Biology subjects

Medina, E. M.

Publications and source records attributed to Medina, E. M..

7 recordsLinked to original sources

bioRxiv2026

Cytokine signaling regulates multiple stages of gammaherpesvirus infection in myeloid cells

The gammaherpesviruses, including Epstein-Barr Virus and Kaposis Sarcoma-associated Herpesvirus, establish lifelong infections by maintaining a balance between lytic and latent infection states. How this balance is regulated in myeloid cells, an important but understudied cell type that can achieve both infection states, remains unclear. Using the murine gammaherpesvirus 68 (MHV68) model, we show that IFNg; and IL-4 reciprocally regulate lytic infection in macrophages in a time- and JAK/STAT signaling dependent-manner, with basal JAK/STAT signaling further restricting lytic infection. Using a combination of MHV68 reporter viruses, cellular and molecular techniques, we demonstrate that IFNg blocks lytic replication through at least two distinct mechanisms, restricting delivery to or stability of the viral genome in the nucleus and potently restricting lytic cycle progression downstream of the immediate-early viral transactivator, RTA. These findings define specific points during lytic infection where IFNg; to enforce restricted, latent-like infection in myeloid cells.

immunology↗

bioRxiv2025

Three-dimensional cellularization in chytrid fungi uses distinct mechanisms from those driving one- and two-dimensional cytokinesis in animals and yeast

Chytrid fungi provide a model for studying foam-like cellularization, where nuclei that are dispersed throughout the cytoplasm are synchronously compartmentalized into daughter cells. This organization poses geometric challenges not faced by cells undergoing conventional cytokinesis or Drosophila monolayer cellularization, where nuclei are organized in linear or planar arrangements with ready-access to the plasma membrane. We use the chytrid Spizellomyces punctatus to show that chytrid cellularization begins with migration of nuclei and their attached centrosomes to the plasma membrane, where centrosome-associated vesicles mark sites of membrane invagination. These vesicles then extend inwards, resulting in tubular furrows that branch and merge to create a honeycomb of polyhedral membrane compartments-a cellularization foam-each with a nucleus and cilium. Using inhibitors and laser ablation, we show that tensile forces produced by actomyosin networks drive aphrogenesis (foam-generation), while microtubules are important for foam patterning and ciliogenesis but are not essential for cellularization. Finally, we suggest that chytrids may have incorporated ancestral mechanisms associated with ciliogenesis to coordinate the association of internal nuclei with membrane furrows to solve the unique geometric challenges associated with aphrogenic cellularization.

cell biology↗

bioRxiv2023

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

The gammaherpesviruses establish a lifelong infection, with the cellular outcome of infection intimately regulated by cell type. Though myeloid cells are an early infection target, infection results in widely divergent outcomes without a clear explanation. Here we use murine gammaherpesvirus 68 to demonstrate that macrophages are readily infectable, resulting in three divergent infection outcomes dictated by viral and host factors. Infection in vivo and in a cell culture model results in a high frequency of viable cells characterized by restricted viral transcription and unexpected transcription of the ORF75 locus, with rare cells initiating but failing to complete lytic replication. Restricted infection can be fully converted to productive lytic replication by pre-treatment with the host cytokine IL-4. Finally, infection with a high viral dose also results in productive infection, but triggers increased cell death. These studies demonstrate stepwise regulation of virus infection and identify virus and host factors that dictate divergent outcomes in a single cell type.

microbiology↗

bioRxiv2022

Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

Gammaherpesvirus ({gamma}HV) non-coding RNAs (ncRNAs) are integral modulators of viral infection. The {gamma}HVs engage RNA polymerase III (pol III)-dependent transcription of both host and viral ncRNAs, which contribute to viral establishment, gene expression, and pathogenesis. Viral ncRNAs, such as the EBV-encoded RNAs (EBERs), reportedly interact with multiple host RNA-binding proteins (RBPs) and contribute to inflammatory responses implicated in the development of malignancies. Here, we examined RBP interactions of the pol III-transcribed tRNA-miRNA encoded non-coding RNAs (TMERs) of murine {gamma}HV68, and the potential contributions of these and the related EBERs to in vivo pathogenesis. Using sequential enzymatic treatments, we found that several TMER1 forms retain a 5-triphosphate, lending the possibility of recognition by the innate immune sensor RIG-I. We further examined the interactions of TMERs and EBERs with host RBPs, and found that multiple TMERs and EBERs interact with the La protein, though minimal interaction was detected with RIG-I during primary virus infection. Finally, we investigated the contributions of the TMERs and EBERs to disease in an immune-compromised mouse model with a series of viral recombinants. We found that expression of multiple single TMERs, or the EBERs expressed in place of the TMERs, was capable of restoring virulence to a viral recombinant lacking expression of all TMERs. Ultimately, these studies demonstrate that divergent pol III-transcribed {gamma}HV ncRNAs share interaction characteristics with two host RBPs and conserved contributions to disease, despite little to no significant sequence conservation. These findings support a model of convergent functions of the sequence-variable pol III-transcribed {gamma}HV ncRNAs. IMPORTANCEViruses manipulate the infected cell and host inflammatory responses through expression of coding and non-coding RNAs. The gammaherpesviruses are a subfamily of herpesviruses associated with chronic inflammatory diseases and malignancies, especially in immune-compromised individuals. Among these, the human Epstein-Barr virus and murine gammaherpesvirus 68 ({gamma}HV68) express highly abundant, RNA polymerase III-dependent, short non-coding RNAs. Whether these sequence-divergent ncRNAs have conserved functional properties is unknown. By using viral recombinants to allow direct comparison of these ncRNAs during primary infection, we find that the sequence-divergent ncRNAs of {gamma}HV68 and EBV share a conserved property to bind to the host RNA binding protein, La, and function interchangeably to facilitate in vivo pathogenesis. These studies demonstrate that abundant, RNA polymerase III-dependent viral ncRNAs can potently function to alter the host cell landscape and promote disease in a sequence-independent manner.

microbiology↗

bioRxiv2020

Optimized detection of acute MHV68 infection with a reporter system identifies large peritoneal macrophages as a dominant target of primary infection

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally-infected cells. Here, we combine detection of a beta-lactamase activated fluorescent substrate with full spectrum flow cytometry to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages by 8 days post-infection, and a concordant increase in small peritoneal macrophages and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh large peritoneal macrophages emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 to 3 days post-infection, MHV68 infects ~5-10% of peritoneal cells, with >75% being large peritoneal macrophages. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in small peritoneal macrophages, with few infected dendritic cells and B cells. MHV68 infection at 3 days post-infection contains both lytic and latent infection, consistent with the identification of cells with active reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection. ImportanceIdentifying virally-infected cells in vivo is key to tracking viral infection and understanding host-pathogen interactions. The ability to further characterize and phenotype virally-infected cells is technically challenging. We use a mouse gammaherpesvirus, MHV68, expressing a reporter gene to identify infected cells during primary infection via flow cytometry. Optimization using this reporter system allowed us to further characterize infected cells via multiparameter full spectrum flow cytometry. Our study provides a technical model for high throughput single-cell immunophenotyping methods in the context of gammaherpesvirus infection. Furthermore, we show that acute MHV68 infection in the peritoneal cavity dramatically changes the immune landscape of this tissue, results in a high number of infected macrophages at early times, and is characterized by both lytic and latent infection within immune cells.

microbiology↗

bioRxiv2019

Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III-dependent promoters to enhance non-coding RNA expression

RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses ({gamma}HVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during {gamma}HV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 ({gamma}HV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with {gamma}HV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCEGammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

microbiology↗

bioRxiv2019

Genetic transformation and live-cell nuclear and actin dynamics during the life cycle of a chytrid

Chytrids are early-diverging fungi that share ancestral features of animals, including cells that crawl and swim. At later stages, chytrid cells resemble fungi with a chitin-based cell wall and hyphal-like structures known as rhizoids. Chytrids are important evolutionary transitional forms, but much remains unknown about their cell biology because we lack genetic tools for the live-cell imaging of their nuclear and cytoskeletal dynamics. Here, we generated stable transgenic lines of the soil chytrid Spizellomyces punctatus, and coupled live-cell microscopy and fluorescent tagging to measure the timing and coordination of growth, the cell cycle, and the actin cytoskeleton. We show that Spizellomyces zoospores rapidly encyst, develop rhizoids, and undergo multiple rounds of synchronous nuclear division in a sporangium, followed by cellularization, to create and release hundreds of zoospores. The life cycle is complete in less than 30 hours. We further demonstrate that crawling zoospores, akin to animal cells, display polymerized actin at the leading edge of amoeboid fronts. After encystment, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into the rhizoid. Actin remains highly dynamic during sporo-genesis with the formation of actin perinuclear shells each cell cycle and the emergence of polygonal territories during cellularization. Spizellomyces is a fast-growing and genetically-tractable organism that should be useful for comparative cell biology and understanding the evolution of fungi and early eukaryotes.

cell biology↗