ABSTRACT
The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8+ T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8+ T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8+ T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8+ T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8+ T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8+ T cells during convalescence.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Immunodominant Epitopes/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Alleles , CD8-Positive T-Lymphocytes/pathology , COVID-19/pathology , Epitopes, T-Lymphocyte/immunology , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Immunodominant Epitopes/chemistry , Immunologic Memory , Lymphocyte Activation , Male , Middle Aged , Polyproteins/immunology , Viral Proteins/immunologyABSTRACT
The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/metabolism , Ubiquitin/metabolism , Animals , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Crystallography, X-Ray , Cytokines/genetics , Drug Evaluation, Preclinical/methods , Drug Repositioning , Fluorescence Polarization , HEK293 Cells , Humans , Kinetics , Models, Molecular , Protease Inhibitors/pharmacology , Protein Conformation , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Ubiquitins/genetics , Vero CellsABSTRACT
The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.