types B and C1. The measurement of these two parameters in plain radiography images may contribute to the selection of a proper hip preservation strategy.
Asunto(s)
Necrosis de la Cabeza Femoral , Cabeza Femoral , Estudios Transversales , Cabeza Femoral/diagnóstico por imagen , Cabeza Femoral/patología , Necrosis de la Cabeza Femoral/diagnóstico por imagen , Necrosis de la Cabeza Femoral/patología , Humanos , Radiografía , Estudios RetrospectivosRESUMEN
N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.
Asunto(s)
Polisacáridos/metabolismo , Alveolos Pulmonares/citología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes , Sitios de Unión , COVID-19/metabolismo , COVID-19/virología , Línea Celular , Células Epiteliales , Glicosilación , Interacciones Huésped-Patógeno/fisiología , Humanos , Mutación , Polisacáridos/química , Alveolos Pulmonares/virología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Acoplamiento ViralRESUMEN
In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.
Asunto(s)
Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Adenosina Trifosfato/metabolismo , Animales , Estabilidad de Enzimas , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Imidazoles/metabolismo , Luciferasas/sangre , Luciferasas/genética , Sustancias Luminiscentes/metabolismo , Ratones Endogámicos BALB C , Neoplasias/diagnóstico por imagen , Pirazinas/metabolismo , Distribución TisularRESUMEN
As an essential element for living organisms, zinc (Zn2+) exerts its biological functions both intracellularly and extracellularly. Previous studies have reported a number of genetically encoded Zn2+ indicators (GEZIs), which have been widely used to monitor Zn2+ in the cytosol and intracellular organelles. However, it is challenging to localize existing GEZIs to the extracellular space to detect secreted Zn2+. Herein, we report two photostable, green fluorescent protein (GFP) based indicators, ZIBG1 and ZIBG2, which respond to Zn2+ selectively and have affinities suited for detecting Zn2+ secretion from intracellular vesicles. In particular, ZIBG2 can be effectively targeted to the extracellular side of plasma membrane. We applied cell surface-localized ZIBG2 to monitor glucose-induced dynamic Zn2+ secretion from mouse insulinoma MIN6 cells and primary mouse and human pancreatic islets. Because Zn2+ is co-released with insulin from ß-cells, the fluorescence of cell surface-localized ZIBG2 was shown to be a strong indicator for the functional potency of islets. Our work here has thus expanded the use of GEZIs to image dynamic Zn2+ secretion in live tissue. Because it is convenient to use genetically encoded indicators for expression over extended periods and for in vivo delivery, we envision future applications of ZIBG2 in development of induced ß-cells or islets to advance cell replacement therapies for diabetes and in direct imaging of Zn2+ secretion dynamics in vivo.
Asunto(s)
Islotes Pancreáticos/metabolismo , Imagen Molecular/métodos , Proteínas Recombinantes/genética , Zinc/análisis , Animales , Calcio/análisis , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Color , Glucosa/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Insulinoma/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratones , Neoplasias Pancreáticas/metabolismo , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Zinc/metabolismoRESUMEN
Protein-protein interactions (PPIs) occur in a vast variety of cellular processes, and many processes are regulated by multiple protein interactions. Identification of PPIs is essential for the analysis of biological pathways and to further understand underlying molecular mechanisms. However, visualization and identification of multiprotein complexes, including ternary complexes in living cells under physiological conditions, remains challenging. In this work, we reported a three-fragment fluorescence complementation (TFFC) by splitting the Venus fluorescent protein for visualizing ternary complexes in living cells under physiological conditions. With this Venus-based TFFC system, we identified the multi-interaction of weak-affinity ternary complexes under physiological conditions. The TFFC system was further applied to the analysis of multi-interactions during the HIV-1 integration process, revealing the important role of the barrier-to-autointegration factor protein in HIV-1 integration. This TFFC system provides a useful tool for visualizing and identifying ternary complexes in living cells under physiological conditions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Luminiscentes/química , Microscopía Fluorescente/métodos , Complejos Multiproteicos/análisis , Fragmentos de Péptidos/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Bacterianas/genética , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fluorescencia , Células HEK293 , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Microscopía Confocal/métodos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/genética , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Vero , Integración Viral/fisiologíaRESUMEN
As a promising energy-storage device, rechargeable Zn-air batteries have attracted considerable interests. Herein, a bifunctional oxygen electrode film prepared by adhering NiCo2 O4 nanosheets to a nitrogen and oxygen dual-doped carbon nanotubes film in a large scale is reported. The resulting self-supporting film electrode is multifunctional, which integrates a porous conducting structure for air diffusion and charge transfer, high-performance catalysts for oxygen reduction and evolution, and novel structural flexibility. The composite film demonstrates excellent oxygen reduction/evolution reaction catalytic activities with low Tafel slopes (50 mV dec-1 for oxygen reduction reaction; 92 mV dec-1 for oxygen evolution reaction). Without any additional current collector, gas diffusion layer, or binder, the obtained bifunctional film performs as an "all-in-one" air electrode in a Zn-air battery. A 50-cm-long cable-shaped Zn-air battery based on such a film air electrode exhibits high operating potentials (≈1.2 V at 0.25 mA cm-2 ), low charging-discharging overpotentials (≈0.7 V), and stable cycling performance. Moreover, the flexible cable Zn-air batteries show excellent stability under different deformation conditions. The proposed concept of constructing scalable, all-in-one, freestanding, and flexible air electrodes would pave the way to develop next-generation wearable and portable energy-storage devices.
RESUMEN
Air electrodes with high catalytic activity are of great importance for rechargeable zinc-air batteries. Herein, a flexible, binder-free composite air electrode for zinc-air batteries is reported, which utilizes a lightweight, conductive, and crosslinked aerogel film of carbon nanotubes (CNTs) functioned as a 3D catalyst-supporting scaffold for bifunctional cobalt (II/III) oxides and as a current collector. The composite electrode shows high catalytic activities for both oxygen reduction reaction and oxygen evolution reaction, resulting from the synergistic effect of nitrogen-doped CNTs and spinel Co3 O4 nanoparticles. Solid-state Zn-air batteries assembled using such free-standing air electrodes (without the need of additional current collectors) are bendable and show low resistances, low charge/discharge overpotentials, and a high cyclic stability.
RESUMEN
Translationally controlled tumor protein (TCTP) is a highly conserved housekeeping protein present in eukaryotic organisms. It is involved in regulating many fundamental processes and plays a critical role in tumor reversion and tumorigenesis. Increasing evidence suggests that TCTP plays a role in the regulation of cell fate determination and is a promising therapeutic target for cancer. To decipher the exact mechanisms by which TCTP functions and how all these functions are integrated, we analyzed the interactome of TCTP in HeLa cells by coimmunoprecipitation (IP) and mass spectrometry (MS). A total of 98 proteins were identified. We confirmed the in vitro and in vivo association of TCTP with six of the identified binding proteins using reciprocal IP and bimolecular fluorescence complementation (BiFC) analysis, respectively. Moreover, TCTP interacted with Y-box-binding protein 1 (YBX1), and their interaction was localized to the N-terminal region of TCTP and the 1-129 amino acid (aa) residues of YBX1. The YBX1 protein plays an important role in cell proliferation, RNA splicing, DNA repair, drug resistance, and stress response to extracellular signals. These data suggest that the interaction of TCTP with YBX1 might cooperate or coordinate their functions in the control of diverse regulatory pathways in cancer cells. Taken together, our results not only reveal a large number of TCTP-associated proteins that possess pleiotropic functions, but also provide novel insights into the molecular mechanisms of TCTP in tumorigenesis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Carcinogénesis/química , Carcinogénesis/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas , Neoplasias/química , Neoplasias/patología , Unión Proteica , Proteína Tumoral Controlada Traslacionalmente 1 , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
To date, it has been a great challenge to design high-performance flexible energy storage devices for sufficient loading of redox species in the electrode assemblies, with well-maintained mechanical robustness and enhanced electron/ionic transport during charge/discharge cycles. An electrochemical activation strategy is demonstrated for the facile regeneration of carbon nanotube (CNT) film prepared via floating catalyst chemical vapor deposition strategy into a flexible, robust, and highly conductive hydrogel-like film, which is promising as electrode matrix for efficient loading of redox species and the fabrication of high-performance flexible pseudosupercapacitors. The strong and conductive CNT films can be effectively expanded and activated by electrochemical anodic oxygen evolution reaction, presenting greatly enhanced internal space and surface wettability with well-maintained strength, flexibility, and conductivity. The as-formed hydrogel-like film is quite favorable for electrochemical deposition of manganese dioxide (MnO2 ) with loading mass up to 93 wt% and electrode capacitance kept around 300 F g(-1) (areal capacitance of 1.2 F cm(-2) ). This hybrid film was further used to assemble a flexible symmetric pseudosupercapacitor without using any other current collectors and conductive additives. The assembled flexible supercapacitors exhibited good rate performance, with the areal capacitance of more than 300 mF cm(-2) , much superior to other reported MnO2 based flexible thin-film supercapacitors.
RESUMEN
The first critical step in making vertically aligned carbon nanotube (VACNT)-based thermal interface materials is to transfer the VACNTs on a large scale. Although VACNTs have been transferred by several methods, they were only transferred inadvertently in most cases. Here we report well-controlled weak-oxidation-assisted transfer of VACNTs. Specifically, after a short time of weak oxidation, we found that VACNTs could be easily detached from the native growth substrates, and thus, a freestanding VACNT film was obtained. Then the VACNTs could be assembled onto specific substrates for its real applications. More importantly, the repeated growth-transfer synthesis of VACNT arrays can be realized in one batch by introducing an additional process of weak oxidation in chemical vapor deposition, which makes the strategy more effective. Surprisingly, no degradation in the quality was observed before and after the weak oxidation according to thermogravimetric analysis and Raman spectra of VACNTs. Enhanced thermal and mechanical properties were achieved after reactive ion etching (RIE) and subsequent metallization of the surfaces of the VACNTs, and this might be due to the removal of impurities such as amorphous carbon and entangled CNTs by RIE. These findings provide an efficient approach for transferring VACNTs, which is important for the application of VACNTs in thermal management.
RESUMEN
Bimolecular fluorescence complementation (BiFC) and its derivative molecular biosensor systems provide effective tools for visualizing biomolecular interactions. The introduction of red and near-infrared fluorescence emission proteins has expanded the spectrum of signal generating modules, enabling BiFC for in vivo imaging. However, the large size of the signal module of BiFC can hinder the interaction between proteins under investigation. In this study, we constructed the near-infrared BiFC and TriFC systems by splitting miRFP670nano, the smallest cyanobacteriochrome-evolved phytochrome available. The miRFP670nano-BiFC sensor system identified and enabled visualization of protein-protein interactions in living cells and live mice, and afforded a faster maturation rate and higher photostability and cellular stability when compared with those of reported near-infrared BiFC systems. We used the miRFP670nano-BiFC sensor system to identify interactions between the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular stress granule proteins in living cells and found that the N protein downregulated the expression level of granule protein G3BP1. With the advantages of small size and long wavelength emission of the signal module, the proposed molecular biosensor system should be suitable for various applications in cell imaging studies.
RESUMEN
SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Polisacáridos , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
The ongoing coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health and social stability. There is an urgent need for understanding the nature and infection mechanism of the virus. Owing to its high infectivity and pathogenicity and lack of effective treatments, live SARS-CoV-2 has to be handled in biosafety level 3 laboratories, which has impeded research into SARS-CoV-2 and the development of vaccines and therapeutics. Pseudotyped viruses that lack certain gene sequences of the virulent virus are safer and can be investigated in biosafety level 2 laboratories, providing a useful virological tool for the study of SARS-CoV-2. In this review, we will discuss the construction of SARS-CoV-2 pseudoviruses based on different packaging systems, current applications, limitations, and further explorations.
Asunto(s)
COVID-19/virología , SARS-CoV-2/fisiología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Humanos , SARS-CoV-2/inmunología , Empaquetamiento del Genoma ViralRESUMEN
Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Ensamble de Virus , Células HEK293 , Humanos , Fosfoproteínas/metabolismo , Mapas de Interacción de ProteínasRESUMEN
Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein-protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems-iRFP-BiFC and IFP1.4-BiFC-the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.
Asunto(s)
Diagnóstico por Imagen , Animales , Fluorescencia , Proteínas Luminiscentes , RatonesRESUMEN
The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.
Asunto(s)
Colorantes Fluorescentes/química , Alveolos Pulmonares/virología , SARS-CoV-2/fisiología , Internalización del Virus , Enzima Convertidora de Angiotensina 2/metabolismo , Endocitosis , Células Epiteliales/virología , Fluorescencia , Células HEK293 , VIH-1/genética , Humanos , Mucosa Nasal/virología , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Imaging RNA-protein interaction in the cellular space with single molecule sensitivity is attractive for studying gene expression and regulation, but remains a challenge. In this study, we reported a photoactivatable trimolecular fluorescence complementation (TriFC) system based on fluorescent protein, mIrisFP, to identify and visualize RNA-protein interactions in living mammalian cells. We also combined this TriFC system with photoactivated localization microscopy (PALM), named the TriFC-PALM technique, which allowed us to image the RNA-protein interactions with single molecule sensitivity. Using this TriFC-PALM technique, we identified the actin-bundling protein, FSCN1, specifically interacting with the HOX Transcript Antisense RNA (HOTAIR). The TriFC-PALM imaging acquired a higher resolution compared with the traditional method of total internal reflection (TIRF) imaging. The TriFC-PALM thus provides a useful tool for imaging and identifying the RNA-protein interactions inside cells at the nanometer scale.