Altered ISGylation drives aberrant macrophage-dependent immune responses during SARS-CoV-2 infection.

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.

Preventing an Antigenically Disruptive Mutation in Egg-Based H3N2 Seasonal Influenza Vaccines by Mutational Incompatibility.

Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation.