There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes ...angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.
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•CR3022 binds the RBD of SARS-CoV-2 and shows strong neutralization•Neutralization is by destroying the prefusion spike conformation•CR3022 binds a highly conserved epitope that is inaccessible in prefusion spike protein•CR3022 could have therapeutic potential alone or in synergy with a receptor blocker
Huo et al. find that the antibody CR3022 binds tightly to the receptor binding domain of the SARS-CoV-2 spike at a site different to that used by the receptor. CR3022 effectively neutralizes the virus, and cryo-EM reveals that it disrupts the spike. Such antibodies could have potential as COVID-19 therapeutics.
Cryo electron microscopy (cryo-EM) is used by biological research to visualize biomolecular complexes in 3D, but the heterogeneity of cryo-EM reconstructions is not easily estimated. Current ...processing paradigms nevertheless exert great effort to reduce flexibility and heterogeneity to improve the quality of the reconstruction. Clustering algorithms are typically employed to identify populations of data with reduced variability, but lack assessment of remaining heterogeneity. Here we develope a fast and simple algorithm based on spatial filtering to estimate the heterogeneity of a reconstruction. In the absence of flexibility, this estimate approximates macromolecular component occupancy. We show that our implementation can derive reasonable input parameters, that composition heterogeneity can be estimated based on contrast loss, and that the reconstruction can be modified accordingly to emulate altered constituent occupancy. This stands to benefit conventionally employed maximum-likelihood classification methods, whereas we here limit considerations to cryo-EM map interpretation, quantification, and particle-image signal subtraction.
The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two ...and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the ...human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K
of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from ...COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K
of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
Immune-mediated myocardial injury following Severe Acute Respiratory Syndrome Coronavirys-2 (SARS-CoV2) infection has been described in adults and children. Cases of myocarditis following ...immunization for SARS-CoV2 have recently been documented, mostly associated with mild severity and spontaneous recovery. We herein report two cases of fulminant myocarditis following BNT162b2 mRNA Covid-19 vaccination associated with systemic hyperinflammatory syndrome and refractory shock requiring support with veno-arterial extracorporeal membrane oxygenation.
•Fulminant myocarditis may occur following SARS-CoV2 mRNA vaccination requiring mechanical cardiac support.•It is important to recognize features of systemic hyperinflammatory syndrome following SARS-CoV2 mRNA vaccination.
Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and ...visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.
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•Cryoelectron tomography enabled characterization of rotavirus assembly intermediates•Subtomogram averaging provided detailed descriptions of the intermediates•Tri-lobed conformation of intact attachment protein VP4 described in enveloped stage•RNA-dependent RNA polymerase, VP1, has a role in recruiting genomes to particles
Shah et al. used cryo-tomography to characterize the assembly pathway of rotavirus inside infected cells. The use of flash frozen samples preserved the assembly stages in their native state and relative proportions, and subtomogram averaging revealed their molecular structures, to near atomic detail in the best case.
Commentary: Data—what is it good for? Shah, Pranav R.; Butterworth, John F.
The Journal of thoracic and cardiovascular surgery,
February 2022, 2022-02-00, 20220201, Letnik:
163, Številka:
2
Journal Article
Summary
Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini‐organelles called virus factories. On the basis of the serotype of the virus, the virus factories can ...manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high‐pressure frozen and freeze‐substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule‐dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule‐independent strain, progeny virions lacked organisation. Conversely to the microtubule‐dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome‐less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule‐dependent strain resulted in a significant increase of genome‐less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.
Nanomedicines exhibit unbelievable capability in overcoming the hurdles faced in biological applications. Carbon nanotubes (CNTs), graphene-family nanomaterials and fullerenes are a class of ...engineered nanoparticles that have emerged as a new option for possible use in drug/gene delivery for life-threatening diseases. Their adaptability to pharmaceutical applications has opened new vistas for biomedical applications. Successful applications of this family of engineered nanoparticles in various fields may not support their use in medicine due to inconsistent data on toxicity as well as the lack of a centralized toxicity database. Inconsistent toxicological studies and lack of mechanistic understanding have been the reasons for limited understanding of their toxicological aspects. These nanoparticles, when underivatized or pristine, are considered as safe, however less reactive. The derivatized forms or functionalization changes their chemistry significantly to modify their biological effects including toxicity. They can cause acute and long term injuries in tissues by penetration through the the blood-air barrier, blood-alveolus barrier, blood-brain barrier, and blood-placenta barrier. and by accumulating in the lung, liver, and spleen . The toxicological effects are manifested through inflammatory response, DNA damage, apoptosis, autophagy and necrosis. Other factors that largely influence the toxicity of carbon nanotubes, graphenes and fullerenes are the concentration, functionalization, dimensional and surface topographical factors. Thus, a better understanding of the toxicity profile of CNTs, graphene-family nanomaterials and fullerenes in humans, animals and the environment is of significant importance, to improve their biological safety, to facilitate their wide biological application and for the successful commercial application. The exploration of appropriate cell lines to investigate specific receptors and intracellular targets as well as chronic toxicity beyond the proof-of-concept is required.
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