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1.
Sci Rep ; 11(1): 780, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436939

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.


Assuntos
/métodos , Carga Viral , /diagnóstico , Humanos , Mucosa Nasal/virologia , RNA Viral/química , RNA Viral/genética , RNA Viral/normas , /isolamento & purificação , Sensibilidade e Especificidade
2.
Biochem Biophys Res Commun ; 541: 50-55, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477032

RESUMO

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.


Assuntos
Trifosfato de Adenosina/metabolismo , Extração Líquido-Líquido , RNA Viral/metabolismo , /metabolismo , Trifosfato de Adenosina/química , Sítios de Ligação , /genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Viral/química , RNA Viral/genética , Motivos de Ligação ao RNA/genética , /química
3.
Virology ; 554: 75-82, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33387787

RESUMO

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Antivirais/química , Antivirais/uso terapêutico , Coronavirus/crescimento & desenvolvimento , Coronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Regulação Viral da Expressão Gênica , Humanos , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , /genética , /fisiologia , Replicação Viral
5.
PLoS One ; 16(1): e0245280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33449976

RESUMO

rfaRm is an R package providing a client-side interface for the Rfam database of non-coding RNA and other structured RNA elements. The package facilitates the search of the Rfam database by keywords or sequences, as well as the retrieval of all available information about specific Rfam families, such as member sequences, multiple sequence alignments, secondary structures and covariance models. By providing such programmatic access to the Rfam database, rfaRm enables genomic workflows to incorporate information about non-coding RNA, whose potential cannot be fully exploited just through interactive access to the database. The features of rfaRm are demonstrated by using it to analyze the SARS-CoV-2 genome as an example case.


Assuntos
RNA não Traduzido/genética , Análise de Sequência de RNA/métodos , Software , Bases de Dados Genéticas , Humanos , RNA não Traduzido/química , RNA Viral/química , RNA Viral/genética , /genética
6.
Talanta ; 224: 121850, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379066

RESUMO

In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.


Assuntos
/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /instrumentação , DNA Viral/análise , Reações Falso-Negativas , Humanos , Estabilidade de RNA , RNA Viral/química , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , /genética
7.
Clin Chim Acta ; 514: 54-58, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33316217

RESUMO

BACKGROUND AND AIMS: Immediate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for preventing the spread of coronavirus disease 2019 (COVID-19). The LabTurbo AIO 48 system is an automated platform that allows nucleic acid extraction and sample analysis on the same instrument, producing faster results without affecting their accuracy. We aimed to independently evaluate the LabTurbo AIO 48 (all-in-one system) for SARS-CoV-2 detection. MATERIALS AND METHODS: Comparative limit of detection (LOD) was assessed on both the LabTurbo AIO 48 and current standard detection system based on real-time reverse transcriptase polymerase chain reaction (RT-PCR), using SARS-CoV-2 RNA control. Additional 125 primary clinical samples were assessed using both the protocols in parallel. RESULTS: The turnaround time from sample to results for 48 samples analyzed on LabTurbo AIO 48 was approximately 2.5 h, whereas that analyzed using the in-house RT-PCR protocol was 4.8 h. LabTurbo AIO 48 also demonstrated higher sensitivity than our reference RT-PCR assay, with a LOD of 9.4 copies/reaction. The overall percentage agreement between both the methods for 125 samples was 100%. CONCLUSION: LabTurbo AIO 48 is a robust detection option for SARS-CoV-2, allowing faster results and, consequently, aiding in better control and prevention of COVID-19.


Assuntos
/métodos , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , /diagnóstico , Humanos , Limite de Detecção , RNA Viral/química , Padrões de Referência , Sensibilidade e Especificidade , Carga Viral
8.
Sci Rep ; 10(1): 19004, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149153

RESUMO

Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT-PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~ 10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA extraction to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~ 50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.


Assuntos
Técnicas de Laboratório Clínico/métodos , Custos e Análise de Custo , Nanopartículas de Magnetita/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Coronavirus/diagnóstico , Países em Desenvolvimento , Humanos , Nanopartículas de Magnetita/economia , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia
9.
Nat Commun ; 11(1): 5885, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208793

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic ß-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Vesículas Citoplasmáticas/virologia , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/metabolismo , Células Vero , Vírion/química , Vírion/metabolismo , Montagem de Vírus
10.
Nat Commun ; 11(1): 5531, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139729

RESUMO

Biomolecules form dynamic ensembles of many inter-converting conformations which are key for understanding how they fold and function. However, determining ensembles is challenging because the information required to specify atomic structures for thousands of conformations far exceeds that of experimental measurements. We addressed this data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformation library. Refinement of this library with NMR residual dipolar couplings provided an atomistic ensemble model for HIV-1 TAR, and the model accuracy was independently supported by comparisons to quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through designed ensemble redistribution via atomic mutagenesis. Applications to TAR bulge variants and more complex tertiary RNAs support the generality of this approach and the potential to make the determination of atomic-resolution RNA ensembles routine.


Assuntos
Quimioinformática/métodos , HIV-1/química , Dobramento de RNA , RNA Viral/ultraestrutura , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/ultraestrutura , Modelos Químicos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , RNA Viral/química , RNA Viral/genética
11.
J Phys Chem Lett ; 11(21): 9408-9414, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33104327

RESUMO

Chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as COVID-19, correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. We propose novel descriptors based on a COSMO-RS (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ML). The descriptors' performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. The COSMO-RS-based descriptors account for both chemical reactivity and structure, and are more effective for ML-based screening than fingerprints based on molecular structure and simple physical/chemical properties. The descriptors are evaluated using principal component analysis, an unsupervised ML technique. Our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug EIDD-2801 with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. The COSMO-RS-based descriptors could help accelerate drug discovery for the treatment of emerging diseases.


Assuntos
Aprendizado de Máquina , Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Nucleotídeos/metabolismo , Nucleotídeos/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Análise de Componente Principal , Teoria Quântica , RNA Viral/química , RNA Viral/metabolismo , /genética , /metabolismo
12.
mBio ; 11(6)2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127861

RESUMO

The ultimate outcome of the coronavirus disease 2019 (COVID-19) pandemic is unknown and is dependent on a complex interplay of its pathogenicity, transmissibility, and population immunity. In the current study, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was investigated for the presence of large-scale internal RNA base pairing in its genome. This property, termed genome-scale ordered RNA structure (GORS) has been previously associated with host persistence in other positive-strand RNA viruses, potentially through its shielding effect on viral RNA recognition in the cell. Genomes of SARS-CoV-2 were remarkably structured, with minimum folding energy differences (MFEDs) of 15%, substantially greater than previously examined viruses such as hepatitis C virus (HCV) (MFED of 7 to 9%). High MFED values were shared with all coronavirus genomes analyzed and created by several hundred consecutive energetically favored stem-loops throughout the genome. In contrast to replication-associated RNA structure, GORS was poorly conserved in the positions and identities of base pairing with other sarbecoviruses-even similarly positioned stem-loops in SARS-CoV-2 and SARS-CoV rarely shared homologous pairings, indicative of more rapid evolutionary change in RNA structure than in the underlying coding sequences. Sites predicted to be base paired in SARS-CoV-2 showed less sequence diversity than unpaired sites, suggesting that disruption of RNA structure by mutation imposes a fitness cost on the virus that is potentially restrictive to its longer evolution. Although functionally uncharacterized, GORS in SARS-CoV-2 and other coronaviruses represents important elements in their cellular interactions that may contribute to their persistence and transmissibility.IMPORTANCE The detection and characterization of large-scale RNA secondary structure in the genome of SARS-CoV-2 indicate an extraordinary and unsuspected degree of genome structural organization; this could be effectively visualized through a newly developed contour plotting method that displays positions, structural features, and conservation of RNA secondary structure between related viruses. Such RNA structure imposes a substantial evolutionary cost; paired sites showed greater restriction in diversity and represent a substantial additional constraint in reconstructing its molecular epidemiology. Its biological relevance arises from previously documented associations between possession of structured genomes and persistence, as documented for HCV and several other RNA viruses infecting humans and mammals. Shared properties potentially conferred by large-scale structure in SARS-CoV-2 include increasing evidence for prolonged infections and induced immune dysfunction that prevents development of protective immunity. The findings provide an additional element to cellular interactions that potentially influences the natural history of SARS-CoV-2, its pathogenicity, and its transmission.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Coronavirus/genética , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , Animais , Sequência de Bases , Evolução Molecular , Genoma Viral , Humanos , Conformação de Ácido Nucleico , Pandemias , Alinhamento de Sequência
13.
Nat Commun ; 11(1): 5496, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127896

RESUMO

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.


Assuntos
Fenômenos Bioquímicos , Exorribonucleases/genética , Exorribonucleases/metabolismo , RNA Viral/química , Zika virus/genética , Anisotropia , Humanos , Magnésio/metabolismo , Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Viral/genética , Infecção por Zika virus/virologia
14.
BMC Bioinformatics ; 21(1): 431, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33008363

RESUMO

BACKGROUND: This paper describes a web based tool that uses a combination of sonification and an animated display to inquire into the SARS-CoV-2 genome. The audio data is generated in real time from a variety of RNA motifs that are known to be important in the functioning of RNA. Additionally, metadata relating to RNA translation and transcription has been used to shape the auditory and visual displays. Together these tools provide a unique approach to further understand the metabolism of the viral RNA genome. This audio provides a further means to represent the function of the RNA in addition to traditional written and visual approaches. RESULTS: Sonification of the SARS-CoV-2 genomic RNA sequence results in a complex auditory stream composed of up to 12 individual audio tracks. Each auditory motive is derived from the actual RNA sequence or from metadata. This approach has been used to represent transcription or translation of the viral RNA genome. The display highlights the real-time interaction of functional RNA elements. The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. Functional RNA motifs such as transcription regulatory sequences and stem loop regions have also been sonified. Using the tool, audio can be generated in real-time from either genomic or sub-genomic representations of the RNA. Given the large size of the viral genome, a collection of interactive buttons has been provided to navigate to regions of interest, such as cleavage regions in the polyprotein, untranslated regions or each gene. These tools are available through an internet browser and the user can interact with the data display in real time. CONCLUSION: The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Furthermore, the tool has been used as an algorithmic based audio generator. These audio tracks can be listened to by the general community without reference to the visual display to encourage further inquiry into the science.


Assuntos
Betacoronavirus/genética , Genoma Viral , Software , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Genômica , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo
15.
Sci Rep ; 10(1): 16577, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024223

RESUMO

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Exonucleases/metabolismo , Pneumonia Viral/tratamento farmacológico , Pró-Fármacos/farmacologia , RNA Viral/efeitos dos fármacos , Sofosbuvir/farmacologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , Pró-Fármacos/uso terapêutico , RNA Viral/química , RNA Viral/metabolismo , /metabolismo , Sofosbuvir/química , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 117(37): 22823-22832, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868439

RESUMO

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.


Assuntos
Vírus da Dengue/genética , Ouro/química , Nanopartículas Metálicas/química , RNA Viral/química , RNA Viral/genética , Vírus da Dengue/química , Espalhamento a Baixo Ângulo , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Poliproteínas , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
18.
Nat Commun ; 11(1): 4775, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963221

RESUMO

Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.


Assuntos
Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Sítios Internos de Entrada Ribossomal/fisiologia , Replicação Viral/fisiologia , Regiões 5' não Traduzidas , Linhagem Celular , Infecções por Enterovirus/virologia , Regulação Viral da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , RNA Viral/química , Proteínas Virais/metabolismo
19.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
20.
PLoS Pathog ; 16(9): e1008825, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886709

RESUMO

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.


Assuntos
Alphavirus , Genoma Viral , Conformação de Ácido Nucleico , RNA Viral , Proteínas Virais , Alphavirus/química , Alphavirus/genética , Alphavirus/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , /genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
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