Biology subjects

Boura, E.

Publications and source records attributed to Boura, E..

22 records · Page 2Linked to original sources

bioRxiv2020

Substrate specificity of SARS-CoV-2 nsp10-nsp16 methyltransferase

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2) encodes all viral enzymes. In this work, we focus on one particular methyltransferase (MTase), nsp16, which in complex with nsp10 is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered five types of capped RNA (m7Gp3A(G)-, Gp3A(G)- and Gp4A-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin (286 {+/-} 66 nM) to illustrate the value of our approach for inhibitor screening. In the future, this approach can be used for screening inhibitors of any type of 2'-O-MTase.

molecular biology↗

bioRxiv2020

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation

COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.

molecular biology↗

bioRxiv2020

Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus disease 2019 (COVID-19) which is currently negatively affecting the population and disrupting the global economy. SARS-CoV-2 belongs to the +RNA virus family that utilize single-stranded positive-sense RNA molecules as genomes. SARS-CoV-2, like other coronaviruses, has an unusually large genome for a +RNA virus that encodes four structural proteins - the matrix (M), small envelope (E), spike (S) and nucleocapsid phosphoprotein (N) - and sixteen nonstructural proteins (nsp1-16) that together ensure replication of the virus in the host cell. The nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Its N-terminal RNA binding domain (N-NTD) captures the RNA genome while the C-terminal domain anchors the ribonucleoprotein complex to the viral membrane via its interaction with the M protein. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD lined with arginine residues suggesting a putative RNA binding site. Next, we performed an NMR titration experiment using an RNA duplex. The observed changes in positions of signals in the N-NTD NMR spectra allowed us to construct a model of the N-NTD in complex with RNA.

biochemistry↗

bioRxiv2019

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

Phosphatidylinositol (PtdIns) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of how the dynamic regulation of PtdIns synthesis and transport contributes to the turnover of PPIn pools. To address these shortcomings, we capitalized on the substrate selectivity of a bacterial enzyme, PtdIns-specific PLC, to establish a molecular toolbox for investigations of PtdIns distribution and availability within intact cells. In addition to its presence within the ER, our results reveal low steady-state levels of PtdIns within the plasma membrane (PM) and endosomes as well as a relative enrichment of PtdIns within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membranes. Kinetic studies also demonstrate the requirement for sustained PtdIns supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.\n\nSummaryPemberton et al. characterize a molecular toolbox for the visualization and manipulation of phosphatidylinositol (PtdIns) within intact cells. Results using these approaches define the steady-state distribution of PtdIns across subcellular membrane compartments as well as provide new insights into the relationship between PtdIns availability and polyphosphoinositide turnover.

cell biology↗