(E) Quantification of the luciferase gene expression from the pScalps Luciferase Zsgreen reporter plasmid with and without the presence of N protein. (D) Representative flow cytometric analysis of the effect of N protein on Zsgreen expression, using populations transfected with either the reporter plasmid alone (red fraction) or along with the N protein (cyan fraction). (C) Microscopy images for the infected cells with indicated conditions (scale 100 μm). (B) p24 levels from the virus-producing cells for all the SARS CoV-2 genes were detected by Western blotting. Background signal levels are indicated by the non-enveloped (Non. Vector normalized values for both readout methods has been shown, such as to indicate the percentage change relative to the baseline (indicated by the dotted line). Results from the screen of SARS CoV-2 genes, either by quantifying luciferase activity (left panel) or GFP positive cell count (right panel). SARS CoV-2’s N protein enhances retroviral spike-pseudotyped particle infectivity. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.ĪCE2-Fc SARS-CoV-2 nucleocapsid spike lentiviral pseudotyping virus neutralization.Ĭopyright © 2021 Mishra, Sreepadmanabh, Ramdas, Sahu, Kumar and Chande. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities.
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