Non-canonical proteolytic activation of RNase L by SARS-CoV-2 3CLpro offsets inactivation of OAS1 antiviral signaling

Upon sensing viral dsRNA, interferon-stimulated oligoadenylate synthetases (OAS) synthesize 2´-5´oligoadenylates that activate ribonuclease L (RNase L) to restrict viral replication. The predominant protective OAS during SARS-CoV-2 infection is the membrane-anchored OAS1 isoform p46. We show that SARS-CoV-2 3C-like main protease (3CL pro /M pro ) cleaves p46 at 381 STLQ 384 ↓A, removing the prenylated Cys 397 anchor, releasing p46 into the cytosol, and decreasing antiviral restriction. Unexpectedly, RNase L continues to restrict SARS-CoV-2 replication in p46-null cells. We identify RNase L as a second substrate within the OAS–RNase L pathway. Cleavage at 406 SCLQ 409 ↓S removes an inhibitory N-terminal domain, generating RNase L (410–741), a proteolytically activated product that restricts viral replication. A non-cleavable RNase L (Gln409Ala) mutant lacks antiviral activity in p46-null cells, confirming this non-canonical activation mechanism. RNA-seq revealed that the proteolytically activated RNase exhibits altered specificity toward cellular mRNA targets and that 3CL pro indirectly suppresses OAS3 transcripts without directly cleaving OAS3. We propose that the conserved 3CL pro -sensitive motif in RNase L functions as a failsafe tripwire that constrains complete loss of antiviral restriction despite simultaneous evasion of OAS1 p46. Thus, SARS-CoV-2 replication dynamics are intrinsically balanced by viral proteolysis within the OAS–RNase L axis, sufficient for polyprotein processing but countered by protease-triggered activation of RNase L and downstream antiviral restriction.