Biology subjects

Ruzsics, Z.

Publications and source records attributed to Ruzsics, Z..

4 recordsLinked to original sources

bioRxiv2026

M-CSF drives alveolar macrophage plasticity during development and cytomegalovirus infection

Alveolar macrophages (AM), the most frequent resident immune cells of the lung, are at the first line of defence against respiratory pathogens and instruct structural lung cells, e.g. in tissue repair. They are long-lived and receive their terminal phenotypic imprint through signals originating from the unique location at the tissue-air interface, as well as through cytokines like granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-{beta} (TGF-{beta}). However, the regulatory mechanisms governing their phenotypic plasticity, which is conceptually critical for their positioning and differentiation in early life and for their functional adaptation during infection, remain poorly defined. Here we explored respiratory tract infection with cytomegalovirus (CMV), which is closely linked to mammalian immune evolution. Complementary host-pathogen fate-mapping strategies revealed AM to constitute the bottleneck for efficient mouse (M)CMV infection. MCMV infection induced macrophage colony-stimulating factor (M-CSF) in the alveolar space, and culturing of AM in M-CSF led to a profound remodelling of morphology, immunophenotype, and transcriptional identity, e.g. it increased the expression of interferon-stimulated genes (ISG), which modulated susceptibility to infection. Notably, already at baseline recently differentiated neonatal AM across species retained an M-CSF-associated transcriptional program. This was linked to reduced permissiveness to respiratory MCMV infection in vivo. Overall, our findings identify the role of M-CSF-dependent signalling in conferring plasticity to AM, when it is most needed, particularly during early-life establishment and in response to viral infection.

immunology↗

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↗

bioRxiv2023

Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high diversity adenoviral libraries

While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based rAd-libraries employing the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of [~]2.5x104 modified rAd per cm2 of transfected cell culture. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/566979v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db61a7org.highwire.dtl.DTLVardef@1e85764org.highwire.dtl.DTLVardef@c17178org.highwire.dtl.DTLVardef@a299_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

The landscape of SARS-CoV-2 RNA modifications

In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.

genetics↗