期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 14, 页码 -出版社
MDPI
DOI: 10.3390/ijms23147826
关键词
COVID-19; SARS-CoV-2; coronavirus; protease PLpro; live cell imaging; genetically encoded probes; FRET; translocation
资金
- Russian Foundation for Basic Research [20-04-60370]
The researchers designed genetically encoded fluorescent sensors for real-time detection of PLpro activity in live cells of SARS-CoV-2. These sensors can be used to detect early stages of virus propagation and screen enzyme inhibitors.
In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Forster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target-green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker-were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.
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