Biology subjects

Kobiler, O.

Publications and source records attributed to Kobiler, O..

10 recordsLinked to original sources

bioRxiv2026

HSV-1 hijacks the DNA repair protein RAD51 at gene promoters to drive viral transcription

Productive infection by Herpes Simplex Virus type 1 (HSV-1) requires initiation of efficient viral gene expression. Upon nuclear entry, HSV-1 genomes are associated with several cellular factors, including DNA repair proteins. It is unclear how these cellular factors impact viral processes at early stages of infection. In this study, we investigate the role of RAD51, a core homologous recombination (HR) factor. We measured nascent viral transcription and show that RAD51 plays a pro-viral role in infection by promoting immediate-early viral gene expression. We demonstrate that RAD51 binds GC-rich gene promoter of ICP4 and directly promotes gene expression. We also reveal a previously unknown interaction between RAD51 and ADNP, a subunit of the ChAHP complex, known for its role in transcription regulation. We propose a model where RAD51 binds incoming genomes at promoter regions regulating the genome landscape and allowing for efficient transcription initiation.

cell biology↗

bioRxiv2026

Disruption of the Brain-Spleen Axis Impairs Monocyte-Microglia Communication and Accelerates Disease Progression in a Model of Amyloidosis

Alzheimers disease (AD) is characterized by a prolonged asymptomatic phase before cognitive decline emerges, yet the mechanisms driving symptom onset remain unclear. Here, we hypothesized that the transition from asymptomatic to symptomatic disease is linked to dysfunction of brain-immune communication. Retrograde neuronal tracing in the 5xFAD mouse model of amyloidosis revealed reduced brain-spleen connectivity at advanced disease stages. To probe the functional role of the brain-spleen axis in coping with disease, we denervated the splenic nerve at an early presymptomatic stage. This intervention accelerated cognitive decline, impaired splenic hematopoiesis, diminished monocyte recruitment to the brain, disrupted monocyte-microglia signaling networks, and reduced the transition of microglia from a homeostatic to the disease-associated (DAM) state. Conversely, enhancing splenic noradrenergic input increased hematopoiesis, restored monocyte homing to the brain, and delayed cognitive impairment. The protective role of splenic monocytes was independently validated in a retinal cytotoxic injury model, in which splenic denervation impaired post-insult retinal ganglion cell survival. Together, these findings identify an active brain-spleen circuit in regulating monocyte recruitment and establish peripheral monocytes as key drivers of microglial state transitions and disease progression.

neuroscience↗

bioRxiv2025

Partial Inhibition of Viral Replication Machinery Enhances Recombination in Herpes Simplex Viruses

Herpes simplex viruses (HSV-1 and HSV-2) are widespread human pathogens, most commonly causing oral and genital lesions. These DNA viruses use recombination as a major driver of evolution. Intragenomic and intergenomic recombination events can be detected both in vivo and in vitro. As viral recombination is tightly linked to replication, deciphering mechanisms that specifically effect recombination remains a challenge. Here, we employed a triple-fluorescent color recombination assay to identify homology mediated and non-canonical recombination events between co-infecting HSV-1 strains. We developed a deep learning model that detects and classifies progeny plaques according to their colors. This setup enabled us to perturb the infection process using small molecule inhibitors targeting either viral or host proteins. We identified that inhibitors that reduce infectious viral progeny, increased viral homology mediated recombination. The antiviral drugs, including acyclovir, also increased recombination between HSV-1 and HSV-2 and aberrations in the progeny viral genomes. Taken together our results indicate that commonly used anti-herpes drugs increase intraspecies and interspecies recombination rates and genetic rearrangements. SignificanceHerpes simplex viruses cause significant morbidity in all human populations. Current therapies rely primarily on antivirals that target viral replication. Viral recombination plays a key role in replication and evolution of these dsDNA viruses. To better characterize the viral recombination process, we introduce a fluorescence-based HSV-1 recombination assay coupled with deep learning-based plaque classification, enabling high-throughput quantification of recombination rates. We found that clinically relevant moderate concentrations of commonly used antiviral replication inhibitors, enhance homology-mediated intraspecies recombination, HSV-1/HSV-2 interspecies recombination, as well as the accumulation of defective genomes. We conclude that partial inhibition of the herpes replication complex can promote viral diversification, with potential implications for HSV evolution and drug resistance.

microbiology↗

bioRxiv2025

Potent Neutralization by Antibodies Targeting the Mpox A28 Protein

Mpox is the most pathogenic Poxvirus in circulation. While several antigens have been identified as targets for neutralizing antibodies, many proteins remain unexplored. We isolated and characterized four monoclonal antibodies (mAbs) targeting the Mpox A28 (OPG153), a virulence factor present on mature Mpox virions. The antibodies were isolated from convalescent individuals, alongside 14 additional mAbs targeting the A35 and H3 proteins. Anti-A28 mAbs potently neutralized Mpox and Vaccinia virus (VACV) through complement-dependent mechanisms involving C1q and C3 deposition. High resolution crystal structures of Anti-A28 mAbs 10M2146 and 8M2110 in complex with VACV A26 revealed two proximal epitopes within the N-terminal domain. Passive transfer of 8M2110 attenuated disease in infected mice. Moreover, immunization with A28 elicited antigen-specific B cells and robust neutralizing antibody responses and provided complete protection against lethal VACV challenge. These findings support Mpox A28 as a promising target for the induction of neutralizing antibodies and antiviral interventions.

immunology↗

bioRxiv2023

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes.

Critical stages of lytic Herpes simplex type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 also persists in neuronal tissues via a non-replicative, transcriptionally repressed infection called latency. Understanding the regulation of lytic and latent transcriptional profiles provides focused insight into HSV-1 infection and disease. We sought a fluorescence-based approach to observe temporal progression of HSV-1 infection at the single-cell level. We constructed and characterized a novel HSV-1 recombinant that reports IE and L gene expression by fluorescent protein detection. The dual-reporter HSV-1 visualizes IE gene expression by a CMV promotor-driven YFP, and L gene expression by an endogenous mCherry-VP26 fusion. We confirmed that viral gene expression, replication and spread of infection in epithelial cells is not altered by the incorporation of the fluorescent reporters. Interference with viral DNA polymerase activity abolishes VP26-mCherry detection late in HSV-1 infection, visually reporting the failure to complete viral replication. Viral replication in primary neurons is not altered, but retrograde-directed inoculation of the dual-reporter HSV-1 exhibits a modest reduction in titer, compared to unlabeled HSV-1. Low-dose axonal inoculation in the presence of small molecule modulation of neuronal signaling results in divergent outcomes of YFP and mCherry detection, suggesting different states of latent and lytic replication. Rigorous characterization of this dual-reporter HSV-1 recombinant has demonstrated the utility of temporal observation of HSV-1 replication in live cells and the potential for further insight into the dynamics of infection. ImportanceHerpes simplex virus-type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We constructed and characterized a dual-fluorescent HSV-1 recombinant that enables visualization of IE and L gene expression. The recombinant HSV-1 is used to observe the progression and outcome of infection. We demonstrate that drug treatments targeting cellular pathways can manipulate latent HSV-1 infection in neurons to achieve divergent outcomes of infection.

microbiology↗

bioRxiv2020

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

immunology↗

bioRxiv2020

Conserved interactions required for in vitro inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50M concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

microbiology↗

bioRxiv2018

Recombination between co-infecting herpesviruses occurs where replication compartments coales

Homologous recombination (HR) is considered a major driving force of evolution since it generates and expands genetic diversity. Evidence of HR between co-infecting herpesvirus DNA genomes can be found frequently, both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when co-infecting viral genomes interact. To study the spatiotemporal requirements for inter-genomic recombination, we developed an assay with dual-color fluorescence in situ hybridization which enables detection of HR between different pairs of co-infecting HSV-1 genomes. Our results revealed that when viral RCs enlarge towards each other, there is detectable overlap between territories of genomes from each virus. Infection with paired viruses that allow visualization of HR correlates with increased overlap of RCs. Further, inhibition of RC movement reduces the rate of HR events among co-infecting viruses. Taken together, these findings suggest that inter-genomic HR events take place during replication of HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce. Our observations have implications on understanding the recombination restrictions of other DNA viruses and cellular DNA.

microbiology↗

bioRxiv2016

Histone deacetylase inhibitors reduce the number of herpes simplex virus-1 genomes initiating expression in individual cells.

Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACis). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the infection. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may down regulate the number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.

microbiology↗

bioRxiv2016

Dynamic proteomics of HSV1 infection reveals molecular events that govern non-stochastic infection outcomes

Viral infection is usually studied at the level of cell populations, averaging over hundreds of thousands of individual cells. Moreover, measurements are typically done by analyzing a few time points along the infection process. While informative, such measurements are limited in addressing how cell variability affects infection outcome. Here we employ dynamic proteomics to study virus-host interactions, using the human pathogen Herpes Simplex virus 1 as a model. We tracked >50,000 individual cells as they respond to HSV1 infection, allowing us to model infection kinetics and link infection outcome (productive or not) with the cell state at the time of initial infection. We find that single cells differ in their preexisting susceptibility to HSV1, and that this is partially mediated by their cell-cycle position. We also identify specific changes in protein levels and localization in infected cells, attesting to the power of the dynamic proteomics approach for studying virus-host interactions.

systems biology↗