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 (3CLpro/Mpro) efficiently cleaves p46 at 381STLQ384↓A, excising the prenylated-Cys397 membrane anchor, which releases p46 from endomembranes and thereby antagonizes antiviral restriction. We further show that 3CLpro cleaves RNase L at 406SCLQ409↓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 3CLpro reduces OAS3 transcripts. We propose that the conserved RNase L 3CLpro cleavage motif functions as a failsafe "tripwire" that preserves antiviral restriction despite p46 evasion. Thus, 3CLpro not only processes viral polyproteins but also directly activates RNase L, triggering antiviral restriction and revealing an intrinsic evolutionary constraint on immune evasion.