Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Abstract

Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra-high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles able to transduce susceptible cell types, thus expanding utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS-CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies

“…SARS-CoV-2 replicons (SARS-2R_mNG_NeoR_NL) of WA1 and Omicron BA.1 were constructed either in the WT background (WT WA1 , WT Om_BA.1 ) or in the presence of putative drug-resistance mutations at the 50, 132, 142, 166, and 167 sites of nsp5 as previously described [34]. All sequences were validated by long-read sequencing (Plasmidsaurus, Eugene, OR, USA).…”

“…All sequences were validated by long-read sequencing (Plasmidsaurus, Eugene, OR, USA). Construction of the N expression vectors was as previously described [34].…”

“…SARS-CoV-2 replicons (SARS-2R_mNG_NeoR_NL) of WA1 and Omicron BA.1 strains were constructed in the absence of mutations (WT WA1 , WT Om_BA.1 ) or in the presence of mutations at the 50, 132, 142, 166, and 167 sites of nsp5 as we have previously described [34]. HEK293T/17 cells seeded in a 6-well plate were transfected with 1 µg replicon plasmid (SARS-2R) using jetPRIME transfection reagent (Polyplus transfection, Illkirch-Graffenstaden, France).…”

“…Replicon fitness and dose response. SARS-CoV-2 replicons (SARS-2R_mNG_NeoR_NL) of WA1 and Omicron BA.1 strains were constructed in the absence of mutations (WTWA1, WTOm_BA.1) or in the presence of mutations at the 50, 132, 142, 166, and 167 sites of nsp5 as we have previously described [34].…”

“…Mutants were generated by site directed mutagenesis using QuikChange II (Agilent, Santa Clara, CA, USA) and validated via Sanger sequencing (GeneWiz, Chelmsford, MA, USA). SARS-CoV-2 replicons (SARS-2R_mNG_NeoR_NL) of WA1 and Omicron BA.1 were constructed either in the WT background (WTWA1, WTOm_BA.1) or in the presence of putative drug-resistance mutations at the 50, 132, 142, 166, and 167 sites of nsp5 as previously described [34]. All sequences were validated by longread sequencing (Plasmidsaurus, Eugene, OR, USA).…”