[Design and functional validation of a chimeric E3 ubiquitin ligase targeting the spike protein S1 subunit of SARS-CoV-2].

Abstract

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.