Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy

Casasanta, Michael A.; Jonaid, G. M.; Kaylor, Liam; Luqiu, William Y.; Solares, Maria J.; Schroen, Mariah L.; Dearnaley, W. J.; Wilson, Joseph S.; Dukes, Madeline J.; Kelly, Deborah F.

doi:10.1039/d1nr00388g

Cited by 15 publications

(22 citation statements)

References 47 publications

(54 reference statements)

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“…In the current study, we detected N-protein in fatal COVID-19 infections using mouse anti-SARS-CoV-1-N 46-4 antibody, which recognizes an epitope located in the region between amino acids 50 to 200 [80], which is likely the RNA binding domain. A cryo-EM study demonstrated that the C-terminal region of the N-protein, is recognized and decorated by antibodies, and thus, is a potential immunogenic interaction site [81]. Further investigations of the stability of the various nsps and structural proteins of SARS-CoV-2 might explain the observed differences in localisation in lung.…”

Section: Discussionmentioning

confidence: 99%

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

Grootemaat¹,

Niet²,

Scholl³

et al. 2021

Preprint

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The trafficking of coronaviruses in lung of COVID-19 patients is not well understood and virus particles are difficult to find. Here we have visualized virus particles in SARS-CoV-2 infected cells by focusing on viral protein detection, in combination with ultrastructure. We studied how the virus is altering the cell morphology and determined that in Vero cells, lipid filled compartments contained various viral proteins. In these cells, also membrane enclosed multi-virus bodies were visible that contain a different set of viral proteins. We demonstrated that lipid filled compartments are novel viral induced compartments, as no known cellular marker such as lipid droplet or lysosomal marker was present. Using this knowledge, we then studied lung tissue from patients with a fatal SARS-Cov-2 infection, processed in a similar manner. Again we detected lipid filled compartments with viral proteins nsp4 and the stable nucleocapsid N-protein. The presence of these lipid filled compartments with viral proteins induced by SARS-CoV-2 infections, could be why the immune response of the COVID-19 patients is so strong, resulting in a fatal infection, and should be considered for new therapeutic strategies.

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Section: Discussionmentioning

confidence: 99%

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

Grootemaat¹,

Niet²,

Scholl³

et al. 2021

Preprint

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“…Cryo-EM structural investigations of the SARS-CoV-2 N protein (48 kDa) have revealed their interactions with human antibodies. However, the N proteins in other coronaviruses such as OC43, HKU1, NL63, and 229E show structural divergences [19].…”

Section: Genomic Variantsmentioning

confidence: 99%

Classical and Next-Generation Vaccine Platforms to SARS-CoV-2: Biotechnological Strategies and Genomic Variants

Simões

Rodrı́guez-Lázaro

2022

IJERPH

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Several coronaviruses (CoVs) have been identified as human pathogens, including the α-CoVs strains HCoV-229E and HCoV-NL63 and the β-CoVs strains HCoV-HKU1 and HCoV-OC43. SARS-CoV, MERS-CoV, and SARS-CoV-2 are also classified as β-coronavirus. New SARS-CoV-2 spike genomic variants are responsible for human-to-human and interspecies transmissibility, consequences of adaptations of strains from animals to humans. The receptor-binding domain (RBD) of SARS-CoV-2 binds to receptor ACE2 in humans and animal species with high affinity, suggesting there have been adaptive genomic variants. New genomic variants including the incorporation, replacement, or deletion of the amino acids at a variety of positions in the S protein have been documented and are associated with the emergence of new strains adapted to different hosts. Interactions between mutated residues and RBD have been demonstrated by structural modelling of variants including D614G, B.1.1.7, B1.351, P.1, P2; other genomic variants allow escape from antibodies generated by vaccines. Epidemiological and molecular tools are being used for real-time tracking of pathogen evolution and particularly new SARS-CoV-2 variants. COVID-19 vaccines obtained from classical and next-generation vaccine production platforms have entered clinicals trials. Biotechnology strategies of the first generation (attenuated and inactivated virus–CoronaVac, CoVaxin; BBIBP-CorV), second generation (replicating-incompetent vector vaccines–ChAdOx-1; Ad5-nCoV; Sputnik V; JNJ-78436735 vaccine-replicating-competent vector, protein subunits, virus-like particles–NVX-CoV2373 vaccine), and third generation (nucleic-acid vaccines–INO-4800 (DNA); mRNA-1273 and BNT 162b (RNA vaccines) have been used. Additionally, dendritic cells (LV-SMENP-DC) and artificial antigen-presenting (aAPC) cells modified with lentiviral vector have also been developed to inhibit viral activity. Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.

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“…This structure also cannot provide more information on the interaction between N protein and other structural proteins like M and E [ 127 ]. Recently, a 4.3 Å cryo-EM map of full-length SARS-CoV-2 N protein has been released in the Electron Microscopy Data Bank, but the corresponding structural model is still unavailable [ 130 ]. This N protein structure of moderate resolution may provide valuable insights into RNP assembly of SARS-CoV-2.…”

Section: In Situ Structure Of Sars-cov-2 Virionsmentioning

confidence: 99%

“…Although the high-resolution crystal structures of NTD and CTD of N protein have been solved, and a low resolution full-length N protein structure has been available recently [ 124 , 125 , 130 ], the structural basis for the assembly of RNPs has not been clarified. It has been proposed that the assembly of RNPs enables efficient genome packaging in a nucleosome-like manner [ 126 ].…”

Section: Summary and Perspectivesmentioning

confidence: 99%

Structure genomics of SARS-CoV-2 and its Omicron variant: drug design templates for COVID-19

Yin

Jiang

et al. 2022

Acta Pharmacol Sin

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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought an unprecedented public health crisis and persistently threatens to humanity. With tireless efforts from scientists around the world, understanding of the biology of coronavirus has been greatly enhanced over the past 2 years. Structural biology has demonstrated its powerful impact on uncovering structures and functions for the vast majority of SARS-CoV-2 proteins and guided the development of drugs and vaccines against COVID-19. In this review, we summarize current progress in the structural biology of SARS-CoV-2 and discuss important biological issues that remain to be addressed. We present the examples of structure-based design of Pfizer’s novel anti-SARS-CoV-2 drug PF-07321332 (Paxlovid), Merck’s nucleotide inhibitor molnupiravir (Lagevrio), and VV116, an oral drug candidate for COVID-19. These examples highlight the importance of structure in drug discovery to combat COVID-19. We also discussed the recent variants of Omicron and its implication in immunity escape from existing vaccines and antibody therapies.

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Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy

Abstract: Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress,...

Cited by 15 publications

References 47 publications

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

Classical and Next-Generation Vaccine Platforms to SARS-CoV-2: Biotechnological Strategies and Genomic Variants

Structure genomics of SARS-CoV-2 and its Omicron variant: drug design templates for COVID-19

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