Biology subjects

Ploss, A.

Publications and source records attributed to Ploss, A..

11 recordsLinked to original sources

bioRxiv2025

Metadomain and metaloop genome interactions in mammalian T cells

Recent studies have advanced our understanding of chromosomal organization and its principal role in gene regulation. However, most analyses have focused on short-range interactions (<2 Mb), limiting insight into broader regulatory architecture. In particular, the relationships between topologically associating domains (TADs), sub-TAD loops, long-range cross-TAD interactions, and higher-order chromosomal compartmentalization remain poorly understood. Here, we identify extensive multi-megabase and interchromosomal interactions (metaloops) in T lymphocytes, which organize into larger meta-TAD associations (metadomains). Metaloops bridge distal promoters and regulatory elements of key T cell-specific genes such as Ctla4, Ikzf2, Il2ra, Ets1, Lef1, Runx1, Bach2, Foxo1 and others, and are both shared and cell type-specific across functionally distinct T cell lineages. Reanalysis of published data confirms the reproducibility of these interactions in both mouse and human T cells and their dependence on superenhancers. Genome-wide clustering of metadomains reveals three interchromosomal hubs with distinct epigenomic profiles, including a superenhancer-enriched hub associated with T cell-specific gene activation. By integrating a compendium of new and public T cell epigenomic data, we infer distinct architectural factors associated with short-range loops and long-range metaloops. Altogether, our study reveals new features of T cell-specific 3D genome organization across scales, and our computational framework is broadly applicable to analyses of chromatin architecture across different cell types and experimental systems. Highlights- Ultra-long-range (>2 Mb) chromatin interactions linked to gene regulation in T cells - A new algorithm identifies distal and interchromosomal meta-TADs and metaloops - An interchromosomal T cell-specific active hub emerges from metadomain clustering - Epigenomic compendium implicates TFs associated with long-range interactions

genomics↗

bioRxiv2025

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗

bioRxiv2024

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

molecular biology↗

bioRxiv2023

Highly multiplexed mRNA quantitation with CRISPR-Cas13

RNA quantitation tools are often either high-throughput or cost-effective, but rarely are they both. Existing methods can profile the transcriptome at great expense or are limited to quantifying a handful of genes by labor constraints. A technique that permits more throughput at a reduced cost could enable multi-gene kinetic studies, gene regulatory network analysis, and combinatorial genetic screens. Here, we introduce quantitative Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (qCARMEN): an RNA quantitation technique which leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 to address this challenge by quantifying over 4,500 gene-sample pairs in a single experiment. Using qCARMEN, we studied the response profiles of interferon-stimulated genes (ISGs) during interferon (IFN) stimulation and flavivirus infection. Additionally, we observed isoform switching kinetics during epithelial-mesenchymal transition. qCARMEN is a simple and inexpensive technique that greatly enhances the scalability of RNA quantitation for novel applications with performance similar to gold-standard methods.

molecular biology↗

bioRxiv2023

Persistent hepatitis B virus and HIV coinfections in dually humanized mice engrafted with human liver and immune system

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including HIV and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection and preclinical testing of novel immunotherapeutics.

microbiology↗

bioRxiv2023

Hepatitis delta virus RNA decline post inoculation in human NTCP transgenic mice is biphasic

Background and AimsChronic infection with hepatitis B and hepatitis delta viruses (HDV) is considered the most serious form of viral hepatitis due to more severe manifestations of and accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no FDA-approved treatment for HDV and current interferon-alpha treatment is suboptimal. We characterized early HDV kinetics post inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. MethodsWe analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor - human sodium taurocholate co-transporting peptide (hNTCP). ResultsKinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 18 minutes (standard error, SE: 2.4), binds to non-specific cells with a rate of 0.06 hour-1 (SE: 0.03), and returns as free virus with a rate of 0.23 hour-1 (SE: 0.03). ConclusionsUnderstanding early HDV-host kinetics will inform pre-clinical therapeutic kinetic studies on how the efficacy of anti-HDV therapeutics can be affected by early kinetics of viral decline. LAY SUMMARYThe persistence phase of HDV infection has been studied in some animal models, however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics. Understanding the kinetics of viral clearance in the blood can aid pre-clinical development and testing models for anti-HDV therapeutics.

microbiology↗

bioRxiv2022

Sfrp2 is a multifunctional regulator of rodent color patterns

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we compare the coats of the African striped mouse (Rhabdomys pumilio) and the laboratory mouse (Mus musculus) to study the molecular mechanisms controlling stripe pattern formation. By combining transcriptomics, mathematical modeling, and mouse transgenics, we show that Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing and employing in vivo gene editing experiments in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants also exhibit changes in coat color, revealing an additional function of Sfrp2 in regulating hair color. Thus, a single factor controls coat pattern formation by acting both as an orienting signaling mechanism and a modulator of pigmentation. By uncovering a multifunctional regulator of stripe formation, our work provides insights into the mechanisms by which spatial patterns are established in developing embryos and the molecular basis of phenotypic novelty.

developmental biology↗

bioRxiv2022

Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEVs open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; further, the "pPCP" domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together our work provides a comprehensive model of the structure and function of HEV ORF1. Significance StatementThe development of non-teratogenic and potent antiviral therapies against HEV have been hindered by an incomplete understanding of the viral replication cycle. Our work provides a mechanistic insight into the complex replicative cycle of this understudied human pathogen and identifies a novel domain-domain interaction that is vital for replicative fitness.

microbiology↗

bioRxiv2021

Macrophages govern antiviral responses in human lung tissues protected from SARS-CoV-2 infection

The majority of SARS-CoV-2 infections among healthy individuals result in asymptomatic to mild disease. However, the immunological mechanisms defining effective lung tissue protection from SARS-CoV-2 infection remain elusive. Unlike mice solely engrafted with human fetal lung xenograft (fLX), mice co-engrafted with fLX and a myeloid-enhanced human immune system (HNFL mice) are protected against SARS-CoV-2 infection, severe inflammation, and histopathology. Effective control of viral infection in HNFL mice associated with significant macrophage infiltration, and the induction of a potent macrophage-mediated interferon response. The pronounced upregulation of the USP18-ISG15 axis (a negative regulator of IFN responses), by macrophages was unique to HNFL mice and represented a prominent correlate of reduced inflammation and histopathology. Altogether, our work shed light on unique cellular and molecular correlates of lung tissue protection during SARS-CoV-2 infection, and underscores macrophage IFN responses as prime targets for developing immunotherapies against coronavirus respiratory diseases. HIGHLIGHTSO_LIMice engrafted with human fetal lung xenografts (fLX-mice) are highly susceptible to SARS-CoV-2. C_LIO_LICo-engraftment with a human myeloid-enriched immune system protected fLX-mice against infection. C_LIO_LITissue protection was defined by a potent and well-balanced antiviral response mediated by infiltrating macrophages. C_LIO_LIProtective IFN response was dominated by the upregulation of the USP18-ISG15 axis. C_LI

microbiology↗

bioRxiv2020

SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen [~]6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses. HighlightsO_LICell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19 C_LIO_LIDrug screen reveals critical role for membrane lipid composition in fusion C_LIO_LISpikes unusual membrane-proximal cysteines and aromatics are essential for fusion C_LIO_LICholesterol tunes relative infectivity of SARS-CoV-2 viral particles C_LI

cell biology↗

bioRxiv2019

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the post-fusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFV{Delta}SK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFV{Delta}SK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E expression; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. YFV{Delta}SK/Nluc gene expression was also shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, as well as valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a post-fusion, pre-translation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a post-fusion step in virus entry.\n\nIMPORTANCEFlaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here we show that the yellow fever virus nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

microbiology↗