Bridging the antiviral gap: A BAC-based non-infectious SARS-CoV-2 replicon reveals potent antisense gapmers

To facilitate studies of SARS-CoV-2 replication and antiviral drug discovery, we developed a non-infectious SARS-CoV-2 replicon, pBAC-SARS-CoV-2_S/eGFP_ΔE, based on a bacterial artificial chromosome vector. The viral genome was modified to ensure non-infectivity while allowing replication monitoring: the spike (S) protein and envelope (E) protein open reading frames (ORF) were deleted, and ORF S was replaced with an enhanced GFP (eGFP) reporter. Upon transfection, the pBAC-SARS-CoV-2_S/eGFP_ΔE was replication-competent in HEK293T cells and was inhibited by remdesivir, validating the replicon utility for studying SARS-CoV-2 replication and antiviral screening. RNA structures regulate the SARS-CoV-2 replication cycle by controlling genome stability, expression, and interactions with viral and host proteins. Using the pBAC-SARS-CoV-2_S/eGFP_ΔE, we evaluated antisense gapmers targeting RNA structural motifs within the untranslated regions (UTRs) and nucleocapsid (N) and membrane (M) genes. Screening identified five potent gapmers: one targeting the 5' UTR, three targeting the N gene, and one targeting the M gene. The M-directed gapmer was the most active, with an EC₅₀ of 6.3 nM, highlighting the M coding region as a promising antisense target. Four of the identified gapmers, including the lead, recognize accessible motifs present in both genomic and subgenomic RNAs, suggesting that dual targeting may be an effective antiviral strategy.