ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. Upon purification of the recombinant SARS-CoV-2 Nsp13 protein expressed using a eukaryotic cell-based system, we biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA during loading to unwind with greater efficiency a partial duplex helicase substrate. The binding affinity of Nsp13 to nucleic acid was confirmed through electrophoretic mobility shift assays (EMSA) by determining that Nsp13 binds to DNA substrates with significantly greater efficiency than RNA. These results demonstrate strand-specific interactions of SARS-CoV-2 Nsp13 that dictate its ability to load and unwind structured nucleic acid substrates. We next determined that Nsp13 catalyzed unwinding of double-stranded (ds) RNA forked duplexes on substrates containing a backbone disruption (neutrally charged polyglycol linker (PGL)) was strongly inhibited when the PGL was positioned in the 5' ssRNA overhang, suggesting an unwinding mechanism in which Nsp13 is strictly sensitive to perturbation of the translocating strand sugar-phosphate backbone integrity. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 helicase to disrupt a high-affinity nucleic acid-protein interaction, i.e., a streptavidin tetramer bound to biotinylated RNA or DNA substrate, in a uni-directional manner and with a preferential displacement of the streptavidin complex from biotinylated ssDNA versus ssRNA. In contrast to the poorly hydrolysable ATP-gamma-S or non-hydrolysable AMP-PNP, ATP supports Nsp13-catalyzed disruption of the nucleic acidprotein complex, suggesting that nucleotide binding by Nsp13 is not sufficient for protein-RNA disruption and the chemical energy of nucleoside triphosphate hydrolysis is required to fuel remodeling of protein bound to RNA or DNA. Our results build upon structural studies of the SARS-CoV-2 RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. Experimental evidence from our studies demonstrate that Nsp13 helicase efficiently remodels a large high affinity protein-RNA complex in a manner dependent on its intrinsic ATP hydrolysis function. We proposed that this novel biochemical activity of Nsp13 is relevant to its role in SARS-CoV-2 RNA processing functions and replication. It was proposed that Nsp13 facilitates proofreading during coronavirus replication when a mismatched base is inadvertently incorporated into the SARS-CoV-2 genome during replication to reposition the RTC so that the proofreading nuclease complex (Nsp14-Nsp10) can gain access and remove the nascently synthesized nucleotide to ensure polymerase fidelity. Our findings implicate a direct catalytic role of Nsp13 in protein-RNA remodeling during coronavirus genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into the proofreading mechanism. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.
ABSTRACT
Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. Nonetheless, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied "piggyback" transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to actualize the promise of mRNA-based therapeutics.
Subject(s)
Adenoviridae Infections , COVID-19 , Humans , Adenoviridae/genetics , Genetic Vectors/genetics , Gene Transfer Techniques , Polylysine , RNA, Messenger/genetics , RNA, Messenger/metabolism , Pandemics , Capsid Proteins/genetics , Capsid Proteins/metabolism , BiologyABSTRACT
Background-aim: With recent emergences in new infectious diseases and their variants, there is a need to develop a faster and more specific analytical tool to detect different respiratory infectious diseases such as SARS-CoV-2 or influenza viruses. Not only their symptoms are similar at early stages, but also, they are both enveloped viruses with several common biological properties, often leading to challenges in disease identification. Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved, less post-translational modifications possessed, and mostly specific for each infectious disease virus types. Therefore, targeting NP could be more advantageous to the method development, achieving much simpler and robust method with minimal subsequent modifications. This study describes a targeted approach for simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple viruses, SARS-CoV-2, influenza virus A and B types, respiratory syncytial virus, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. Methods: Sample collected via nasopharyngeal swabs in viral transport media was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin). The IP purified samples were then digested using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data processing was performed using TraceFinder™ LDT software 1.0. Results: Combining IP and LC-MS/MS resulted in a highly targeted approach with the high sensitivity and specificity. The method detected sub tens to hundreds amol of peptides on LC column. Also, it simplified the overall sample preparation process eliminating prior protein precipitation and post sample clean-up. Since the NPs mostly remain unchanged or less modified regardless of variants, the method doesn’t need tremendous alterations once established. Conclusions: This targeted approach can be applied to other enveloped viruses’ detection. Automated IP method is available with KingFisher system so it could lead to a faster turn-around time and higher throughput of the method.
ABSTRACT
Introduction: Biotin is a vitamin-B supplement and is sold over-the-counter alone or in combination with other vitamins and minerals to improve skin, nail, and hair health. Exogenous biotin intake is becoming a common cause of apparent abnormal thyroid function tests in endocrine practice. We present a case report. Case Description: A 55-year-old lady was referred to endocrinology clinic for low TSH but normal fT4. The only symptom she had was abnormal smell of ‘smoke’. She had no ongoing symptoms of thyrotoxicosis or hypothyroidism apart from hair thinning. She had COVID-19 infection with pyrexia and constitutional symptoms a year ago. There was no other past medical or family history of note. On direct questioning, she admitted taking biotin 300ug every day in preceding six months for hair loss. Examination revealed euthyroidism and no goitre. TFTs prior to commencing biotin were normal. Further TFTs confirmed low TSH but normal fT4 and fT3 and thyroid receptor antibody was negative. TFTs off biotin for three days were normal and she was reassured. Discussion: There is no in vivo relationship between biotin and thyroid hormones, but high dose biotin affects thyroid function test in vitro when assessed with immunoassays using streptavidin-biotin immobilizing system. Apparently low TSH with or without raised T4 suggests thyrotoxicosis or thyroxine over-replacement. All patients presenting with such abnormal thyroid function tests should be questioned about biotin intake along with iodine as many multivitamins contain high dose biotin and patients are often unaware. This can avoid unnecessary investigations, patient anxiety, and healthcare costs. American Thyroid Association advises stopping biotin two days before thyroid function test. Patients can continue to take biotin except temporarily before thyroid blood tests.
ABSTRACT
Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2.
ABSTRACT
The proceedings contain 38 papers. The topics discussed include: longitudinal assessment of SARS-CoV-2 nucleocapsid antigenemia in patients hospitalized with COVID-19;machine learning-based automated selection of regions for analysis on bone marrow aspirate smears;direct detection of beta-lactamase mediated antibiotic resistance in clinical specimens;convalescent plasma does not provide adequate replacement of C1-Inhibitor and complements in COVID-19 patients;mapping cell-to-cell mitochondria transfer in obesity using high-dimensional spectral flow cytometry;capture the tag: mitigation of biotin interference in ELISA and automated immunoassays by pre-conjugating biotinylated antibodies to the streptavidin surface;and assessment of coagulation tests in hospitalized COVID19 patients;challenging coagulopathies.
ABSTRACT
As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin-biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.
ABSTRACT
Since the COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO) in March 2020, ongoing efforts have been made to develop sensitive diagnostic platforms. Detection of viral RNA provides the highest sensitivity and specificity for detection of early and asymptomatic infections. Thus, this work aimed at developing a label-free genosensor composed of graphene as a working electrode that could be embedded into a flex printed circuit board (FPCB) for the rapid, sensitive, amplification-free and label-free detection of SARS-CoV-2. To facilitate liquid handling and ease of use, the developed biosensor was embedded with a user-friendly reservoir chamber. As a proof-of-concept, detection of a synthetic DNA strand matching the sequence of ORF1ab was performed as a two-step strategy involving the immobilization of a biotinylated complementary sequence on a streptavidin-modified surface, followed by hybridization with the target sequence recorded by the differential pulse voltammetric (DPV) technique in the presence of a ferro/ferricyanide redox couple. The effective design of the sensing platform improved its selectivity and sensitivity and allowed DNA quantification ranging from 100 fg/mL to [Formula: see text]/mL. Combining the electrochemical technique with FPCB enabled rapid detection of the target sequence using a small volume of the sample (5-[Formula: see text]). We achieved a limit-of-detection of 100 fg/mL, whereas the predicted value was ~33 fg/mL, equivalent to approximately [Formula: see text] copies/mL and comparable to sensitivities provided by isothermal nucleic acid amplification tests. We believe that the developed approach proves the ability of an FPCB-implemented DNA sensor to act as a potentially simpler and more affordable diagnostic assay for viral infections in Point-Of-Care (POC) applications.