Biology subjects

Kotova, E.

Publications and source records attributed to Kotova, E..

6 recordsLinked to original sources

bioRxiv2026

CAPRIN1 localizes to CD63-positive MVB-like SARS-CoV-2 egress compartments and limits cytopathic effects

Stress granules (SGs) are cytoplasmic condensates that assemble under cellular stress, including viral infection. The best-characterized evasion strategy in SARS-CoV-2 infection involves the viral nucleocapsid (N) protein hijacking the SG core protein G3BP1, thereby preventing SG assembly. By performing network analysis of N protein interactomes, we identified CAPRIN1 as an underexplored candidate, despite its central role alongside G3BP1 in SG assembly. Here, we provide the first high-resolution spatial and functional data on CAPRIN1 in SARS-CoV-2 infection. Using ultrastructure expansion microscopy (U-ExM), we localized CAPRIN1 and viral egress markers to enlarged, CD63- and LAMP1-positive, multivesicular body (MVB)-like egress compartments, refining the current model of SARS-CoV-2 egress. CAPRIN1 was found on both the limiting membrane of these compartments and within the intraluminal vesicles, a pattern distinct from G3BP1. Knockout of CAPRIN1 increased infection-associated cell death, promoted an aberrant cell-death phenotype, and enhanced syncytia formation. Together, our data reveal distinct activities of CAPRIN1 during infection and identify the egress compartments as MVB-like, raising the possibility that SARS-CoV-2 repurposes more than one cellular pathway for egress.

molecular biology↗

bioRxiv2025

The nuclear egress complex of MHV-68 is not essential for nuclear egress but mediates C-capsid specificity

Herpesvirus capsids must exit the nucleus to undergo additional maturation steps in the cytoplasm, such as secondary envelopment. This process is orchestrated by the nuclear egress complex (NEC), a conserved heterodimer that deforms the inner nuclear membrane and facilitates capsid egress. While NEC proteins are critical for the release of capsids into the cytoplasm and, therefore, efficient replication in [a]-, {beta}-, and {gamma}-herpesviruses, residual infectivity has been observed in NEC-deficient mutants of certain herpesviruses, including the [a]-herpesvirus Pseudorabies virus (PrV). To investigate the role of the NEC in murine gammaherpesvirus 68 (MHV-68), a mutant virus lacking most of the nucleoplasmic NEC component ORF69 ({Delta}ORF69) was generated. Unlike previous studies relying on transfection-based systems, this study demonstrates that NEC-deficient MHV-68 remains replication-competent, albeit with a significant replication defect. Despite the absence of ORF69, the mutant virus produced infectious progeny in non-complementing cells. Quantitative electron microscopy revealed regular nuclear capsid assembly and DNA packaging. However, while the parental virus primarily exported mature, genome-filled C-capsids, the {Delta}ORF69 mutant exhibited cytoplasmic and extracellular emergence of all capsid forms. These findings indicate that the MHV-68 NEC functions as a quality control checkpoint, selectively facilitating the nuclear egress of mature capsids. In the absence of ORF69, some capsids still egress, likely through alternative, less efficient pathways. Inhibition of cyclin-dependent kinases reduced parental and mutant virus spread, suggesting that {gamma}-herpesviruses exploit host-dependent mechanisms for an alternative nuclear egress route. ImportanceGammaherpesviruses, such as Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus, establish lifelong infections and can contribute to cancer. A crucial step in the viral life cycle depends on the nuclear egress of capsids, a process mediated by the viral nuclear egress complex (NEC). Surprisingly, murine gammaherpesvirus 68 (MHV-68) can translocate capsids from the nucleus to the cytoplasm even when a key NEC component, nucleoplasmic ORF69, is disrupted. Deleting most of ORF69 reduced viral replication but did not entirely block nuclear egress. Instead, the mutant virus allowed both mature and immature capsids to leave the nucleus, suggesting that the NEC functions as a quality control checkpoint for mature capsids. Furthermore, host cell kinases seem to enable alternative routes for egress that do not rely on the NEC. These findings further support the notion that NEC is not strictly necessary and suggest the potential exploitation of host-virus interactions for antiviral approaches.

microbiology↗

bioRxiv2024

Histone Tetrasome Dynamics Affects Chromatin Transcription

During various DNA-centered processes in the cell nucleus, the minimal structural units of chromatin organization, nucleosomes, are often transiently converted to hexasomes and tetrasomes missing one or both H2A/H2B histone dimers, respectively. However, the structural and functional properties of the subnucleosomes and their impact on biological processes in the nuclei are poorly understood. Here, using biochemical approaches, molecular dynamics simulations, single-particle Forster resonance energy transfer (spFRET) microscopy and NMR spectroscopy, we have shown that, surprisingly, removal of both dimers from a nucleosome results in much higher mobility of both histones and DNA in the tetrasome. Accordingly, DNase I footprinting shows that DNA-histone interactions in tetrasomes are greatly compromised, resulting in formation of a much lower barrier to transcribing RNA polymerase II than nucleosomes. The data suggest that tetrasomes are remarkably dynamic structures and their formation can strongly affect various biological processes.

molecular biology↗

bioRxiv2023

Zinc-dependent Nucleosome Reorganization by PARP2

Poly(ADP-ribose)polymerase 2 (PARP2) is a nuclear protein that acts as a DNA damage sensor; it recruits the repair enzymes to a DNA damage site and facilitates formation of the repair complex. Using single particle Forster resonance energy transfer microscopy and electrophoretic mobility shift assay (EMSA) we demonstrated that PARP2 forms complexes with a nucleosome containing different number of PARP2 molecules without altering conformation of nucleosomal DNA both in the presence and in the absence of Mg2+or Ca2+ions. In contrast, Zn2+ ions directly interact with PARP2 inducing a local alteration of the secondary structure of the protein and PARP2-mediated, reversible structural reorganization of nucleosomal DNA. AutoPARylation activity of PARP2 is enhanced by Mg2+ ions and modulated by Zn2+ ions: suppressed or enhanced depending on the occupancy of two functionally different Zn2+ binding sites. The data suggest that Zn2+/PARP2-induced nucleosome reorganization and transient changes in the concentration of the cations could modulate PARP2 activity and the DNA damage response. Significance StatementPARP2 recognizes and binds DNA damage sites, recruits the repair enzymes to these sites and facilitates formation of the repair complex. Zn2+-induced structural reorganization of nucleosomal DNA in the complex with PARP2, which is reported in the paper, could modulate the DNA damage response. The obtained data indicate the existence of specific binding sites of Mg2+ and Zn2+ ions in and/or near the catalytic domain of PARP2, which modulate strongly, differently and ion-specifically PARylation activity of PARP2, which is important for maintaining genome stability, adaptation of cells to stress, regulation of gene expression and antioxidant defense.

biochemistry↗

bioRxiv2022

Mechanism of Curaxin-dependent Nucleosome Unfolding by FACT

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT- dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The structures of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

cancer biology↗

bioRxiv2020

Detailed temporal dissection of an enhancer cluster reveals two distinct roles for individual elements

Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. Yet, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that form a previously identified enhancer cluster and activate the Fgf5 locus during exit from naive murine pluripotency. Four elements are located downstream of the Fgf5 gene and form a super-enhancer. Each of these elements contributes to Fgf5 induction at a distinct time point of differentiation. The fifth element is located in the first intron of the Fgf5 gene and contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. This amplifier element strongly accumulates paused RNA Polymerase II but does not give rise to a mature Fgf5 mRNA. By transplanting the amplifier to a different genomic position, we demonstrate that it enriches for high levels of paused RNA Polymerase II autonomously. Based on our data, we propose a model for a mechanism by which RNA Polymerase II accumulation at a novel type of enhancer element, the amplifier, contributes to enhancer collaboration.

molecular biology↗