Interferon redundancy counteracts proteolytic inactivation by SARS-CoV-2 3CL ^pro main protease

Interferons (IFNs) are secreted during virus infection and induce antiviral responses through receptor-mediated phosphorylation of signal transducer and activator of transcription (STAT) proteins, leading to IFN-stimulated gene expression with antiviral activity. We previously reported that the SARS-CoV-2 main protease, 3CL ^pro , is secreted from infected cells through gasdermin D/E pores and retains proteolytic activity in human serum against extracellular substrates. Here, we show that 3CL ^pro selectively cleaves glycosylated IFN-λ1, IFN-λ2, and a rare naturally occurring variant of IFN-γ (Arg160Gln), but does not cleave wild-type IFN-γ, IFN-λ3, IFN-λ4, IFN-α proteins or IFN-β. We identified sites of O-linked glycosylation of IFN-λ1 at Thr ¹³⁷ and one or more threonines or serine in the sequence ²⁴ TSKPTTT ³⁰ , and N-linked glycosylation at Asn ⁶⁵ that were indispensable for signaling. Unexpectedly, O-glycosylation was also required for the cleavage and inactivation of IFN-λ1 by 3CL ^pro at two sites. Cleavage reduced STAT1 phosphorylation and impaired the induction of the IFN-stimulated proteins MX1, OAS2, and IFIT1. Although 3CL ^pro cleaved IFN-λ2 proximal to its N-terminus at ARLH ³² ↓GALP, cleavage neither disrupted signaling nor antiviral activity against SARS-CoV-2 and vesicular stomatitis virus. We further show that matrix metalloproteinases (MMPs) 2, 7, 8, and 12 degrade 3CL ^pro , whereas 3CL ^pro shows no activity against these MMPs.