Turelli, P. | Search bioRxiv

bioRxiv2026

Antigenic landscape of a highly mutated SARS-CoV-2 Spike in ongoing viral evolution

The ongoing emergence of SARS-CoV-2 variants in an increasingly immune-experienced population is largely enabled by the plasticity of the Spike protein, which lies at the frontline of host immune pressure. Here, we investigated how extensive remodeling of the N-terminal domain (NTD) in Spike influences its antigenic properties. Using BA.2.87.1, a variant heavily mutated in this domain, we found that even large deletions do not substantially disrupt overall Spike structure or pseudovirus infectivity. However, our structural and binding analyses revealed that the NTD exhibits increased flexibility and that interaction with a heme-metabolite, contributing to assembly of an immunodominant epitope, is lost. These conformational effectscoupled with mutations, compromised the ability of human convalescent antibodies to engage this domain, contributing to their reduced neutralizing capacity. Consequently, BA.2.87.1-like variants may escape recognition by the pre-existing NTD-targeting antibodies, potentially reducing protection. Together, our results highlight the intrinsic adaptability of the Spike beyond the receptor-binding domain, with important implications for immune escape during viral evolution. TeaserStructural flexibility of the Spike and immune escape of a SARS-CoV-2 variant with public health implications.

immunology↗

bioRxiv2025

Cancer cells subvert the primate-specific KRAB zinc finger protein ZNF93 to control APOBEC3B

ZNF93 is a primate-restricted KRAB zinc finger protein responsible for repressing 20- to 12-million-year-old L1 transposable elements. Here, we reveal that ZNF93 also regulates the key cancer driver APOBEC3B--a mutagenic enzyme linked to tumorigenesis and cancer progression. ZNF93 depletion impairs DNA synthesis, activates replication and DNA damage checkpoints, and triggers proinflammatory phenotypes. Conversely, its overexpression enhances resistance to exogenous genotoxic stress, mirroring the effects observed with APOBEC3B depletion. ZNF93 expression correlates with cell proliferation rates and is overexpressed in many cancer types. These findings suggest that ZNF93 serves as a critical guardian of genome integrity, co-opted by cancer cells to counterbalance APOBEC3B-induced and L1-derived genomic instability and inflammation.

genetics↗

bioRxiv2023

ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys

The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares [~]93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding escape mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.

microbiology↗

bioRxiv2023

KRAB zinc finger proteins ZNF587/ZNF417 protect lymphoma cells from replicative stress-induced inflammation

Heterochromatin loss and genetic instability enhance cancer progression by favoring clonal diversity, yet uncontrolled replicative stress can lead to mitotic catastrophe and inflammatory responses promoting immune rejection. KRAB-containing zinc finger proteins (KZFPs) are epigenetic modulators, which for many control heterochromatin at transposable element (TE)-embedded regulatory sequences. We identified a cluster of 18 KZFPs associated with poor prognosis in diffuse large B cell lymphoma (DLBCL). We found their upregulation to correlate with increased copy number alterations and suppression of immune responses in tumor samples. Upon depleting two that target evolutionarily recent TEs, the primate-specific ZNF587 and ZNF417 paralogs, the proliferation of DLBCL cell lines was drastically impaired and replicative stress abruptly induced with marked alterations of the chromatin landscape and multiplication of DNA replication origins. Furthermore, ZNF587/417 knockdown upregulated interferon/inflammatory-related genes through activation of the cGAS-STING DNA sensing pathway, augmented the susceptibility of tumor cells to macrophage-mediated phagocytosis, and modified their immunogenicity through an increased surface expression of HLA-I and reshuffling of their immunopeptidome. ZNF587 and ZNF417 are thus pro-oncogenic factors allowing for higher degrees of genetic instability through attenuation of replicative stress and secondary inflammation, an influence that likely facilitates the clonal expansion, diversification, and immune evasion of cancer cells.

cancer biology↗

bioRxiv2023

Genetic features and genomic targets of human KRAB-Zinc Finger Proteins

Kruppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) are one of the largest groups of transcription factors encoded by tetrapods, with 378 members in human alone. KZFP genes are often grouped in clusters reflecting amplification by gene and segment duplication since the gene family first emerged more than 400 million years ago. Previous work has revealed that many KZFPs recognize transposable element (TE)-embedded sequences as genomic targets, and that KZFPs facilitate the co-option of the regulatory potential of TEs for the benefit of the host. Here, we present a comprehensive survey of the genetic features and genomic targets of human KZFPs, notably completing past analyses by adding data on more than a hundred family members. General principles emerge from our study of the TE-KZFP regulatory system, which point to multipronged evolutionary mechanisms underlaid by highly complex and combinatorial modes of action with strong influences on human speciation.

genomics↗

bioRxiv2022

Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2022

P2G3 human monoclonal antibody neutralizes SARS-CoV-2 Omicron subvariants including BA.4 and BA.5 and Bebtelovimab escape mutants

The rapid evolution of SARS-CoV-2 has led to a severe attrition of the pool of monoclonal antibodies still available for COVID-19 prophylaxis or treatment. Omicron subvariants notably escape most antibodies developed so far, with Bebtelovimab last amongst clinically approved therapeutic antibodies to display still good activity against all of them including the currently dominant BA.4/BA.5. We recently described P2G3, a broadly active SARS-CoV-2 monoclonal antibody, which targets a region of Spike partly overlapping with the site recognized by Bebtelovimab. Here, we reveal that P2G3 efficiently neutralizes SARS-CoV-2 omicron subvariants including BA.4/BA.5. We further demonstrate that P2G3 neutralizes Omicron BA.2 and BA.4 mutants escaping Bebtelovimab blockade, whereas the converse is not true. FundingEU COVICIS program; private foundation advised by CARIGEST SA.

microbiology↗

bioRxiv2022

De novo design of site-specific protein interactions with learned surface fingerprints

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1, and CTLA-4. Several designs were experimentally optimized while others were purely generated in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

bioengineering↗

bioRxiv2022

SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one anothers escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

immunology↗

bioRxiv2021

Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2021

S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S-acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

cell biology↗

bioRxiv2020

Ongoing evolution of KRAB zinc finger protein-coding genes in modern humans

BackgroundKruppel-associated box (KRAB) zinc finger proteins (KZFPs) constitute the largest and fastest evolving family of gene regulators encoded by the human genome. Recent data indicate that many KZFPs serve as repressors of transposable element-embedded regulatory sequences (TEeRS) and that the evolutionary turnover of KZFP genes is mainly attributable to the changing transposable element (TE) load of their hosts. However, how natural selection and genetic variation are shaping this process is still poorly defined. MethodsGenetic information was collected from nine primate species and 138,500 human genomes. Gene-wide as well as functional amino acid position specific constraint was calculated across all human KZFPs. ResultsWe found that the most conserved KZFPs, some of which go back close to 400 million years, have been subjected to marked negative selection in the evolutionarily recent past and are very homogeneous within the human population. In contrast, younger, largely primate-restricted family members present evidence of less negative selection than the rest of genome and lower levels of coding constraint, particularly within the sequences encoding the functional sites of their zinc finger (ZF) arrays. We defined 33 sets of KZFP paralogs, which pairwise displayed a broad range of coding constraints differentials, with more recently emerged paralogs usually displaying a higher frequency of putatively deleterious mutations and missense variants within the functional sites of their ZF arrays than their source gene. Finally, we identified three KZFP genes more constrained in the genomes of individuals of African ancestry than in Europeans, with their modes of expression or DNA targets pointing to possible links between these inter-populational genetic differences and regional differences in the prevalence of some diseases. ConclusionsThis work shows how the ongoing selection of KZFPs contributes to modern human genetic variation, in particular through the constraint of putatively deleterious- and missense variants in functional protein sites, and how ongoing interplays between environment and KZFP genes might be impacting the biology of modern humans.

genomics↗

bioRxiv2020

Endogenous retroviruses drive KRAB zinc-finger family protein expression for tumor suppression

Numerous genes are aberrantly expressed in tumors, but its cause remains unclear. Human endogenous retroviruses (HERVs) are repetitive elements in the genome and have a potential to work as enhancers modulating adjacent genes. Since numerous HERVs are activated epigenetically in tumors, their activation could alter gene expression globally in tumors and change the tumor characteristics. Here, we show the HERV activation in tumors is associated with the upregulation of hundreds of transcriptional suppressors, Kruppel-associated box domain-containing zinc-finger family proteins (KZFPs). KZFP genes are preferentially encoded nearby the activated HERVs in tumors and transcriptionally regulated by the adjacent HERVs. Increased HERV and KZFP expression in tumors was associated with better disease conditions. Many KZFPs could suppress the progressive characteristics of cancer cells by downregulating genes related to the cell cycle and cell-matrix adhesion. Our data suggest that HERV activation in tumors drives the concerted expression of KZFP genes for tumor suppression.

cancer biology↗

bioRxiv2019

Primate-restricted KRAB zinc finger proteins and target retrotransposons control gene expression in human neurons

In the first days of embryogenesis, transposable element-embedded regulatory sequences (TEeRS) are silenced by Kruppel-associated box (KRAB)-zinc finger proteins (KZFPs). Many TEeRS are subsequently coopted in transcription networks, but how KZFPs influence this process is largely unknown. We identify ZNF417 and ZNF587 as primate-specific KZFPs repressing HERVK (human endogenous retrovirus K) and SVA (SINE-VNTR-Alu) integrants in human embryonic stem cells (ESC). Expressed in specific regions of the human developing and adult brain, ZNF417/587 keep controlling TEeRS in ESC-derived neurons and brain organoids, secondarily influencing the differentiation and neurotransmission profile of neurons and preventing the induction of neurotoxic retroviral proteins and an interferon-like response. Thus, evolutionarily recent KZFPs and their TE targets partner up to influence human neuronal differentiation and physiology. One Sentence SummaryYoung transposable elements and their protein controllers team up to regulate the differentiation and function of human neurons.

evolutionary biology↗

bioRxiv2019

The KZFP/KAP1 system controls transposable elements-embedded regulatory sequences in adult T cells

Transposable elements-embedded regulatory sequences (TEeRS) are subjected to early embryonic repression through sequence-specific recruitment of KRAB zinc finger proteins (KZFPs), their cofactor KAP1/TRIM28 and associated chromatin modifiers. This modulates the TEeRS-mediated regulation of gene expression in embryonic stem cells (ESCs) and leads to DNA methylation-induced silencing. However, KZFPs are broadly expressed in adult tissues, suggesting that they control TEeRS throughout life. Confirming this hypothesis, we reveal here that the KZFP/KAP1 system exerts a highly dynamic control of TEeRS in adult human CD4+ T lymphocytes. First, we observed that in these cells many TEs are still bound by KAP1, the recruitment of which is dynamically regulated upon T cell receptor stimulation. Second, we found that KAP1 depletion induces broad transcriptional alterations in T cells, with de-repression of TE-based regulatory elements leading to the illegitimate activation of nearby genes. Finally, we show that the tissue-restricted expression of KZFPs correlates with KAP1-mediated lineage-specific chromatin signatures and transcriptional repression. These data support a model where TE-targeting KZFPs and KAP1 are important regulators of gene expression in adult human cells.

genomics↗