Fig. 1
Identification of the E3 ubiquitin ligase RNF5 as a factor involved in degradation of the E protein of SARS-CoV-2. a The proteasomal inhibitor MG132 increased the stability of the E protein. b MG132 enhanced the half-life of the E protein in cells treated with cycloheximide (CHX). HEK293T cells transfected to express E-HA for 24 h were treated 10 µM MG132 for 10 h. For CHX treatment, cells were harvested at the indicated time points after adding the inhibitor at 50 μg/mL. Cleared lysates resolved by SDS-PAGE were probed by immunoblotting (IB). c Immunoprecipitation (IP) of the E protein after MG132 treatment. HEK293T cells transfected to express E-HA were treated with MG132 for 10 h prior to harvest. Lysates were subjected to HA IP and eluents were analyzed by mass spectrometry. d Volcano plot of binding proteins with RNF5 in eluents. Black circles represent proteins that were not significantly changed. Red circles represent proteins that potentially interact with the E protein, blue circles represent non-binding proteins. e Knockdown of RNF5 increased E stability. f mRNA level of silenced RNF5 by RT-qPCR. g The E3 ubiquitin ligase activity of RNF5 is required for E degradation. RNF5-silenced HEK293T cells transfected with RNF5-Flag wild-type (WT) or its C42S mutant for 36 h were harvested and analyzed by IB. h Knockdown of RNF5 prolonged the half-life of the E protein. HEK293T cells receiving RNF5 silencing or the negative control pLKO.1 were transfected with the E-HA plasmid, and then treated with 50 μg/ml of CHX prior to being harvested at the indicated time points
