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1.
PLoS Pathog ; 17(11): e1009820, 2021 11.
Article in English | MEDLINE | ID: covidwho-1528735

ABSTRACT

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.


Subject(s)
COVID-19/virology , Furin/metabolism , Nuclear Receptor Coactivators/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Cell Line , Endosomes/metabolism , Furin/genetics , Gene Expression , Humans , Immune Evasion , Interferons/metabolism , Lysosomes/enzymology , Nuclear Receptor Coactivators/genetics , Protein Isoforms , Proteolysis , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
2.
Nat Microbiol ; 6(11): 1433-1442, 2021 11.
Article in English | MEDLINE | ID: covidwho-1469971

ABSTRACT

COVID-19 vaccine design and vaccination rollout need to take into account a detailed understanding of antibody durability and cross-neutralizing potential against SARS-CoV-2 and emerging variants of concern (VOCs). Analyses of convalescent sera provide unique insights into antibody longevity and cross-neutralizing activity induced by variant spike proteins, which are putative vaccine candidates. Using sera from 38 individuals infected in wave 1, we show that cross-neutralizing activity can be detected up to 305 days pos onset of symptoms, although sera were less potent against B.1.1.7 (Alpha) and B1.351 (Beta). Over time, despite a reduction in overall neutralization activity, differences in sera neutralization potency against SARS-CoV-2 and the Alpha and Beta variants decreased, which suggests that continued antibody maturation improves tolerance to spike mutations. We also compared the cross-neutralizing activity of wave 1 sera with sera from individuals infected with the Alpha, the Beta or the B.1.617.2 (Delta) variants up to 79 days post onset of symptoms. While these sera neutralize the infecting VOC and parental virus to similar levels, cross-neutralization of different SARS-CoV-2 VOC lineages is reduced. These findings will inform the optimization of vaccines to protect against SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19/therapy , COVID-19/virology , COVID-19 Vaccines , Female , Humans , Immunization, Passive , Immunoglobulin G , Immunoglobulin M , Male , Middle Aged , Mutation , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Young Adult
3.
Immunity ; 54(6): 1276-1289.e6, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1163900

ABSTRACT

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , Epitopes/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , COVID-19/diagnosis , Cross Reactions/immunology , Epitopes/chemistry , Epitopes/genetics , Humans , Models, Molecular , Mutation , Neutralization Tests , Protein Binding/immunology , Protein Conformation , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship
4.
J Virol ; 95(9)2021 04 12.
Article in English | MEDLINE | ID: covidwho-1075938

ABSTRACT

The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs.IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.


Subject(s)
Interferon Type I/metabolism , Membrane Proteins/metabolism , Proteolysis , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Virus Replication , A549 Cells , Humans , Interferon Type I/genetics , Membrane Proteins/genetics , Spike Glycoprotein, Coronavirus/genetics
5.
Nat Microbiol ; 5(12): 1598-1607, 2020 12.
Article in English | MEDLINE | ID: covidwho-892039

ABSTRACT

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/pathology , Female , Humans , Kinetics , Longitudinal Studies , Male , Middle Aged , Seroconversion , Severity of Illness Index , Young Adult
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