Ut Southwestern Medical Center

NIH Research Projects · FY 2026 · 2016-08

PROJECT SUMMARY Copper (Cu) is a vital nutrient required by many essential enzymes. Cu-transporting ATPases, ATP7A and ATP7B, play pivotal roles in delivering Cu to proteins in the secretory pathway or to vesicles that mediate extracellular excretion of Cu. In response to varying levels in cellular Cu, ATP7A and B undergo dynamic movement between the trans-Golgi network and the endolysosomal system. Whole body Cu homeostasis is maintained by the liver, which is responsible for Cu excretion through ATP7B-mediated Cu delivery to the biliary canaliculus. Impaired Cu excretion, which can occur as a result of loss-of-function mutations in ATP7B, leads in Cu accumulation and tissue injury in the liver and brain, a condition known as Wilson’s disease. In addition, alterations in cellular systems that control ATP7A/B trafficking across the endolysosomal system can lead to altered Cu balance. An example of the latter is Cu toxicosis resulting from loss-of-function mutations in the COMMD1 gene, encoding an essential component of a larger complex known as the COMMD / CCDC22 / CCDC93 or CCC complex, which our group identified. The CCC complex sits at the apex of a hierarchy of regulatory systems that play essential roles in endosomal regulation, including endosomal recycling of transporters, such as ATP7A/B. Studies supported by the prior competitive period of this grant defined the principles by which the CCC complex regulates early endosomal function and recycling of endocytosed surface proteins back to the plasma membrane (PM), including elucidating the structural organization of the CCC complex. Interestingly, ATP7A/B trafficking to the plasma membrane has been shown to also involve late endolysosomal vesicles, implicating the CCC complex in their regulation through hitherto unknown mechanisms. In this proposal, we seek to uncover how the CCC complex regulates Cu excretion, focused on CCC-interacting factors in the late endolysosomal compartment. The project includes two specific aims, and each aim integrates biochemical and cellular studies, with animal models. In Aim 1 we will examine the role of Rab27a in supporting CCC-dependent trafficking of ATP7B. This aim is founded on preliminary studies showing that Rab27a binds to CCC directly and regulates ATP7B trafficking. Rab27a is known to be associated with late endolysosomal vesicles and to promote vesicle fusion events with the PM. In Aim 2, we will examine the contribution of DENND10 to Cu excretion. DENND10 is a CCC-binding protein of unknown function. We find that a proportion of DENND10 is localized on late endolysosomes and that DENND10 deficiency leads to an accumulation of ATP7B in these vesicles, indicating that DENND10 participates in regulating ATP7B trafficking. Altogether, the studies proposed in this project will close key knowledge gaps in Cu metabolism. More broadly, this work explores an unappreciated intersection between the CCC complex and the regulation of late endolysosomal vesicles, with potential implications to multiple other fields.

NIH Research Projects · FY 2026 · 2016-06

PROJECT SUMMARY Funded by this R01, we have used random germline mutagenesis coupled with automated meiotic mapping (AMM) in mice for real-time discovery of point mutations that cause abnormalities of immune function. To date, we have attributed 20,577 FACS and/or adaptive immune response phenotypes to 3,394 non-synonymous mutations in 1,344 genes. Many of the causative mutations affected proteins that were not previously known to support immunity. In most instances sequence orthology, subsequent genetic discoveries, and/or in vitro experiments suggested functional conservation with the human proteins. AMM offers speed and efficiency unmatched by any other forward genetic approach: when presented with a phenotype, one is informed of the genetic cause simultaneously. Often, the cause is a novelty. Beginning in 2020, AMM was strikingly enhanced by an artificial intelligence program (Candidate Explorer; CE) to evaluate the likelihood of causation. Trained on thousands of experiments in which candidate germline mutations were re-created with CRISPR/Cas9 on clean backgrounds, expanded into large pedigrees and re-tested for phenotype, CE utilizes 67 features of primary genetic data to make its assessments, constantly weighing data from new alleles of all genes that are struck, disambiguating closely linked mutations, exonerating many genes, and solidly implicating others. Leveraging discovery science with hypothesis-driven cell biological, biochemical, and structural investigations, we solved the mechanisms by which many novel mutations support immune cell development and function. Further, we hypothesized that several mutations causing focal impairment of immune cell ontogeny or survival might dramatically affect the growth of particular tumors, using both genetically engineered mouse models (GEMMs) and transplantable syngeneic tumors. Remarkably strong suppression of cancer cell proliferation (either elite control or outright cure) emanated from one or more alleles of seven different genes studied to date. We understand the basis of suppression in detail for each mutation. In two instances an immune mechanism of tumor control has been clearly established; in the other six, non-immune mechanisms predominate. The existence and relative abundance of strong cancer suppressor mutations might partly explain different rates of cancer progression among human patients who suffer from cancers driven by equivalent oncogene combinations. Strong suppressor mutations may also inspire therapies by presenting novel targets. These considerations impelled us not only to extend our immunological screens, but to directly (rather than secondarily) screen for cancer suppressor mutations by mutagenizing on the same background (C57BL/6J) modified by gene cassettes that create a highly penetrant, inducible B cell leukemia. We now propose to examine the effects of over 60,000 non-synonymous coding/splicing changes, in both homozygous and heterozygous states, on FACS phenotypes, leukemic B cell numbers, and survival time. Many novel suppressors are expected, and will likely operate within cancer cells themselves or via augmentation of immunity.

NIH Research Projects · FY 2025 · 2016-05

Cell-cell communication plays a central role in embryonic development and adult tissue homeostasis and its deregulation leads to human diseases including birth defects and cancers. Therefore, understanding how extracellular signals are transduced and integrated to control cell proliferation and differentiation during development, tissue homeostasis and regeneration is of central importance in biomedical research. The overarching goal of this team is to understand how signaling networks control organ development and regeneration, with an emphasis on Hedgehog (Hh), Hippo, and BMP signaling pathways. Hh signaling controls many key developmental processes in species ranging from Drosophila to human and its abnormal activity has been implicated in numerous human cancers including medulloblastoma and basal cell carcinoma. Hh acts through a conserved signaling cascade emanating from the GPCR family receptor Smoothened (Smo) to the Zn-finger transcription factor Ci/Gli but how Smo activates Ci/Gli is still poorly understood. In this proposal, the team will combine genetics, biochemistry, cell biology, and biophysical approaches to explore conserved mechanisms governing Smo trafficking, phosphorylation-dependent and - independent activation of Ci/Gli, and the molecular links between Smo and Ci/Gli. The Hippo pathway was initially discovered in Drosophila and plays a conserved role in the control of tissue growth and organ size by simultaneously regulating cell proliferation and survival. Deregulation of Hippo signaling has also been implicated in many types of human cancer. Despite its central importance in development and diseases, the mechanisms underlying Hippo pathway regulation under physiological conditions or deregulation under pathological conditions remain incompletely understood. The team has developed a genetic modifier screen allowing them to identify novel and evolutionarily conserved Hippo pathway regulators. The team will continue to investigate the mechanisms by which the newly identified components regulate Hippo pathway activity in organ size control and tissue regeneration. The team has pioneered the use of Drosophila adult intestine as a model system to investigate how stem cell self-renewal, proliferation, and differentiation are regulated during tissue homeostasis and regeneration, and identified Hippo, Hh and BMP signaling as essential for the regulation of stem cell activity. In the previous funding period, the team has also established Drosophila adult intestine as a model to study in vivo reprogramming after injury and began to explore the molecular underpinning. In the proposed study, the team will investigate how other cell extrinsic and intrinsic factors acts in conjunction with BMP signaling to promote stem cell self-renewal and explore the genetic and cellular mechanisms that control the reprogramming of fully differentiated cells to adult stem cells in response to injury. The knowledge gained from this study will have important implications for developmental biology, cancer biology, and regenerative medicine.

NIH Research Projects · FY 2025 · 2016-04

ABSTRACT This proposal will be focused on the understanding of mechanisms of two fundamental biological phenomena in eukaryotes: the circadian clock and codon usage bias. Circadian clocks control diverse cellular, physiological, and behavioral processes in eukaryotic organisms. Our long-term goal is to understand the molecular and biochemical mechanisms that permit the measurement of time and the output of circadian rhythms in eukaryotic circadian clocks. Our previous studies made fundamental contributions to the understanding of the eukaryotic circadian clock mechanisms. Synonymous codons are not used with equal frequencies in all genomes examined, a phenomenon called codon usage bias. Even though the phenomenon of codon usage bias has been known for several decades, the functions and mechanisms of codon usage bias are unclear. Our previous work demonstrate that codon usage is a novel layer of the genetic code that can determine both gene expression levels and protein structures. Our lab uses Neurospora, Drosophila and mammalian systems to study these two phenomena. For the circadian clock project, we propose to focus on several key aspects of the circadian oscillator mechanism in both Neurospora and mammalian clock systems. We will determine the role and mechanism of FRQ-CK1a interaction in circadian period determination in Neurospora. In addition, we will expand our study into a mammalian system by determining the role of the PERIOD-CK1 interaction in the mammalian circadian clock. These studies will establish a conserved mechanism for period determination in fungi and animals. Although FRQ in Neurospora and PER proteins in animals are not considered homologous, most of the domains in both proteins are predicted to be intrinsically disordered and both are progressively phosphorylated. We will determine how FRQ and PER function in the circadian clock using biochemical and molecular methods. For the codon usage project, we will build on our ground-breaking discoveries on the roles and mechanisms of codon usage biases in determining gene expression and protein structures. We will determine the mechanism of the codon usage effect on gene transcription in Neurospora based on a previously performed large-scale genetic screen. This study will reveal the mechanisms that underlie the conserved effect of codon usage on gene transcription. We will evaluate how codon usage influences gene expression in mice by creating an in vivo codon usage reporter. This study will establish the mechanism that contributes to effects of codon usage on tissue- and cell type-specific gene expression in mammals. In addition, we will develop a method to modulate translation elongation speed based on the role of codon usage in regulating protein folding that will have potential for use in treatment of many diseases. Together, these studies will address fundamental questions that are critical for our understanding of these two biological phenomena in eukaryotes.

NIH Research Projects · FY 2026 · 2016-04

Abstract Nucleic acid therapeutics are an increasingly successful modality for drug discovery and development. Clinical success is becoming routine. Drugs range from compounds designed to treat just one patient, to agents that are effective for the treatment of rare (1 in ~10,000) disease, to an approved treatments with the potential to treat millions. Despite this progress, it remains unclear whether nucleic acid drugs will have an impact on therapy that approaches the large and sustained impact of small molecules or antibodies. Maintaining the momentum of the field will require increased understanding of mechanisms of gene regulation by nucleic acids and expanding the pool of targets for nucleic acid drugs. Achieving this understanding will involve asking fundamental questions of basic science. My goal for the next five years is to explore mechanisms for RNA-mediated recognition of cellular targets. We will use this information to better understand cellular gene regulation as well as expand our insights into disease targets. Small binding RNAs (sbRNAs) are duplex RNAs that regulate gene expression by mimicking the action of miRNAs. Rather than induce cleavage of RNA targets, sbRNAs affect gene expression by binding to them. We will apply sbRNAs broadly to test their potential as an alternative mechanism for silencing gene expression. One specific target for sbRNAs is the expanded CUG trinucleotide repeat that causes Fuchs Corneal Endothelial Dystrophy (FECD). We will optimize anti-CUG sbRNAs for potency and their ability to function in vivo. While mammalian RNAi is well known to control translation in the cytoplasm, miRNAs and protein RNAi factors are also present in cell nuclei and can affect transcription splicing, and potentially other nuclear gene regulatory mechanisms. We will examine the molecular mechanism of nuclear RNAi. We will use the insights we gain to identify endogenous targets for gene regulation by nuclear miRNAs and develop strategies for controlling the expression of genes involved in disease.

NIH Research Projects · FY 2025 · 2016-03

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma and lymphoproliferative diseases primarily in immunocompromised patients. Like all herpesviruses, KSHV uses the host gene expression machinery to carefully control the timing and abundance of viral mRNAs in its latent and lytic phases. While emphasis has been placed on transcriptional control, recent reports suggest two distinct interactions between KSHV and host RNA quality control (QC) pathways that degrade nuclear transcripts. The goal of this proposal is to define the molecular mechanisms at the interface of host nuclear RNA QC pathways and KSHV gene expression. The aims will define mechanisms that KSHV uses to protect from or exploit host RNA decay pathways. In addition, some of the host pathways remain poorly understood, so the viral mechanisms will be harnessed to uncover fundamental aspects of human molecular biology. In one pathway studied here, the host RNA QC machinery targets viral transcripts for nuclear decay. To protect its RNAs, the KSHV ORF57 protein counters that pathway likely through other host factors including an RNA-binding protein called ALYREF. In Aims 1 and 2 of the current proposal, the mechanisms of ORF57-mediated protection and the RNAs targeted by specific host factors will be determined in molecular detail. Interestingly, ALYREF and other ORF57 binding proteins are host mRNA export factors. In Aim 3, their proposed roles in viral RNA stability will be tested on host RNAs to determine whether they reflect a general activity of these proteins. In addition to protection from decay, KSHV has been reported to exploit the PABPN1-PAPα/γ mediated RNA decay (PPD) pathway to control temporal expression of late genes. Aim 4 of the current proposal seeks to define the mechanisms of PPD- mediated regulation of viral gene expression and to better define the components of the PPD pathway. Taken together, the proposed KSHV studies investigate the molecular mechanisms involved in nuclear RNA QC pathways to better understand the basic gene expression mechanisms of the human pathogen KSHV and its human host cell.

NIH Research Projects · FY 2025 · 2016-02

Schistosomiasis ranks second (behind malaria) as the world’s most devastating parasitic disease. Despite this, treatment relies on a single drug, Praziquantel (PZQ), that has marginal efficacy. This disease is caused by Schistosoma flatworms (schistosomes) that live in the vasculature, producing eggs that spur a variety of chronic pathologies that are exacerbated by the fact that schistosomes can survive in the blood for decades. How these parasites thrive in this hostile environment remains an open question. Our group discovered that adult schistosomes possess a population of somatic stem cells, neoblasts, that are critical for tissue renewal. During our studies of these cells we made a surprising discovery: mechanical injury induces a massive increase in neoblast proliferation at the site of wounding. Because this mirrors what happens in highly regenerative free-living planarian flatworms, we reasoned that schistosomes may possess an uncharacterized regenerative capacity. Whole-body regeneration (i.e., regenerating amputated heads) is not known to occur in schistosomes; yet, classic studies suggest that treatment of adult worms with sub-lethal doses of PZQ results in extensive tissue damage that the worms can repair. Likewise, we find that sublethal concentrations of PZQ induce neoblast proliferation in adult parasites. Furthermore, rapidly growing juvenile schistosomes, which have a massive number of neoblasts, are refractory to PZQ. Thus, PZQ sensitivity and neoblast number are inversely correlated. Given these data, we hypothesize that neoblasts fuel regenerative responses in the worm and we predict these regenerative responses are critical to the parasite’s ability to respond to insults in vivo, including PZQ treatment. To test this hypothesis, we propose two specific aims. In Specific Aim 1, we will use single cell RNA sequencing to describe the cellular lineages that operate during parasite development and determine whether these linages programs are “reactivated” in adult worms following injury. In Specific Aim 2, we will evaluate how well schistosomes are able to restore form and function to their tissues following injury and the extent to which tissue repair relies on neoblasts. We will additionally determine whether neoblast-driven regenerative responses are essential for juvenile and adult parasite survival following PZQ administration in vivo. Together, these studies will be the first to explore schistosome regenerative responses on a molecular level. Because we predict that neoblasts mediate tissue repair following PZQ-induced damage, these studies could also suggest that targeting neoblasts may enhance the efficacy of PZQ, thereby transforming how we treat this disease.

NIH Research Projects · FY 2024 · 2015-09

Project Summary/Abstract for the Overall Component The techniques of CRISPR/Cas9-mediated genomic editing and the ability to generate induced pluripotent stem cells (iPSCs) from a sample of a patient’s blood have placed medicine on the brink of a revolution in our ability to treat, and perhaps even cure, a broad range of genome-based diseases. The overall goal of the UT Southwestern Wellstone Muscular Dystrophy Specialized Research Center is to improve the treatment provided to Duchenne muscular dystrophy (DMD) patients by developing a new therapeutic strategy called “myoediting”. The Center has been built around five integral components. These include: two inter-related research projects (1) one that will work to optimize the tools for application of CRISPR/Cas9-mediated DMD exon skipping to permanently restore dystrophin function, and the other (2) that will identify genetic and biomarker associations with cardiac phenotypes in patients with dystrophinopathies [i.e. DMD and Becker muscular dystrophy (BMD)] and serve as a primary source for human iPSCs. These projects will be complemented and supported by three Cores (A) an Administrative Core, that will also direct patient outreach and education, (B) a Myoediting Scientific Research Resource Core, which will generate DMD/BMD iPSCs from DMD/BMD patients and differentiate them into iPSC-derived cardiomyocytes as well as house the DMD/BMD Biobank, which will store relevant clinical data as well as validated guide RNAs for each genetic mutation, and (C) a Training Core, which will enhance the educational environment in order to recruit, train, and maintain the next generation of transformative investigators focused on addressing the challenges of muscular dystrophy. We firmly believe myoediting will offer an innovative therapeutic modality for the treatment of many thousands of DMD patients and offer a long awaited hope to these patients and their families devastated by DMD.

NIH Research Projects · FY 2024 · 2015-07

Project Summary/Abstract 22q11.2 deletion syndrome (22q11.2del) is the most com~mon human microdeletion syndrome known, affecting some 1/4000 individuals. Most patients have a 3 Mb deletion on one copy of chromosome 22q11.2, resulting in a haploinsufficiency of over 107 genes, 46 protein coding and the remainder noncoding RNAs and pseudogenes. Children born with this deletion can have a range of congenital anomalies that often include three that are developmentally linked; hypoplasia of the thymus, congenital heart defects (CHD), and hypoparathyroidism. Approximately 60-70% of the patients have an immunodeficiency due to reduced T cell output from a hypoplastic thymus and are often clinically referred to as having DiGeorge syndrome. The underlying mechanisms causing the defective formation/patterning of the thymic tissue remains poorly understood. Our results, obtained during the previous cycle of this grant, suggest a defect among the neural crest derived mesenchymal cells, which form the thymic capsule and vasculature and regulate the expansion of the thymus. In our first aim, the developmental abnormalities of the pharyngeal apparatus leading to the formation of a hypoplastic thymus will be determined. Specifically, the role of the neural crest-derived mesenchymal cells in regulating the development and expansion of the thymus will be studied. Embryonic thymii from mouse models of 22q11.2del will be used in reaggregate fetal thymic organ cultures to define the role of mesenchymal cells in the process of thymus expansion. RNA sequencing approaches, including single cell RNA sequencing will be used to determine what mesenchymal transcripts are involved in this process. These experiments will be complemented with a characterization of human thymii from 22q11.2del patients and normal controls. In humans with 22q11.2del along with the mouse models, there is a post-natal miRNA dysregulation noted. In aim 2, we will explore the consequence of these miRNA changes using a combination of longitudinal studies in humans and diverse mouse models. This will reveal whether the dysregulation of miRNAs impacts immune functions pertaining to the thymus. Results from the two aims will enable us to develop better strategies for restoring thymus functions in various clinical settings resulting in the hypoplasia of this tissue.

NIH Research Projects · FY 2024 · 2015-07

Project Summary The T32-funded Cardiovascular Training Program at UT Southwestern has a long and illustrious history of fostering emergence of capable and productive investigators ideally positioned for independence and success. Indeed, our Program has an impressive track record, producing a large number of capable researchers who continue to have substantial impact. Over this period, our institution and the Division of Cardiology have evolved from a research emphasis heavily weighted towards basic science to one that now includes broad strength and representation in translational science, clinical trials, population-based research, and health services and outcomes research. Four years ago, we completely revamped our T32 Training Program, shrinking its size, limiting it to postdoctoral trainees, sharpening the focus of our oversight and management, and becoming more “data driven” in evaluating our trainees and our Program. Now, 4 years later, we can say that the transition has been a huge success. Our trainees are now uncommonly productive in their research endeavors. The program is evenly balanced between basic and clinical/translational research. Our program is now reflective of a) the growth of highly successful programs in clinical and translational cardiovascular research at UT Southwestern over the past decade, b) our goal to prepare the best investigators across the full spectrum of cardiovascular research, and c) the reality of the current funding environment for basic research. Importantly, this shift in focus allowed us to better recruit MD investigators who are co-enrolled in clinical training programs. Of note, the Internal Medicine Residency and Cardiology Fellowships at UT Southwestern are among the strongest in the country. Our Cardiovascular Training Program is just that, a training program, and not a funding mechanism. Our primary objective is to develop the next generation of transformative cardiovascular investigators by recruiting highly talented and motivated individuals and preparing them for success in an increasingly competitive and resource-challenged environment. A secondary objective is to expand the cadre of women and minorities pursuing careers in cardiovascular research. Our faculty is diverse, spanning a spectrum of expertise from molecule to cell, organ system, organism, and population. Members of our faculty are distributed across the full spectrum of faculty rank, gender, and ethnic background. In summary, our Program is designed to foster the emergence of the next generation of investigators, teachers, and mentors at a time when the need for such has never been greater.

NIH Research Projects · FY 2025 · 2015-04

Abstract Obesity remains a major health problem in the United States and causes metabolic complications such as type 2 diabetes mellitus, dyslipidemia, hepatic steatosis and insulin resistance. Similar complications also occur in patients with familial lipodystrophies characterized by partial (familial partial lipodystrophy, FPLD) or almost complete (congenital generalized lipodystrophy, CGL) lack of body fat. In the last two decades, several causal genes have been discovered for lipodystrophy syndromes including AGPAT2, BSCL2, CAV1 and CAVIN1 for CGL; LMNA, PPARG, ADRA2A, AKT2, CIDEC, LIPE, MFN2, PCYT1A and PLIN1 for FPLD; LMNA and ZMPSTE24 for mandibuloacral dysplasia (MAD); PSMB8 for autoinflammatory lipodystrophy; PIK3R1 for short stature, hyperextensibility/hernias, ocular depression, Rieger anomaly and teething delay (SHORT) syndrome; POLD1 for MDP (mandibular hypoplasia, deafness and progeroid features) syndrome; and FBN1, CAV1, and POL3RA for Weidemann-Rautenstrauch syndrome (WRS). Our laboratory has been at the forefront of these studies and identified AGPAT2, PPARG, ZMPSTE24, and PSMB8 genes for various types of lipodystrophies. In addition, during the last five years, we have identified novel lipodystrophy genes, such as ADRA2A, POLR3A, PRRT3, MTX2, TOMM7, COL3A1 and NOTCH3; and novel variants, such as heterozygous p.R571S and homozygous p.R545H in LMNA, and heterozygous p.Q142* and p.F160* in CAV1 associated with unique lipodystrophy syndromes. However, the genetic basis of about 210 extremely rare patients with various subtypes of genetic lipodystrophies, including 179 pedigrees with FPLD phenotype, remains unknown. Thus, the first aim of this proposal is to identify novel gene(s)/variants involved in adipocyte biology, development and differentiation that cause lipodystrophies and to determine their function in adipocyte biology by using cellular model system. We will use the state-of-the-art whole genome sequencing combined with tissue transcriptome analysis to identify the molecular defects. The second aim is to ascertain relationships between molecular defects in lipodystrophy genes with metabolic derangements using well-phenotyped probands and families. We will conduct deep phenotyping using skinfold thickness measurements, dual-energy X-ray absorptiometry for regional body fat, whole-body magnetic resonance imaging for body fat distribution, and biochemical parameters for metabolic complications. These studies will unravel molecular mechanisms involved in causation of lipodystrophy, and insulin resistance and its associated morbidities. This new knowledge may provide targets for developing novel drugs for treating metabolic complications of obesity including diabetes, dyslipidemias and hepatic steatosis.

NIH Research Projects · FY 2026 · 2015-01

PROJECT SUMMARY Although HIV-1 infection can be controlled through long-term treatment with anti-retroviral therapy (ART), a true cure has been elusive. Reservoir cells persist over time and support latent HIV-1 reactivation upon therapy cessation, yet little is known about the underlying mechanisms. Our lab has recently identified previously unknown facets in the “core” HIV-1 transcriptional program that we will explore in this proposal to help fill this knowledge gap, and may offer key insights into HIV-1 biology as well as cure strategies. The major goal of this grant application is to understand transcriptional regulatory mechanisms shaping HIV-1 latency maintenance and reactivation. We will accomplish this goal by leveraging genetic, genomic and microscopy approaches to explore HIV-1 transcription at high-resolution in several immortalized and primary models of latency as well as in aviremic participants samples. We will focus on the cycle of RNA polymerase II (Pol II) transcription, which is essential for the process of latency reactivation in reservoir cells. Specifically, we will explore the hypothesis that HIV-1 transcription is initially activated by de novo Pol II recruitment through the action of viral enhancers and eukaryotic transcription factors -TFs- (host phase) and later sustained through Tat-dependent Pol II re-initiation (viral phase). We propose to build on our recent findings to gain a deeper understanding of how the viral enhancers promote HIV-1 transcription for latency reactivation in the host and viral phases of the HIV-1 transcriptional program. These goals are reflected in two Specific Aims: to define the viral enhancers that drive HIV-1 transcription and latency reactivation (Aim 1), and to determine how eukaryotic TFs and Tat function with the viral enhancers to facilitate HIV-1 transcription and latency reactivation. If successful, this project will yield a better understanding of the molecular mechanisms by which eukaryotic TFs and Tat converge to promote efficient HIV-1 transcription and latency reactivation, collectively having a sustained impact in the field. In keeping with NIAID’s mission of ending the HIV-1 epidemic, our long-term objective is to leverage the basic discoveries to devise novel and alternative cure strategies. As such, the fundamental knowledge gained by this research could be used in the future studies beyond the scope of this focused grant application, to exploit an enhancer-based strategy to permanently silence HIV-1 to achieve the long-awaited HIV-1 remission.

NIH Research Projects · FY 2025 · 2014-08

Project Summary The GEF-H1-IKKε-IRF5 signaling axis is an essential pathway for the recognition of peptidoglycans and enables host defense responses to cope with intracellular pathogens such as Listeria monocytogenes. Macrophages contain a reservoir of inactive GEF-H1 bound to microtubules that is activated by dephosphorylation to form signaling platforms for the control of innate and adaptive immunity. The GEF-H1 pathway is well positioned to allow the intracellular detection of cell-invasive pathogens. As many pathogens have developed mechanisms for intracellular survival and immune avoidance, the GEF-H1 pathway may have evolved to allow critical immune detection of microbial effectors that target cytoskeletal components. However, it is unclear how microbial effectors and pattern recognition receptors regulate microtubule dynamics for the activation of GEF-H1. This application seeks support for studies designed to understand the key role of GEF-H1 in microbial detection and to define the precise requirements for the activation of immune responses through microtubule based microbial pattern recognition.

NIH Research Projects · FY 2025 · 2014-07

PROJECT SUMMARY Rapid developments in neuroscientific approaches, technologies, and analytics to understand the pathophysiology and treatment of mental illnesses have resulted in major advances that serve as a strong base toward the translation of cutting-edge research into personalized treatments for psychiatric conditions. In order to best leverage these advances, it is imperative that the next generation of researchers in psychiatry receive focused training that integrates translational research with clinical training. Psychiatric training programs that provide this focus, in conjunction with dedicated research time, and leadership and related skill development, are greatly needed. While there is concern in the field of a profound shortage of physician- scientists being trained, there is also reason for optimism, in that the number of MD/PhD medical school graduates applying for psychiatric residencies has increased. Our clinical and translational research training program, Translational Research Activities In Neuropsychiatry (TRAIN) is designed to provide exceptional training and mentoring to psychiatry residents with the express goal of generating clinical and translational researchers in psychiatry who will go on to become independent physician-scientists. This program focuses on the relationship between disruptions in underlying genetics and neurobiology and how they relate to adaptive behavior, as in the NIMH Research Domain Criteria (RDoC) framework, as well as the emergence of biomarkers that can inform clinical care. The TRAIN program harnesses the expertise and processes our faculty employ to bridge existing gaps in the field. This includes focused work and expertise in critical content areas, populations, and settings, as well as the use of approaches that span all ranges of translational science from the bench to the community. Our structured program provides an exceptional mentoring plan with experiential opportunities to participating residents coupled with timely didactics. The TRAIN program is specifically designed to train the next generation of researchers so that they can translate neuroscience and genetics research into advancements of our understanding of a wide range of mental health problems and treatments. TRAIN facilitates team science mentoring and training that is needed for translational research, with an emphasis on productivity through the conduct of a mentored research project, as well as deliverables such as networking and development of funding proposals to equip trainees for their next steps in training and academic research. TRAIN also focuses on wellness and guarding against burnout to encourage early adoption of self-care and enhance resilience.

NIH Research Projects · FY 2026 · 2014-07

PROJECT SUMMARY Exercise induces a wider-range of physiological responses, such as the changes in skeletal muscle, hepatic glucose metabolism and intermediary metabolism. These responses involve a coordinated multi-organ crosstalk between the brain, liver and skeletal muscle. A region in the brain knowns as the ventromedial hypothalamus (VMH) plays a critical role in the regulation of metabolism and the metabolic responses to exercise .The VMH is thought to act primarily through the sympathetic nervous system (SNS) and the release of catelcholamines, which then bind to adrenergic receptors in target tissues. We hypothesize that the VMH mediates some of the metabolic benefits of exercise via the β2AR isoform in skeletal muscle, and the α1bAR isoform in liver. The goal of this application is to investigate the contributions of these tissue-specific isoforms to the metabolic benefits of exercise using novel genetically engineered mice and novel metabolic flux analysis methods. We anticipate that the results from our investigation will provide novel insights into potential therapies to combat metabolic disease.

NIH Research Projects · FY 2024 · 2014-07

ABSTRACT: An over-arching goal of the Department of Dermatology at The University of Texas Southwestern Medical Center is to produce scientific leaders in skin biology and skin disease. To sustain this goal, we seek to renew our T32 Dermatology Research Training Program, which primarily recruits MD/PhD or MD candidates from dermatology residency applicants as early as the residency match. The program’s success can be measured by our graduates’ achievements in acquiring federal grants as principal investigators (over 60% of graduates to date) and leadership positions in academia and industry. These accomplishments validate current policies: (1) selecting candidates with proven scientific achievements and strong potential for independent investigation; (2) continuing a physician-scientist track that trains 2 yrs. in clinical dermatology and then 2 yrs. in research (historically fundamental and/or translational, but with recent expansion into clinical); and (3) preceptorship under outstanding faculty from our Graduate School of Biosciences. The program will continue to be administered: by the same director and 2 co- directors (a senior one and a junior one who is a program alumnus) plus a new (internationally renowned) PhD scientist, by a coordinator, and by 3 advisors who hold Dean-ship positions at Southwestern. We gathered 38 potential preceptors (including a Nobel laureate and 9 members of the National Academy of Science), all of whom have received NIH R01 grants and are effective mentors and meaningful interactors with dermatology personnel. In the aggregate, our preceptors’ publications rate a high mean H-index of 64. For the next funding cycle, we have already recruited 6 outstanding post-doc trainees. The program was given exceptional reviews by Dennis Roop PhD in 2018 and by Alice Pentland MD in 2019, with Dr. Pentland judging the program in the context of comments from the Study Section.

NIH Research Projects · FY 2025 · 2014-06

Project Summary/Abstract Kidney disease constitutes a significant, increasing burden on the United States healthcare system. Although kidney disease related research has made significant advances on our understanding of the various conditions that lead to acute or chronic renal insufficiency, little progress has been made towards finding cures. Focused, concerted efforts by researchers need to be implemented to advance current knowledge towards translational goals. Such efforts will be aided by recruiting the best and brightest minds into this field. The UTSW Summer Undergraduate Research Institute for the Study of Kidney Diseases (SURISKD) strives to introduce undergraduate students with an interest in biomedical research to the exciting field of kidney research. Participants spend 10 weeks learning about the kidney and kidney disease by taking courses from and performing kidney disease-related research in the labs of some of the leading kidney disease researchers in the world. This program seeks to expose, educate, train and recruit the future generation of kidney disease researchers.

NIH Research Projects · FY 2023 · 2014-05

Project Summary Despite the expanding array of new targeted agents to treat castration-resistant prostate cancer (CRPC), the disease remains incurable with nearly half of men with this form of PC developing bone metastases at two years. Metastatic bone disease carries a one-year survival rate of ~40%. Targeting the prostate-specific membrane antigen (PSMA) with small molecules for imaging and radionuclide therapy (RT) of prostate and other cancers has revitalized the field of nuclear medicine. Novartis has recently acquired [177Lu]R2, developed by us, and [177Lu]PSMA-617, two PSMA-targeted RT labeled with the β-particle emitter 177Lu that are in multi-center clinical trials. In Europe, there have been preliminary trials using the α-particle emitting agent [225Ac]PSMA-617 that have shown substantial treatment effects, even in patients that became resistant to the corresponding 177Lu- labeled compound. However, these encouraging responses to targeted α-particle RT (TAT) often came at the expense of immediate ablation of the salivary and lacrimal glands, with long-term toxicities unknown. Accordingly, despite widespread efforts, translational RT for PC is at a crucial stage, having yet to identify an agent that provides durable responses without compromising quality of life. The approach that we shall take in this competing renewal is to extend our basic work focusing on 211At, which emits a single α-particle per decay, to a low-dose, pharmacokinetic clinical trial. We hypothesize that 211At will provide an intermediate between the minimally toxic, but less effective 177Lu and the more powerful but potentially damaging 225Ac, which emits a total of four α-particles per decay that are difficult to control in vivo and promote the aforementioned toxicity. Our goal is to have an agent with an optimal therapeutic index by combining the high linear energy transfer (LET) tumor cell kill of 211At with greater control of toxicity through molecular design for salutary pharmacokinetics and dosing strategies. Preliminary in vivo data with our lead 211At-labeled compound, [211At]VK-02-90-Lu, indicates much lower off-target toxicity than for a related 225Ac-labeled adduct, confirmed by immunohistochemistry, with similar survival characteristics. The current program is intended to provide the experimental rationale and data for an IND-enabling therapeutic study.

NIH Research Projects · FY 2026 · 2013-07

White Adipose Tissue Physiology, Mitochondrial Function and Adiponectin Metabolic dysregulation with all of it pathophysiological sequelae, including diabetes, cardiovascular disease and cancer, continues to be on the rise. We need to identify new areas that can be targeted for improvements in systemic metabolism. Over the last funding period, we have made significant progress towards defining key processes in the white adipocyte that lead to mitochondrial challenges and consequently, impaired adipose tissue function. These include challenges to the mitochondrial import machinery through overexpression of Amyloid Precursor Protein , loss of mitochondrial DNA and a dysregulation in iron metabolism through overexpression of mitoNEET and mitoferritin. We have applied many of these challenges to other cell types of relevance in adipose tissue, such as macrophages and adipocyte precursor cells. Here, we propose to integrate these challenges and observe how they feed into the Integrated Stress Response of mitochondria. We will analyze how endoplasmic reticulum (ER) stress and the mitochondrial stress pathways result in altered cellular physiology of the white adipocyte. We can carefully time and titrate these mitochondrial manipulations, then probe them either in the presence or absence of the integral mitochondrial stress sensor, DELE1. We will examine these phenomena in the following areas: A) at the cellular level in the mature adipocyte; B) in the microenvironment at the level of whole adipose tissue physiology; C) at the whole-body level, assessing the systemic effects of the Integrated Stress Response. Specifically, we propose to address the underlying mechanisms with the following hierarchical approaches: In Aim 1, we will measure and manipulate mitochondrial stress. In Aim 2, we will manipulate mitochondrial matrix iron levels and assess the impact on the Integrated Stress Response. In Aim 3, we will address the interplay between the mitochondrial and ER stress phenomena. In Aim 4, we will determine the role that the Integrated Stress Response has in activating the removal and packaging of sub-mitochondrial particles through exosomes. Combined, this proposal will provide significant novel insights into the downstream consequences of mitochondrial dysfunction, ER stress and the Integrated Stress Response in the white adipocyte in vivo. We are uniquely positioned to address these aims with mouse models that we have generated and characterized during the initial ten years of this grant. In adipocytes, these interventions will severely tip the balance of lipid storage, lipid oxidation and carbohydrate metabolism, thereby contributing directly to the development of obesity and insulin resistance. These experiments will also enable us to carefully dissect the effects of altered mitochondrial function on the production of the critical adipokine, adiponectin. These are the first comprehensive in vivo experiments assessing the role of the Integrated Stress Response in the white adipocyte. As such, this will enhance our understanding of how we define a properly functioning adipocyte.

NIH Research Projects · FY 2025 · 2013-01

Project Summary. Malaria puts at risk 50% of the world’s population and is responsible for nearly 600,000 yearly deaths, mostly in children under the age of five in Africa. While a large portfolio of anti-malarial agents has been used to combat the disease, drug resistance has compromised the effectiveness of most clinically approved drugs, and the recent identification of resistance alleles against the current front line artemisinin combinations in Africa threatens current disease control programs. Thus, the identification of new drugs to combat drug resistant malaria is essential to continued progress against the disease. Our group in collaboration with Medicines for Malaria Venture (MMV) validated dihydroorotate dehydrogenase as a clinically valuable drug target for the treatment of malaria through studies on triazolopyrimidine DSM265, which advanced to phase II clinical development before the project was stopped due to discovery of off-target toxicity in preclinical species. In this current proposal we are working to identify new generation DHODH inhibitors by focusing on a different chemical series from DSM265, thus not expected to share an overlapping toxicity profile. Secondly, we plan structure-based approaches to identify inhibitors that will have reduced resistance risk compared to DSM265, which selected for resistance in 2 patients treated in the Phase II study. In aim one we plan to complete lead optimization of three related pyrazole-based DHODH inhibitor series, identified by scaffold hop using computational approaches (in collaboration with Schrödinger) from a pyrrole series we completed work on during the current fund period. Compounds from our pyrazole series have demonstrated high potency (sub nanomolar to low micromolar), and a reduced propensity to select for resistant parasites in vitro. We have a strong understanding of the SAR around these series, including the potency drivers, and the metabolic hot spots, and we plan mix and match chemistry to identify compounds with improved metabolic stability that will support human half-life (>100 h) and dosing targets (< 500 mg) set out by MMV. In aims 2 and 3 we use a combination of experimental and computational approaches (Schrödinger) to define the enzyme:ligand kinetic and thermodynamic binding properties that are associated with reduced resistance risk, as well as to correlate resistance risk to compound physical chemical properties. Computational models and measured thermodynamic/kinetic parameters will inform design and synthesis of new compounds predicted to have reduced resistance risk. The DHODH program is ideally suited to study the contribution of binding energetics to resistance propensity, as we have a wealth of structural information over three different chemical series with different physical chemical properties and alternative binding modes to the enzyme active site. Successful completion of these aims will allow identification of the strongest DHODH candidate for further clinical development, and it will provide key learnings on how to navigate resistance issues in target-based drug discovery programs for proliferative diseases in general, increasing the impact of our studies.

NIH Research Projects · FY 2024 · 2012-05

PROJECT SUMMARY Fuchs’ endothelial corneal dystrophy (FECD) is an age-related degenerative disorder resulting in corneal edema and loss of vision. FECD occurs in 4% of whites over the age of 40 years and is the leading indication for corneal transplantation in the U.S. Over 70% of cases are caused by a CTG triplet repeat expansion in the TCF4 gene. Expanded CUG repeat RNA (CUGexp) transcripts expressed from this gene locus accumulate as nuclear foci in the corneal endothelium of patients. The CUGexp foci bind and functionally sequester the splicing factors MBNL1 and MBNL2 to trigger mis-splicing in FECD endothelial tissue. To examine the endothelial cell-type specificity for FECD, we will determine if somatic mutations in the post-mitotic corneal endothelium of FECD patients results in a larger triplet repeat expansion than in their blood. We will examine if other anterior segment cell-types including corneal epithelium, stromal keratocytes, and trabecular meshwork cells are prone to accumulation of CUGexp foci and associated molecular defects. To test the MBNL sequestration hypothesis, we will examine if the knockdown of MBNLs in healthy donor endothelial tissue is sufficient to recapitulate the mis-splicing and upregulation of extracellular matrix (ECM) genes found early in the disease course. In early and late FECD, we observed a marked overexpression of the cochlin gene that produces a secreted ECM protein also capable of recruiting immune cells. The cochlin protein was previously detected in the trabecular meshwork of patients with primary open angle glaucoma (POAG), and our preliminary data indicate that the protein is present in the aqueous humor and trabecular meshwork of FECD subjects. We will examine levels of the cochlin protein in corneal tissue, aqueous humor samples, and trabecular meshwork tissue of FECD patients for its possible contribution to corneal disease findings and the increased risk for glaucoma in these patients. Additionally, we will examine the prevalence of POAG in our large UTSW FECD cohort and determine if there is a correlation with the triplet repeat length. We will use single-cell RNA sequencing combined with IHC to identify the immune cells and their contribution to late-stage FECD. We have characterized the efficacy of 45 antisense oligonucleotides (ASOs) and duplex RNAs that block disease-causing CUGexp foci. Using patient-derived cells and tissue, we will examine their potency based on their ability to block foci formation and their specificity based on transcriptome-wide assessment of on- and off-target events. We will examine delivery, length of action, and safety using wild-type mice. To enable studies of disease biology and in vivo drug testing, we have generated what we believe is the first mouse model with a knock-in of expanded CTG repeats. In preliminary studies, we show that our knock-in strategy successfully recapitulates CUGexp foci formation in corneal endothelium. We will further characterize this murine model by aging these mice to examine for the transcriptional and histologic alterations of FECD disease, and then use these mice to test our lead CUG-repeat targeting oligonucleotides for therapeutic development.

NIH Research Projects · FY 2025 · 2011-08

Project Summary The goal of this project is to define the metabolic and molecular basis of fatty liver disease (FLD), a burgeoning health problem with few therapeutic options. Fatty liver disease has been a major focus of our laboratory since 2004, when we undertook the first survey of hepatic triglyceride (TG) content (HTGC) in a population-based sample of different ancestries, the Dallas Heart Study (DHS). Hepatic steatosis was found to be strongly influenced by ancestry (Hispanics>European>African) and adiposity, but varied widely even among individuals who were matched for ancestry and body mass index (BMI). We used human genetics to identify the first and most clinically impactful genetic risk factor variant for FLD: PNPLA3(148M). This variant confers susceptibility to the full spectrum of both alcoholic and nonalcoholic FLD. In the same study in which we identified PNPLA3(148M), we also identified another variant in PNPLA3, S453I, that is associated with reduced HTGC; this variant is present almost exclusively in individuals of African descent, the group with the lowest prevalence of FLD. These two variants together account for ~70% of ancestry-related differences in HTGC. Despite having made significant progress elucidating the pathogenic mechanism of the 148M variant, and having performed successful proof-of-concept studies in mice of potential therapeutic avenues to combat the effects of 148M, important questions regarding the pathobiology of the variant and how it is related to FLD remain unanswered or disputed. Accordingly, we will focus this application on three critical questions: 1) How does PNPLA3(148M) evade ubiquitylation and degradation? 2) How does the 148M variant impair TG hydrolysis? and 3) How does PNPLA3-S453I lower hepatic TG content and protect against FLD? Each of these questions constitutes a Specific Aim. We will take advantage of cutting edge technologies to overcome important limitations in prior methods used by us and others to address these questions. In AIM 1 we will use a CRISPR/Cas9 inactivation screen to identify the E3-ligase that ubiquitylates PNPLA3. In AIM 2 we will use a highly tunable system to control protein expression at the level of translation, and a sensitive, luciferase reconstitution assay to biochemically define the interactions among PNPLA3, ATGL, and ABHD5 at physiologically relevant concentrations in cells. In AIM 3 we will develop the first mouse model of PNPLA3(S453I) to determine how the variant lowers HTGC. Since the region of PNPLA3 spanning residue 453 is not present in mice, we will replace the mouse gene with a human mini-gene containing the S453I variant using CRISPR-Cas9 technology. These mice will be used to determine how this missense variant results in lower hepatic TG levels. These studies, when taken together, hold the promise of revealing new pathways and processes that can be therapeutically manipulated for the prevention and treatment of PNPLA3-related FLD.

NIH Research Projects · FY 2025 · 2010-09

The Harold C. Simmons Comprehensive Cancer Center (SCCC) is the National Cancer Institute (NCI)-designated cancer center of the UT Southwestern Medical Center (UTSW) and its health system affiliates, Parkland Health & Hospital System, and Children’s Medical Center of Dallas. SCCC’s mission is to ease the burden of cancer through ground-breaking discovery, transdisciplinary research, impactful community engagement, education, and exceptional patient care. Upon arrival as the new Director in 2017, Carlos L. Arteaga, MD, shouldered authority over UTSW cancer activities and recasted a vision for the Center that focuses on the translation of science into the forefront of UTSW’s overall mission. To accomplish this, the SCCC Strategic Plan 2020-2025 prioritizes an increase in cancer-focused funding and infrastructure support for clinical trials, expansion of the clinical capacity for cancer treatment through further developed multidisciplinary cancer care, a new > 300,000 sf. Outpatient Cancer Care Tower, and a renewed emphasis on community outreach and engagement. In addition, SCCC will continue to create educational and training opportunities aimed at empowering a new generation of basic scientists, physician-scientists, clinical investigators, and other healthcare providers to make a difference in cancer discovery, clinical investigation and care, cancer control, and community outreach. The Center’s operations are optimized through a visionary and highly qualified Senior Leadership team and a value-creating administrative infrastructure. Together, leadership, infrastructure, and an engaged Center membership of 227 scientists and clinical investigators are organized into five highly interactive research programs—Cellular Networks in Cancer, Development and Cancer, Chemistry and Cancer, Experimental Therapeutics, and Population Science and Cancer Control. During the current six-year funding cycle, SCCC has secured several large collaborative multi-investigator awards, including a new SPORE in kidney cancer, six new U01s, and two new U54s, and maintains two NCI-funded T32s. As a result of strengthening the clinical trials infrastructure, accruals to interventional clinical trials has more than doubled. The rapid pace of discovery and movement toward effective translational research is supported by six shared resources: Biostatistics, Data Science, High Throughput Screening, Quantitative Light Microscopy, Small Animal Imaging, and Tissue Management. Today, SCCC has $25M in annual direct NCI funding. It has successfully leveraged Cancer Center Support Grant (CCSG) funding during the current period to continue the rapid growth of the Center, as demonstrated by the recruitment of 97 new members. SCCC has invested $210M since 2014 (six years) and has secured another $232M in institutional commitment for the next five years to increase the range of available resources. These impressive developments ensure that catchment area needs and concerns are addressed and SCCC scientists will continue their exemplary track record in advancing understanding of cancer biology while coupling these findings with the development of novel approaches to cancer control, diagnosis, and treatment.

NIH Research Projects · FY 2026 · 2009-07

7. Project Summary/Abstract This proposal focuses on defining the molecular mechanisms of axon navigation and connectivity. A normal functioning human nervous system requires the interconnection of billions of neurons. Improper formation or maintenance of these connections leads to abnormalities that result in mental diseases and disorders. How are these connections assembled and integrated? Research has revealed that the molecular mechanisms of axon guidance and connectivity are well-conserved between simple and complex animals. Simple animals like flies use many of the same guidance signals as mammals. Therefore, as a step towards understanding how complex nervous systems form and properly function, we have pursued a strategy to determine how the simple model fly nervous system is assembled – where we can also apply high-resolution molecular, genetic, biochemical, cellular, and imaging approaches to solve this problem. Indeed, the goal of my research program is to focus on a group of axons within the simple nervous system of the fly embryo and characterize the molecules and mechanisms that guide them to their targets. In particular, elegant studies from multiple labs have now identified numerous extracellular cues and receptors that guide axons, revealing fundamental mechanisms of how axons form connections. Far less is known, however, of the intracellular signaling pathways and mechanisms that link these guidance cues and their receptors to the control of axon navigation. Likewise, these guidance cues work to either attract or repel axons. Yet, how navigating axons choose between these antagonistic signals when they simultaneously encounter them remains far from clear. To answer these questions, we have focused on one of the largest protein families involved in connectivity, the Semaphorins (Semas) and their Plexin cell-surface receptors. Employing these strategies, our work has uncovered critical new molecules and mechanisms directing guidance/connectivity. Namely, we have discovered MICAL family enzymes, and have found that MICAL and SelR enzymes employ a specific reversible biochemical mechanism to control guidance/connectivity. We have also discovered that precise connectivity occurs via direct links between Semas/Plexins and other guidance molecules including Integrin- mediated adhesive receptors, specific growth factors, cyclic nucleotides, adaptors, small GTPases, serine- threonine and tyrosine kinases, and cytoskeletal assemblers and disassemblers. Now, using these strategies, our preliminary results uncover that these specific molecular pathways that provide connectivity to neurons are spatiotemporally and directly instructed by specific molecular pathways that provide metabolic sustenance to neurons. We therefore hypothesize that specific factors that govern neuronal connectivity are directly, locally, selectively, and instructively controlled by specific factors that govern neuronal metabolism. We propose to test this biomedically significant and innovative hypothesis by employing rigorous molecular, genetic, biochemical, cellular, and imaging approaches and the robust Drosophila high-resolution model system.

NIH Research Projects · FY 2025 · 2008-02

PROJECT SUMMARY/ABSTRACT Enteric viruses encounter a vast array of microbes in the mammalian intestine, and microbiota influence their infection efficiency. Previous work has shown that most enteric viruses benefit from the microbiota, and microbiota depletion reduces infection with enteric viruses including poliovirus, coxsackievirus, noroviruses, and one strain of reovirus called T3SA+. However, recent studies indicate that members of the Reoviridae family are outliers in microbiota effects. For example, rotavirus infection is inhibited by bacteria, and most tested strains of reovirus have enhanced replication upon microbiota depletion. Interestingly, a pair of reovirus strains differ by a single amino acid but have opposing effects from microbiota depletion. Microbiota depletion decreases replication of strain T3SA+ but increases replication of strain T3SA-. These viruses differ by a proline-leucine polymorphism in the s1 attachment protein, which confers sialic acid binding to the T3SA+ strain but not the T3SA- strain. Recent studies indicate that these two reovirus strains also differ in intestinal cell tropism and sensitivity to host innate immune responses in mice. Thus, these isogenic viruses provide an unprecedented opportunity to define how microbiota influence viral infection. In this work we will 1) examine the specificity of reovirus-glycan interactions including interactions with microbial glycans, 2) determine how microbiota facilitate infection with reovirus strain T3SA+, and 3) elucidate mechanisms of microbiota inhibition of reovirus strain T3SA-. These studies will provide mechanistic insight into how microbiota influence enteric virus infection in the complex environment of the intestine.