Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
- MeSH
- COVID-19 * MeSH
- glykoprotein S, koronavirus genetika MeSH
- lidé MeSH
- mutace MeSH
- SARS-CoV-2 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
- MeSH
- COVID-19 * MeSH
- fylogeneze MeSH
- glykoprotein S, koronavirus genetika MeSH
- křečci praví MeSH
- lidé MeSH
- rekombinace genetická MeSH
- SARS-CoV-2 genetika MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- SARS-CoV-2 * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN-λ1, type 3 interferon (also known as IL-29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL-28 receptor 1 (IL-28R1) and IL-10 receptor 2 (IL-10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN- λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine-related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN-λ.
- MeSH
- antivirové látky metabolismus MeSH
- cytokiny metabolismus MeSH
- interferony * metabolismus MeSH
- interleukiny * metabolismus MeSH
- lidé MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viruses rapidly co-evolve with their hosts. The 9 million sequenced SARS-CoV-2 genomes by March 2022 provide a detailed account of viral evolution, showing that all amino acids have been mutated many times. However, only a few became prominent in the viral population. Here, we investigated the emergence of the same mutations in unrelated parallel lineages and the extent of such convergent evolution on the molecular level in the spike (S) protein. We found that during the first phase of the pandemic (until mid 2021, before mass vaccination) 31 mutations evolved independently ≥3-times within separated lineages. These included all the key mutations in SARS-CoV-2 variants of concern (VOC) at that time, indicating their fundamental adaptive advantage. The omicron added many more mutations not frequently seen before, which can be attributed to the synergistic nature of these mutations, which is more difficult to evolve. The great majority (24/31) of S-protein mutations under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. Furthermore, among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favoured. Next, we determined the mutation space in the S protein that has been covered by SARS-CoV-2. We found that all amino acids that are reachable by single nucleotide changes have been probed multiple times in early 2021. The substitutions requiring two nucleotide changes have recently (late 2021) gained momentum and their numbers are increasing rapidly. These provide a large mutation landscape for SARS-CoV-2 future evolution, on which research should focus now.
Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.
