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. 2023 Apr 13;8(1):162.
doi: 10.1038/s41392-023-01418-3.

The main protease of SARS-CoV-2 downregulates innate immunity via a translational repression

Weifeng Liang  1   2 , Ming Gu  1   2 , Lingxiang Zhu  2 , Ziqi Yan  2 , Dominik Schenten  3 , Shelby Herrick  2 , Hongmin Li  2 , Subodh Kumar Samrat  2 , Jiapeng Zhu  4   5   6 , Yin Chen  7
Affiliations

The main protease of SARS-CoV-2 downregulates innate immunity via a translational repression

Weifeng Liang et al. Signal Transduct Target Ther. .
No abstract available

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
NSP5 repressed RLR-IFN pathway by affecting the translation. Immunoblotting analysis of p-STAT1, STAT1 when cells were transfected with MDA5 (a), RIG-I (b), MAVS (c), TBK1(d), and IRF3 (e) along with either NSP5 or NSP5 (C145A). Oligomerized MAVS (aggregates) was analyzed by SDD-AGE. Strep-tag (in the control vector, NSP5 or NSP5 (C145A)) was measured to ensure their proper expressions. β-actin was used as a loading control. Cells were transfected with MAVS (f) or TBK1 (g) along with NSP5 or NSP5 (C145A). Total RNA was extracted and analyzed by RT-qPCR with primers specific for IFNβ and ISG15. *: P < 0.05. ns no significance. n = 4. h Immunoblotting analysis of ectopically expressed protein level of MDA5, RIG-I, MAVS, TBK1, IRF3 and STAT1 in 293 T cells along with NSP5 or NSP5 (C145A). i 293 T whole cell extracts (WCEs) were incubated with purified NSP5 at RT for 1 h. Since endogenous MDA5, RIG-I and IFN receptor were low, WCE from FLAG-MDA5, FLAG-RIG-I and IFNαR1 + R2 transfected cells were used. MDA5, RIG-I, MAVS, TBK1, IRF3, STAT1 and IFN-α/βRα were analyzed by immunoblotting. His-tag linked control peptides were spiked in as an internal control. j Fluorescence images of 293 T cells transfected with GFP along with NSP5 or NSP5 (C145A). Scale bar = 100 μm. k A quantification of GFP fluorescence intensity (Excitation 485 nm, Emission 535 nm). l Immunoblotting analysis of GFP expression in the cells transfected with GFP along with NSP5 or NSP5 (C145A). m WCE of cells transfected with GFP was incubated with purified NSP5. His-tag linked control peptides were spiked in as an internal control. n qPCR measurement of GFP RNA in the cells co-transfected with NSP5 or NSP5 (C145A). SUnSET analysis of newly synthesized GFP (o) and MAVS (p). IgL Light chain of IgG. q qPCR measurement of MAVS RNA in the cells co-transfected with vector only (V), NSP5 or NSP5 (C145A). r A screen identified endogenous translation initiation factors that were targeted by an overexpressed NSP5 in 293 T cells. Immunoblotting analysis of translation initiation factors in 293 T cells (s) or in HACEs’ whole cell extracts (t) cleaved by purified NSP5. u Cells were transfected with MAVS, NSP5, PRDIII-Luc, Renilla-Luc and an increasing amount of eIF4G. A dual luciferase assay was performed. v Cells were transfected with MAVS, NSP5, and an increasing amount of eIF4G. Total cellular protein was collected and p-STAT1 was analyzed by immunoblotting analysis. w Immunoblotting analysis of NSP5, eIF4G in HAECs infected with SARS-CoV-2 (MOI = 1)
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References

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