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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329552

ABSTRACT

SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites—a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites, thereby shaping subsequent evolutionary change. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.

2.
PLoS Pathog ; 17(7): e1009715, 2021 07.
Article in English | MEDLINE | ID: covidwho-1315897

ABSTRACT

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/etiology , SARS Virus/physiology , SARS-CoV-2/physiology , Severe Acute Respiratory Syndrome/etiology , Spike Glycoprotein, Coronavirus/physiology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , HEK293 Cells , Humans , Mutation, Missense , Severe Acute Respiratory Syndrome/genetics , Severe Acute Respiratory Syndrome/virology
3.
J Extracell Vesicles ; 10(8): e12112, 2021 06.
Article in English | MEDLINE | ID: covidwho-1272198

ABSTRACT

In late 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. SARS-CoV-2 and the disease it causes, coronavirus disease 2019 (COVID-19), spread rapidly and became a global pandemic in early 2020. SARS-CoV-2 spike protein is responsible for viral entry and binds to angiotensin converting enzyme 2 (ACE2) on host cells, making it a major target of the immune system - particularly neutralizing antibodies (nAbs) that are induced by infection or vaccines. Extracellular vesicles (EVs) are small membraned particles constitutively released by cells, including virally-infected cells. EVs and viruses enclosed within lipid membranes share some characteristics: they are small, sub-micron particles and they overlap in cellular biogenesis and egress routes. Given their shared characteristics, we hypothesized that EVs released from spike-expressing cells could carry spike and serve as decoys for anti-spike nAbs, promoting viral infection. Here, using mass spectrometry and nanoscale flow cytometry (NFC) approaches, we demonstrate that SARS-CoV-2 spike protein can be incorporated into EVs. Furthermore, we show that spike-carrying EVs act as decoy targets for convalescent patient serum-derived nAbs, reducing their effectiveness in blocking viral entry. These findings have important implications for the pathogenesis of SARS-CoV-2 infection in vivo and highlight the complex interplay between viruses, extracellular vesicles, and the immune system that occurs during viral infections.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/therapy , Extracellular Vesicles/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/immunology , COVID-19/virology , Flow Cytometry , HEK293 Cells , Humans , Immunization, Passive , Protein Binding , Spike Glycoprotein, Coronavirus/analysis
4.
Science ; 370(6513): 241-247, 2020 10 09.
Article in English | MEDLINE | ID: covidwho-733186

ABSTRACT

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/physiology , Betacoronavirus/physiology , Coronavirus Infections/immunology , Ebolavirus/physiology , Hemorrhagic Fever, Ebola/immunology , Histocompatibility Antigens Class II/physiology , Host-Pathogen Interactions/immunology , Nuclear Proteins/physiology , Pneumonia, Viral/immunology , Trans-Activators/physiology , Virus Internalization , Antigens, Differentiation, B-Lymphocyte/genetics , COVID-19 , Cell Line, Tumor , Coronavirus Infections/virology , DNA Transposable Elements , Endosomes/virology , Genetic Testing , Hemorrhagic Fever, Ebola/virology , Histocompatibility Antigens Class II/genetics , Host-Pathogen Interactions/genetics , Humans , Nuclear Proteins/genetics , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Trans-Activators/genetics , Transcription, Genetic
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