Building Blocks of Understanding: Constructing a Reverse Genetics Platform for studying determinants of SARS-CoV-2 replication

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Фев. 6, 2024

Abstract To better understand viral pathogenesis, host-virus interactions, and potential therapeutic interventions, the development of robust reverse genetics systems for SARS-CoV-2 is crucial. Here, we present a platform that enables efficient manipulation, assembly, rescue recombinant SARS-CoV-2. The versatility our system was demonstrated by generating viruses. We used this to generate N501Y Y453F spike protein mutants. Characterization studies revealed distinct phenotypic effects, impact on fitness, cell binding, replication kinetics. also investigated recently discovered priming site NSP9, which postulated produce short RNA antisense leader sequence. By introducing U76G mutation into 5’UTR, show necessary correct production genomic subgenomic RNAs, replication. In conclusion, developed provides adaptable generation viruses their comprehensive characterization. Significance statement study, versatile facilitating Demonstrating its adaptability, successfully engineered mutants, each exhibiting effects novel negative sense demonstrating role in This straightforward genetic therefore powerful tool advancing understanding biology.

Язык: Английский

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Structural review of SARS-CoV-2 antiviral targets

Structure, Год журнала: 2024, Номер 32(9), С. 1301 - 1321

Опубликована: Сен. 1, 2024

Язык: Английский

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Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine

Journal of Biological Chemistry, Год журнала: 2025, Номер unknown, С. 108378 - 108378

Опубликована: Март 1, 2025

Язык: Английский

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Inhibition of SARS-CoV-2 Nsp9 ssDNA-Binding Activity and Cytotoxic Effects on H838, H1975, and A549 Human Non-Small Cell Lung Cancer Cells: Exploring the Potential of Nepenthes miranda Leaf Extract for Pulmonary Disease Treatment

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(11), С. 6120 - 6120

Опубликована: Июнь 1, 2024

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores therapeutic potential of miranda leaf extract against nonstructural protein 9 (Nsp9) SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) lines. Nsp9, essential RNA replication, was expressed purified, its interaction with ssDNA assessed. Initial tests myricetin oridonin, known targeting ssDNA-binding proteins respectively, did not inhibit activity Nsp9. Subsequent screenings various N. extracts identified those using acetone, methanol, ethanol as particularly effective disrupting Nsp9's activity, evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol lupenone, major components miranda, inhibitors. The cytotoxic properties were examined across NSCLC lines H1975, A549, H838, focusing on survival, apoptosis, migration. Results showed a dose-dependent effect following order: H1975 > A549 H838 cells, indicating specificity. Enhanced anticancer effects observed when combined afatinib, suggesting synergistic interactions. Flow cytometry indicated that could induce G2 cycle arrest potentially inhibiting cancer proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled tentative identification 19 most abundant compounds miranda. These outcomes underscore dual utility managing infection through Nsp9 inhibition offering benefits carcinoma. results significantly broaden medical applications extract, use only traditional remedies but also prospective treatment pulmonary diseases. Overall, our findings position promising source natural therapeutics antiviral therapies, warranting further investigation into molecular mechanisms clinical applications.

Язык: Английский

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A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS‐CoV‐2—Implications for Future Drug Discovery Efforts

Proteins Structure Function and Bioinformatics, Год журнала: 2024, Номер 92(11), С. 1308 - 1317

Опубликована: Июль 3, 2024

ABSTRACT The ongoing global pandemic of the coronavirus 2019 (COVID‐19) disease is caused by virus SARS‐CoV‐2, with very few highly effective antiviral treatments currently available. machinery responsible for replication and transcription viral RNA during infection made up several important proteins. Two these are nsp12, catalytic subunit polymerase, nsp9, a cofactor nsp12 involved in capping priming RNA. While recent studies have determined structural details interaction nsp9 context capping, biochemical or biophysical In this study, we used combination surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) assays to identify specific residues that critical binding as well RNAylation, both which essential process. Our data indicate dimerization unlikely play significant functional role virus. We confirm set recently discovered peptides inhibit nsp9–nsp12 specifically nsp9; however, find do not impact RNAylation. summary, our results implications future drug discovery efforts combat SARS‐CoV‐2 any newly emerging coronaviruses.

Язык: Английский

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Nanobody against SARS-CoV-2 non-structural protein Nsp9 inhibits viral replication in human airway epithelia

Molecular Therapy — Nucleic Acids, Год журнала: 2024, Номер 35(3), С. 102304 - 102304

Опубликована: Авг. 15, 2024

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve inhibitors of severe acute respiratory syndrome coronavirus s (SARS-CoV-2) entry in the host cell by targeting surface-exposed spike protein. Here we established a pipeline that instead targets highly conserved viral proteins made only after into when SARS-CoV-2 RNA-based genome is translated. As proof principle, designed nanobodies against non-structural protein (Nsp)9, which required replication. One these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and nuclear magnetic resonance spectroscopy epitope mapping, was expressed found block replication specifically. We next encapsulated 2NSP23 nanobody lipid nanoparticles (LNPs) mRNA. show this nanobody, hereby referred LNP-mRNA-2NSP23, internalized translated cells suppresses multiple variants, seen qPCR RNA deep sequencing. These results corroborated three-dimensional reconstituted human epithelium kept at air-liquid interface mimic outer surface lung tissue. observations indicate LNP-mRNA-2NSP23 and, translation, it inhibits Nsp9 living cells. speculate may be an innovative strategy generate novel antiviral drugs efficient across coronaviruses.

Язык: Английский

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A domain of all trades: The enzymatic versatility of the NiRAN domain

Molecular Cell, Год журнала: 2023, Номер 83(21), С. 3758 - 3760

Опубликована: Ноя. 1, 2023

Язык: Английский

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Characterization of the Conformational Hotspots of the RNA-Dependent RNA Polymerase Complex Identifies a Unique Structural Malleability of nsp8

The Journal of Physical Chemistry B, Год журнала: 2024, Номер unknown

Опубликована: Окт. 2, 2024

Several antiviral therapeutic approaches have been targeted toward the RNA-dependent RNA polymerase (RdRp) complex that is involved in viral genome replication. In SARS-CoV-2, although RdRp a multiprotein complex, focus has on ligand binding catalytic core (nonstructural protein nsp12), and not functional dynamics. this study, we conformational ensembles of their modulation by presence RNA, performing comprehensive microsecond-scale atomistic simulations apo- RNA-bound complex. We delineate differential impact constituent proteins, such as polymorphisms, dominant segment-specific fluctuations, switch dynamical crosstalk within distinguish signatures nsp7, nsp8, nsp12 apo-state are reduced appear to "prime" for activity. Importantly, identify unique structural malleability nsp8 with high heterogeneity apo state, especially at three sites (Y71 nsp8A, D52 A66 nsp8B). Our work highlights implications polymorphism structures reveals possibilities development allosteric inhibitors.

Язык: Английский

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Meeting Report of the 37th International Conference on Antiviral Research in Gold Coast, Australia, May 20–24, 2024, organized by the International Society for Antiviral Research

Antiviral Research, Год журнала: 2024, Номер 232, С. 106037 - 106037

Опубликована: Ноя. 13, 2024

Язык: Английский

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