Rapid and quantitative evaluation of vaccinia virus‐induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase

Abstract

Host shutoff, characterized by a global decline of cellular protein synthesis, is commonly observed in many viral infections, including vaccinia virus (VACV). Classic methods measuring host shutoff include the use of radioactive or nonradioactive probes to label newly synthesized proteins followed by radioautography or sodium dodecyl-sulfate polyacrylamide gel electrophoresis to resolve the proteins for follow-up detection. Although these are highly reliable methods, they are time-and labor-consuming. Here, we generated two cell lines stably expressing secreted Gaussia luciferase. These reporter cells allow rapid, quantitative, and consecutive monitoring of host shutoff from a single infection sample. We evaluated host shutoff induced by wild-type and various mutant VACVs using the reporter cell lines. The results validated the utilities of the reporter cells and quantitatively characterized VACV-induced host shutoff at different stages of replication. Notably, the results also indicated additional major unidentified VACV shutoff factors. Our study provides new tools to study host shutoff. The reporter cells are also suitable for high throughput settings and rapid testing of clinically isolated viruses. In combination with classical methods, these tools will greatly facilitate understanding of virusinduced host shutoff, and protein synthesis shutoff caused by other physiologically relevant stresses.

Spatio-temporal analysis of Vaccinia virus infection and host response dynamics using single-cell transcriptomics and proteomics

“…In total, we quantified 8,382 proteins (8,203 human and 179 viral; Supplementary Data 2), several of which are modulated in VV infected cells. In accordance with previous reports, numerous host proteins were downregulated 37 as a consequence of pre- and post-translational regulation, including IFN-related proteins such as the IFITs 38 and IFNGR1, inhibitors of host translation such as EIF4EBP2 and EIF2AK1, and cell cycle checkpoint proteins such as p21 ( CDKN1A gene) and p57 ( CDKN1C gene) whose elimination leads to S and G2 phase accumulation (Fig. 4A) 39 .…”