Biology subjects

King, E. M.

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

7 recordsLinked to original sources

bioRxiv2026

A precision-cut lung slice platform for evaluating respiratory virus replication dynamics

Understanding respiratory virus replication, tropism, and disease mechanisms requires experimental systems that balance physiological relevance with scalability. Although immortalized cell lines remain widely used, they fail to capture the cellular complexity of the lung, while animal models--though informative--are often costly, low-throughput, and limited in their ability to model human disease. Here, we describe a rapid and scalable precision-cut lung slice (PCLS) platform that overcomes many of these limitations. Our workflow generates infection-ready lung tissue slices within 24 hours and maintains tissue viability over extended culture periods. Using influenza A virus as a model pathogen, we demonstrate that PCLS support robust viral replication, recapitulate characteristic infection-associated pathology, and elicit localized immune cell responses within the respiratory epithelium. Together, these features establish PCLS as a versatile and physiologically relevant platform for studying the biology of influenza virus and other respiratory pathogens.

microbiology↗

bioRxiv2026

Efficient replication of influenza D virus in the human airway underscores zoonotic potential

Influenza D virus (IDV), primarily found in livestock species, has demonstrated cross-species transmission potential, yet its threat to humans remains poorly understood. Here, we curated a panel of IDV isolates collected during field surveillance from 2011 to 2020 from swine and cattle to assess their ability to infect human airway cells as a proxy for zoonotic threat assessment. Using lung epithelial cell lines, primary well-differentiated airway epithelial cultures, and precision-cut lung slices, we demonstrated that IDV efficiently propagates in cells and tissues from the human respiratory tract, reaching titers comparable to human influenza A virus (IAV). Infection kinetics in primary porcine airway cultures and respiratory tissues mirrored those from human, suggesting similar infectivity across species. To define host responses to IDV infection, we evaluated innate immune sensing and downstream interferon signaling in human respiratory cells. IDV infection resulted in markedly reduced activation of interferon regulatory factor (IRF) signaling and diminished induction of interferon lambda 1 and interferon-stimulated genes compared to IAV, indicating inefficient activation of innate immune sensing pathways. However, IDV replication was potently restricted in interferon-pretreated cells, demonstrating sensitivity to interferon-mediated antiviral effector mechanisms once an antiviral state was established. Together, these findings show that IDV can efficiently infect the human airway while limiting innate immune sensing, a feature that may facilitate zoonotic spillover. Our study highlights the need for enhanced surveillance of IDV at the animal-human interface and provides a foundation for further investigation into its biology and potential for causing human infection and disease. SIGNIFICANCE STATEMENTInfluenza D virus (IDV) is a poorly understood virus type in the Orthomyxoviridae family. Although initially considered incapable of infecting humans, high seropositivity rates among cattle and swine workers suggest that zoonotic infections may already be occurring. However, the extent of human compatibility--and the potential for spillover--remains poorly understood. Our study demonstrates that IDV replicates efficiently in multiple human respiratory models while largely evading innate immune defenses, raising concern that only minimal evolutionary changes may be required for sustained human transmission. These findings underscore the need for further investigation into IDV biology and zoonotic risk. Such studies are critical for identifying viruses with the potential to adapt to humans before they become public health threats.

microbiology↗

bioRxiv2024

YTHDF1 and YTHDC1 m6A reader proteins regulate HTLV-1 tax and hbz activity

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive neurodegenerative disease. Regulation of viral gene expression plays a key role in viral persistence and pathogenesis. However, the molecular mechanisms underlying this fine-tuned regulation remain poorly understood. Little is known regarding RNA chemical modifications of HTLV-1 RNA and how these affect viral biology and disease development. Posttranscriptional chemical modification of RNA is common in eukaryotes, with N6-methyladenosine (m6A) being the most prevalent. In this study, we investigated the role of m6A RNA modifications on HTLV-1 gene expression. Using MeRIP-Seq, we mapped sites of m6A modification to the 3 end of the viral genome. We found HTLV-1 RNA, as well as viral oncogene transcripts tax and hbz, contained m6A modifications. m6A-depletion in HTLV-1-transformed cells decreased sense-derived viral genes (Tax, Gag, Env) and increased antisense-derived Hbz expression. Tax and hbz transcripts were bound by reader proteins YTHDF1 and YTHDC1 in a panel of HTLV-1 T-cell lines. Using expression vectors and shRNA-mediated knockdown, we found YTHDF1 had opposing effects on viral gene expression - decreasing sense-derived viral genes and increasing antisense-derived Hbz. Upon further study, the YTHDF1 effects on tax abundance were dependent on tax m6A deposition. The nuclear m6A reader protein YTHDC1 affected the abundance of both sense- and antisense-derived viral transcripts and specifically enhanced the nuclear export of tax transcript. Collectively, our results demonstrate global m6A levels and m6A reader proteins YTHDF1 and YTHDC1 regulate HTLV-1 gene expression. ImportanceHuman T-cell leukemia virus type 1 (HTLV-1) persistence and pathogenesis are controlled through tight regulation of viral gene expression. The fate of RNA can be controlled by epigenetic modifications which impact gene expression without altering the DNA sequence. Our study details the impact of N6-methyladenosine (m6A) RNA chemical modifications on HTLV-1 gene expression. We found reductions in global m6A levels affected viral gene expression, decreasing Tax and other sense-derived viral genes, while increasing the antisense-derived Hbz. Our results suggest the oncogenic viral transcripts, tax and hbz, are m6A-modified in cells. We found these viral RNA modifications are interpreted by reader proteins YTHDF1 and YTHDC1, which dictate the fate of the viral RNA. Understanding HTLV-1 RNA chemical modifications offers potential insights into novel therapeutic strategies for HTLV-1-associated diseases.

molecular biology↗

bioRxiv2023

Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

bioengineering↗

bioRxiv2022

Peptides Mimicking RS Repeats Modulate Phase Separation of SRSF1, Revealing a Reliance on Combined Stacking and Electrostatic Interactions

Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing, in vivo phase separation, and a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the proteins RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains spanning the human proteome indicates that RRM domains involved in phase separation have more exposed aromatic residues and that in phase-separating proteins containing RS repeats, such residues are frequently surrounded by acidic residues. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.

biochemistry↗

bioRxiv2022

Uncovering the mechanism for aggregation in repeat expanded RNA reveals a reentrant transition

Repeat expanded RNA molecules aggregate under certain conditions both in vitro and in vivo. Understanding the mechanism for aggregation--including how aggregation properties change with sequence and environmental conditions--would explain and predict the behavior of RNA-based biomolecular condensates, and enable the rational design of RNA-based materials. Here, we introduce an analytical framework to predict aggregation for any repeat RNA sequence, accounting for both intra- and inter-molecular bonding. By enumerating the equilibrium landscape of multimers, we reveal the driving force for aggregation: the increased configurational entropy associated with the multiplicity of ways to form bonds in the aggregate. Our model uncovers rich phase behavior, including a sequence-dependent reentrant phase transition, and repeat parity-dependent aggregation. We validate our results by comparison to a complete computational enumeration of the landscape, and to previously published molecular dynamics simulations. Our work unifies and extends published results, and enables the design of programmable RNA condensates.

biophysics↗

bioRxiv2020

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice

Existing pre-clinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates using an autochthonous small cell lung cancer model, we extrapolated half-life times in the circulation of 50-100 seconds and intravasation rates between 4,000 and 27,000 CTCs/hour - an average daily shedding rate equivalent to [~]0.07% of the total number of primary tumor cells in the lung. Additionally, transfer of 1-2% of daily-shed CTCs from late-stage tumor-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

cancer biology↗