Non-canonical proteolytic activation of RNase L by SARS-CoV-2 3CLpro offsets inactivation of OAS1 p46 antiviral signaling

During SARS-CoV-2 infection, the key protective oligoadenylate synthetase (OAS) that activates RNase L upon sensing viral dsRNA is the membrane-anchored OAS1 isoform p46. We show that SARS-CoV-2 3C-like main protease (3CL pro /M pro ) efficiently cleaves p46 at 381 STLQ 384 ↓A, excising the prenylated-Cys 397 membrane anchor, which releases p46 from endomembranes and thereby antagonizes antiviral restriction. We further show that 3CL pro cleaves RNase L at 406 SCLQ 409 ↓S, removing an inhibitory N-terminal domain and generating RNase L (410–741), a proteolytically activated antiviral effector. Non-cleavable RNase L (Gln409Ala) lacks antiviral activity in infected p46-null cells, confirming this non-canonical activation mechanism. RNA sequencing shows that protease-activated RNase L alters host RNA targeting and that 3CL pro reduces OAS3 transcripts. We propose that the conserved RNase L 3CL pro cleavage motif functions as a failsafe "tripwire" that preserves antiviral restriction despite p46 evasion. Thus, 3CL pro not only processes viral polyproteins but also directly activates RNase L, triggering antiviral restriction and revealing an intrinsic evolutionary constraint on immune evasion.