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

Abstract

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, 3CLpro, is secreted from infected cells through gasdermin D/E pores and retains proteolytic activity in human serum against extracellular substrates. Here, we show that 3CLpro selectively cleaves glycosylated IFN-L1, IFN-L2, and a rare naturally occurring variant of IFN-{gamma} (Arg160Gln), but does not cleave wild-type IFN-{gamma}, IFN-L3, IFN-L4, IFN-alpha proteins or IFN-{beta}. We identified sites of O-linked glycosylation of IFN-L1 at Thr137 and one or more threonines or serine in the sequence 24TSKPTTT30, and N-linked glycosylation at Asn65 that were indispensable for signaling. Unexpectedly, O-glycosylation was also required for the cleavage and inactivation of IFN-L1 by 3CLpro at two sites. Cleavage reduced STAT1 phosphorylation and impaired the induction of the IFN-stimulated proteins MX1, OAS2, and IFIT1. Although 3CLpro cleaved IFN-L2 proximal to its N-terminus at ARLH32{downarrow}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 3CLpro, whereas 3CLpro shows no activity against these MMPs.