Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with ...the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).
SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes ...milder symptoms, especially in vaccinated individuals
. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that ...have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.
Proteins of the bromodomain and exterminal domain (BET) family mediate critical host functions such as cell proliferation, transcriptional regulation, and the innate immune response, which makes them ...preferred targets for viruses. These multidomain proteins are best known as transcriptional effectors able to read acetylated histone and non-histone proteins through their tandem bromodomains. They also contain other short motif-binding domains such as the extraterminal domain, which recognizes transcriptional regulatory proteins. Here, we describe how different viruses have evolved to hijack or disrupt host BET protein function through direct interactions with BET family members to support their own propagation. The network of virus-BET interactions emerges as highly intricate, which may complicate the use of small-molecule BET inhibitors-currently in clinical development for the treatment of cancer and cardiovascular diseases-to treat viral infections.
Turning up the heat on HIV-1 Chen, Irene P.; Ott, Melanie
Proceedings of the National Academy of Sciences - PNAS,
07/2020, Volume:
117, Issue:
28
Journal Article
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a ...possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.
Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of ...this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. ...Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER “degraders” exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
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•Drug candidates optimized for ER degradation can weakly activate ER in cancer cells•“ER degraders” trigger interaction of ER with DNA at canonical binding sites•Impact on chromatin accessibility distinguishes ER antagonists from weak activators•Dramatic slowing of ER mobility drives ER antagonism, and precedes ER turnover
Ligands that limit estrogen receptor mobility exhibit superior anti-proliferative capacity compared to those aimed solely at promoting ER degradation, pointing to a distinct approach for developing treatments for ER+ breast cancer.
Abstract As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an ...adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.
Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly ...emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.
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