Fig 1. VSV-G-Mpro-L construct: molecular mechanism, sequencing workflow, and mutant lineage phylogeny.
(A) Schematic representation of SARS-CoV-2 genome, polyproteins pp1a and pp1ab and the VSV-based mutation selection tool (VSV-Mpro). The InterGenic Region (IGR) between the genes G and L of WT VSV was replaced with the SARS-CoV-2 Mpro Wuhan-1 (also referred to as WT, wild type) sequence and its cognate autocleavage sites. Mpro genome positions in SARS-CoV-2 and in VSV-Mpro are highlighted in light blue. (B) The virus is fully dependent on Mpro for replication. Upon translation of G-Mpro-L, two outcomes are possible: 1. without an inhibitor, Mpro is free to process the polyprotein, and the transcription and replication complexes can assemble; 2. with an inhibitor, Mpro is inhibited, the polyprotein is not processed, and the virus is thus not able to replicate, unless it acquires a mutation rendering the Mpro less susceptible to the inhibitor. Then, the virus can replicate despite the inhibitor. (C) Selection experiments workflow: BHK21 cells are infected with VSV-Mpro and treated with a protease inhibitor (nirmatrelvir), supernatants of cytopathic effect (CPE) positive wells are used to isolate viral RNA, synthesize cDNA and PCR amplify the region of interest (Mpro) that will be sequenced using Nanopore sequencing. 96-well plate was modified from public domain artwork at https://commons.m.wikimedia.org/wiki/File:96-Well_plate.svg. (D) Unrooted phylogenetic tree showing the relationship between original/parental viruses and mutants. Mpro variants belonging to the same parental virus are colored accordingly: WT (black), F305L (sea green), L167F (blue), L167F/F305L (light sea green), L167F/P168S (light blue), Omicron (orange), Omicron/A206T (red). For clarity, only the names of the mutants investigated in this work are displayed. *Previously generated/investigated set of mutants in our first study [35].