Lineage memory shapes viral resistance barriers in human skin

bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-27

Abstract Individual cells within a given population exhibit striking variability in viral susceptibility, but it remains unknown whether this heterogeneity reflects memories encoded into the cellular lineage or true probabilistic variability. We used multi-color lineage tracing in a human primary organotypic skin model to reveal that viral resistance is encoded within specific cellular lineages. These lineages create distinct boundaries that block viral spread. Our lineage analyses in vitro confirmed that viral susceptibility exhibits strong heritability across cell generations, with siblings and cousins displaying remarkably similar infection outcomes. ATAC and proteomics profiling of resistant and susceptible clones revealed distinct epigenomic and proteomic states, with the transcription factor AP-1 emerging as a potential central regulator of lineage-encoded viral resistance. Inducing AP-1 activity with PMA rendered cells resistant to viral infection, suggesting a causative role in mediating resistance memory. Our findings demonstrate that antiviral resistance in human skin cells is encoded within cellular lineages and preserved through cell divisions, revealing how cell memory may shape infection dynamics and viral containment in tissues.

Differences between human and rodent nitric oxide production dictate susceptibility to tick-borne <i>Rickettsia</i>

bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-29 · 3 citations

Abstract Arthropod-borne pathogens cause serious human infections, yet they only cause limited disease in rodent reservoirs. Wild type mice resist infection by tick-borne Rickettsia parkeri, which causes spotted fever in humans, and it remains unclear why humans are vulnerable. Here, we report that whereas mouse type I interferon (IFN-I) or interferon-γ (IFN-γ) dramatically restrict R. parkeri in macrophages, human interferons do not. Differential RNA-seq revealed a significant induction of nitric oxide synthase 2 ( Nos2, encoding inducible nitric oxide synthase, iNOS) in infected mouse but not human macrophages upon interferon treatment. Chemical iNOS inhibition or Nos2 deletion restored IFN-γ-mediated restriction in mouse cells. Human cells treated with cytokine cocktails or with iNOS cofactors and substrates were still unable to restrict R. parkeri . In vivo , whereas wild type mice restricted R. parkeri , infected Nos2 -/- mice developed mild skin eschars, recapitulating a key human disease manifestation. Together, our findings suggest that there is a threshold of NO production required to restrict R. parkeri, which mouse cells reach but human cells do not, and this is a key explanation for why humans develop tick-borne rickettsial diseases while rodents can be tolerant, asymptomatic reservoirs. Differences in NO abundance may provide an evolutionary explanation for human susceptibility to pathogens that propagate themselves in rodent reservoirs.

Lineage Memory Shapes Viral Resistance Barriers in Human Skin

SSRN Electronic Journal · 2025-01-01

Publisher Correction: Herpesviruses mimic zygotic genome activation to promote viral replication

Nature Communications · 2025-03-05 · 1 citations

Herpesviruses mimic zygotic genome activation to promote viral replication

Nature Communications · 2025-01-15 · 15 citations

Zygotic genome activation (ZGA) is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In humans, ZGA is induced by DUX4, a pioneer factor that drives expression of downstream germline-specific genes and retroelements. Here we show that herpesviruses from all subfamilies, papillomaviruses and Merkel cell polyomavirus actively induce DUX4 expression to promote viral transcription and replication. Analysis of single-cell sequencing data sets from patients shows that viral DUX4 activation is of relevance in vivo. Herpes-simplex virus 1 (HSV-1) immediate early proteins directly induce expression of DUX4 and its target genes, which mimics zygotic genome activation. Upon HSV-1 infection, DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for infection, since genetic depletion by CRISPR/Cas9 as well as degradation of DUX4 by nanobody constructs abrogates HSV-1 replication. Our results show that DNA viruses including herpesviruses mimic an embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.

HSV-1 progeny production by individual human keratinocytes spans three orders of magnitude

Journal of Virology · 2025-08-20 · 1 citations

A fundamental property of viruses is their burst size-the amount of progeny produced during one round of infection. Herpes simplex virus 1 (HSV-1) is a widespread pathogen, causing significant morbidity in the human population worldwide. Despite decades of investigation into the biology of HSV-1, its burst size at the single-cell level has not been rigorously assessed. Here, we characterized HSV-1 progeny production by individual human keratinocytes, the physiological target of HSV-1 infection. Using a fluorescently-tagged virus, high-throughput single-cell sorting, and miniaturized plaque assay, we have quantified the progeny released from over a thousand individual cells throughout infection, at two multiplicities of infection. We found that individual keratinocytes harbor a very small number of infectious viruses inside them, which necessitated physical separation of cells to account for both secreted and cell-associated progeny. The total progeny produced by individual cells spans three orders of magnitude, and this variability is observed at both high and low multiplicities of infection, with a small sub-population of super-producer cells responsible for the majority of produced progeny by the entire population. We further found that super-production is not a genetic trait of the virus, as amplification of viruses produced by low and high producer cells results in similar burst sizes. Our findings both shed light on the fundamental process of progeny production during HSV-1 infection and provide a novel target for antiviral development, aimed at blocking super-producer cells.IMPORTANCEViruses are defined by their ability to take over host cells and turn them into factories that produce new progeny. Recent advances in Biology allowed us to study viral infections at the single-cell level, illuminating the vast heterogeneity presented by genetically identical cells during viral infection. Despite these advances, for many viruses, we still lack even basic characterization of progeny production at the single-cell level, due to the highly demanding technical challenges associated with measuring it. Here, we present the first quantification of progeny production by individual human keratinocytes infected by herpes simplex virus 1, revealing most individual cells produce a low level of new progeny, while a few rare cells produce significantly more.

A drug repurposing screen identifies decitabine as an HSV-1 antiviral

Microbiology Spectrum · 2024-09-17 · 14 citations

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a highly prevalent human pathogen that causes a range of clinical manifestations, including oral and genital herpes, keratitis, encephalitis, and disseminated neonatal disease. Despite its significant health and economic burden, there is currently only a handful of approved antiviral drugs to treat HSV-1 infection. Acyclovir and its analogs are the first-line treatment, but resistance often arises during prolonged treatment periods, such as in immunocompromised patients. Therefore, there is a critical need to identify novel antiviral agents against HSV-1. Here, we performed a drug repurposing screen, testing the ability of 1,900 safe-in-human drugs to inhibit HSV-1 infection in vitro . The screen identified decitabine, a cytidine analog that is used to treat myelodysplastic syndromes and acute myeloid leukemia, as a potent anti-HSV-1 agent. We show that decitabine is effective in inhibiting HSV-1 infection in multiple cell types, including human keratinocytes, that it synergizes with acyclovir, and acyclovir-resistant HSV-1 is still sensitive to decitabine. We further show that decitabine causes G &gt; C and C &gt; G transversions across the viral genome, suggesting it exerts its antiviral activity by lethal mutagenesis, although a role for decitabine’s known targets, DNA methyl-transferases, has not been ruled out. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen with a limited arsenal of antiviral agents, resistance to which can often develop during prolonged treatment, such as in the case of immunocompromised individuals. Development of novel antiviral agents is a costly and prolonged process, making new antivirals few and far between. Here, we employed an approach called drug repurposing to investigate the potential anti-HSV-1 activity of drugs that are known to be safe in humans, shortening the process of drug development considerably. We identified a nucleoside analog named decitabine as a potent anti-HSV-1 agent in cell culture and investigated its mechanism of action. Decitabine synergizes with the current anti herpetic acyclovir and increases the rate of mutations in the viral genome. Thus, decitabine is an attractive candidate for future studies in animal models to inform its possible application as a novel HSV-1 therapy.

Dissecting viral infections, one cell at a time, by single-cell technologies

Microbes and Infection · 2023-11-24 · 4 citations

The meteoric rise of single-cell genomic technologies, especially of single-cell RNA-sequencing (scRNA-seq), has revolutionized several fields of cellular biology, especially immunology, oncology, neuroscience and developmental biology. While the field of virology has been relatively slow to adopt these technological advances, many works have shed new light on the fascinating interactions of viruses with their hosts using single cell technologies. One clear example is the multitude of studies dissecting viral infections by single-cell sequencing technologies during the recent COVID-19 pandemic. In this review we will detail the advantages of studying viral infections at a single-cell level, how scRNA-seq technologies can be used to achieve this goal and the associated technical limitations, challenges and solutions. We will highlight recent biological discoveries and breakthroughs in virology enabled by single-cell analyses and will end by discussing possible future directions of the field. Given the rate of publications in this exciting new frontier of virology, we have likely missed some important works and we apologize in advance to the researchers whose work we have failed to cite.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes

Journal of Virology · 2023-11-22 · 8 citations

ABSTRACT Genomic rearrangements contribute to the enhancement of genetic diversity in populations. However, non-canonical rearrangements (NCRs) such as deletions, insertions, and inversions have the potential to trigger genomic instability. In the case of DNA viruses, NCRs can lead to generation of defective viral genomes (DVGs). To study NCRs in herpes simplex virus type 1 (HSV-1) genomes, we enriched DVGs formation by undiluted serial passaging on various cell types. We found that viral passaging on cell type that enables more viral genomes to initiate replication induces higher amplitude and frequency of cyclic patterns associated with DVGs formation. Despite differences in the rates of DVG accumulation, cell lines displayed comparable quantities of distinct NCRs, indicating that fluctuations caused by DVGs may impose bottlenecks on population genetic diversity. These findings propose additional roles for DVGs in modulating viral genetic diversity. Each cell type exhibited a unique population of NCRs, suggesting that NCRs accumulate in a cell type-specific manner. Interestingly, we identified a higher prevalence of short homologies and short reverse complementary in the parental sequences of NCR junction sites across all cell types. These shared sequence characteristics were also observed in NCRs identified in sequences obtained from clinical samples. The fundamental properties of HSV-1 NCR formation uncovered in this study may have broader implications for other DNA viruses. IMPORTANCE Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

Herpesviruses mimic zygotic genome activation to promote viral replication

Research Square · 2023-12-13 · 5 citations