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1.
Int J Mol Sci ; 23(15)2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-1957347

ABSTRACT

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Animals , Antibodies, Monoclonal , Antibodies, Viral , Humans , Mice , Mutation , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
2.
Int J Mol Sci ; 23(11)2022 Jun 04.
Article in English | MEDLINE | ID: covidwho-1884208

ABSTRACT

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing , Antibodies, Viral , Humans , Peptides/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
3.
Int J Biochem Cell Biol ; 145: 106185, 2022 04.
Article in English | MEDLINE | ID: covidwho-1757151

ABSTRACT

Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca2+ flux across ER membranes. Viroporins interact with numerous intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α-ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.


Subject(s)
COVID-19 , Humans , Life Cycle Stages , SARS-CoV-2 , Viroporin Proteins
4.
Eur J Immunol ; 52(5): 770-783, 2022 05.
Article in English | MEDLINE | ID: covidwho-1589126

ABSTRACT

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Humans , Mice , SARS-CoV-2
5.
Journal of Cellular Biochemistry ; n/a(n/a), 2021.
Article in English | Wiley | ID: covidwho-1409859

ABSTRACT

Abstract Variants of concern of the SARS-CoV-2 virus with an asparagine-to-tyrosine substitution at position 501 (N501Y) in the receptor-binding domain (RBD) show enhanced infectivity compared to wild-type, resulting in an altered pandemic situation in affected areas. These SARS-Cov-2 variants comprise the two Alpha variants (B.1.1.7, United Kingdom and B.1.1.7 with the additional E484K mutation), the Beta variant (B.1.351, South Africa), and the Gamma variant (P.1, Brazil). Understanding the binding modalities between these viral variants and the host cell receptor ACE2 allows to depict changes, but also common motifs of virus?host cell interaction. The trimeric spike protein expressed at the viral surface contains the RBD that forms the molecular interface with ACE2. All the above-mentioned variants carry between one and three amino acid exchanges within the interface-forming region of the RBD, thereby altering the binding interface with ACE2. Using molecular dynamics (MD) simulations and decomposition of intermolecular contacts between the RBD and ACE2, we identified phenylalanine 486, glutamine 498, threonine 500, and tyrosine 505 as important interface-forming residues across viral variants. However, especially the N501Y exchange increased contact formation for this residue and also induced some local conformational changes. Comparing here, the in silico generated B.1.1.7 RBD?ACE2 complex with the now available experimentally solved structure reveals very similar behavior during MD simulation. We demonstrate, how computational methods can help to identify differences in conformation as well as contact formation for newly emerging viral variants. Altogether, we provide extensive data on all N501Y expressing SARS-CoV-2 variants of concern with respect to their interaction with ACE2 and how this induces reshaping of the RBD?ACE2 interface.

6.
Front Microbiol ; 12: 692423, 2021.
Article in English | MEDLINE | ID: covidwho-1325543

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus Betacoronavirus, family Coronaviridae. The SARS-CoV envelope protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na+ and high intracellular K+. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC50 values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC50 = 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.

7.
Biomedicines ; 9(5)2021 May 08.
Article in English | MEDLINE | ID: covidwho-1223946

ABSTRACT

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. Amino acid exchanges within the SARS-CoV-2 spike protein variant of B.1.1.7 affect inter-monomeric contact sites within the trimer (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability or ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. Furthermore, we found increased flexibility in direct spatial proximity of the fusion peptide due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7. This study also implies a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7.

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