![\"Jonathan](\"https:\/\/depts.washington.edu\/jaisril\/wordpress\/wp-content\/uploads\/2015\/09\/jon-180x199.png\")
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Jon received his BS from Oregon State University (OSU), where he majored in Life Sciences\/Botany and studied suppressors of RNA silencing encoded by plant viruses, in the laboratory of\u00a0Dr. Valerian Dolja.\u00a0 At OSU, Jon received numerous awards, including two\u00a0HHMI summer undergraduate research fellowships\u00a0and the Outstanding Senior Award at Oregon State University.\u00a0 In 2004, he entered the University of Washington (UW) Molecular and Cellular Biology PhD program, and in 2006, he received an NSF Graduate Research Fellowship\u00a0to pursue his PhD studies on capsid assembly in the Lingappa lab.\u00a0 \u00a0In 2011, he obtained his PhD in Molecular and Cellular Biology and his MS in Epidemiology through a\u00a0joint UW PhD\/MS program.\u00a0 Jon stayed on in the Lingappa lab as a postdoctoral fellow until 2013, when he joined Prosetta Biosciences<\/a> and established the Prosetta satellite lab in Seattle.\u00a0 While at Prosetta, Jon served as Biology Lead and directed two different groups \u2013 one screening for anti-viral compounds that act on the subcellular ribonucleoprotein assembly machines that Jon studied while he was in the Lingappa lab, and one screening for anti-parasitic compounds that act on similar ribonucleoprotein complexes in Plasmodium<\/em>.\u00a0 In 2017, Jon returned to Jaisri Lingappa\u2019s lab at UW as a Senior Research Scientist to continue his studies of antiretroviral compounds in collaboration with Prosetta and to study the basic science of retroviral assembly.<\/p>\n As a PhD student in the Lingappa lab, Jon made a fundamental discovery that reshaped our understanding of HIV-1 virus assembly.\u00a0 He demonstrated that the intermediates (aka assembly machines) that are formed during assembly of the HIV-1 immature capsid are composed of cellular proteins found in RNA granules.\u00a0 RNA granules are important subcellular host ribonucleoprotein complexes involved in all aspects of RNA metabolism except translation, but had not been implicated previously in HIV-1 assembly.\u00a0 Jon also used siRNA knockdown approaches in HIV-1 expressing cells to show that DDX6, one of the host RNA granule proteins in the HIV-1 assembly intermediates, facilitates HIV-1 capsid assembly.\u00a0 In additional experiments, he showed that WT DDX6 rescued the knockdown phenotype, while an enzymatically inactive DDX6 mutant did not.\u00a0 He also confirmed the importance of DDX6 for facilitating assembly of infectious using siRNA knockdowns of primary blood mononuclear cells (PBMC).\u00a0 Jon\u2019s seminal PhD studies in support of a model in which HIV-1 Gag co-opts host RNA granules to form assembly machines were published in the Journal of Cell Biology in 2012, adding to growing evidence that HIV-1 capsid assembly in infected cells is host-catalyzed.<\/p>\n As a postdoctoral fellow, Jon developed RT-qPCR techniques for studying how the \u201cassembly machines\u201d facilitate packaging of the HIV-1 genome into newly assembled capsids. \u00a0These advances later led to a paper, published in PLOS Pathogens in 2018, showing that the RNA-granule-derived HIV-1 assembly intermediates also contain much of the genomic RNA found in the cytoplasm of HIV-expressing cells.\u00a0 Thus, by trafficking to these RNA granules, assembling HIV-1 Gag is able to access host facilitators of assembly (e.g. DDX6 and the host enzyme ABCE1) as well as the HIV-1 genomic RNA that it needs to encapsidate in order to produce virus that is infectious.<\/p>\n As a Senior Research Scientist in the Lingappa Lab, Jon demonstrated that the Gag protein of feline immunodeficiency virus (FIV), a non-primate lentivirus, forms RNA-granule-derived assembly intermediates containing ABCE1 and DDX6, as is the case for HIV-1.\u00a0 This study, published in the Journal of Virology in 2018, also demonstrated that the RNA-granule-derived FIV assembly intermediates contain additional RNA granule proteins, such as DCP-2 (as Jon showed the proximity ligation assay (PLA)), but do not contain the abundant ribosomal protein S6.\u00a0 Thus, these complexes resemble RNA granules but do not resemble ribosomes.\u00a0 Jon\u2019s most recent project describes a novel small molecule (PAV117), discovered in collaboration with Prosetta Biosciences, that inhibits replication of HIV-1 in T cell lines and primary T cells with nanomolar potency, most likely acting on HIV-1 assembly intermediates (manuscript in preparation).<\/p>\n Jon\u2019s contributions to the Lingappa Lab are myriad, and include writing manuscripts, editing grants, supervising junior employees, and developing new methods.\u00a0 He has piloted, optimized, and established a vast number of techniques in the Lingappa lab, including highly sensitive coimmunoprecipitation approaches, quantitative immunogold electron microscopic double labeling, quantitative proximity ligation assay approaches for studying subcellular localization of small molecules, siRNA knockdown and rescue, codon optimization, reverse transcription with quantitative PCR, northern blotting, infectivity assays and other assays for studying efficacy of antiviral small molecules including in primary cells.<\/p>\n <\/p>\n Search PubMed<\/a><\/p>\n Kevin Klein grew up in Madison, WI and received a BS with Distinction in Biochemistry from the University of Wisconsin. He completed his Ph.D. studies in the Lingappa lab and stayed on as a staff scientist. As a PhD student, Kevin was supported by an ARCS Foundation award and an NIH-funded Virology and Oncology training grant. While in the Lingappa lab, Kevin managed a soccer team called the Dominant Negatives and climbed Mt. Kilimanjaro (19,340\u2032) in Africa, Mt. St. Helens (8,365 feet), Mt. Baker (10,778 feet), and Mt. Rainier (14,411 feet).<\/p>\n In 2010, Kevin, a long-standing brewer, started his own nanobrewery,\u00a0Northwest Peaks<\/a>.\u00a0In 2011, he left the Lingappa Lab to focus full time on his brewery.\u00a0At Northwest Peaks, Kevin combines his passion for brewing and climbing peaks around Seattle by making new, creative \u201csmall batch\u201d beers every month, named after his favorite Pacific Northwest climbs.\u00a0In 2014, Kevin started The Bergschrund<\/a>, a great little tap room in Ballard that features Northwest Peaks beers.\u00a0 You can find the Lingappa lab there having \u201clab drinks\u201d on a fairly regular basis!<\/p>\n For his Ph.D. research, Kevin developed a cell-free system for assembling hepatitis C virus (HCV) capsids. At the time, there were no cell culture systems that supported assembly of HCV capsids. Even now, cell culture systems that support assembly of the most common genotypes of HCV are lacking. Kevin utilized this cell-free system to understand mechanisms of capsid formation of HCV and define HCV amino acids required for assembly. The HCV cell-free system established by Kevin is now being used by Prosetta Biosciences to screen for novel inhibitors of HCV assembly that could be used clinically. The success of the HCV cell-free assembly system also led to development of other cell-free assembly systems, including for assembly of alphaviruses.\u00a0 During his time in the Lingappa lab, Kevin\u2019s technical and conceptual insights contributed to numerous advances in our understanding of HIV-1 assembly and restriction.<\/p>\n <\/p>\n Search PubMed<\/a><\/p>\n As a rotation student in the Lingappa lab, Beth established a cell-free system for assembly of Venezuelan equine encephalitis virus (VEEV) capsids. This is the first cell-free system for assembly of an alphavirus capsid.\u00a0 For her Ph.D. dissertation, Beth studied how the enzymatic activities of the cellular restriction factors APOBEC3G and APOBEC3A are regulated in primary human cells.\u00a0 APOBEC3 family members, which are cellular proteins that have antiviral activity against HIV-1 and other viruses, are cellular deaminases.\u00a0 Most studies of APOBEC3 proteins are performed on cells that have been transfected to express APOBEC3 proteins.\u00a0 In contrast, Beth\u2019s studies focused on the enzymatic activity of APOBEC3 proteins expressed endogenously in primary human cells.\u00a0 To carry out these studies, she developed a quantitative, FRET-based high-throughput assay for detecting A3G deaminase activity.\u00a0 Using this assay, she demonstrated that A3G deaminase activity in human T cells is inhibited by an RNase-insensitive inhibitor (PLoS Pathogens 3(9):e135, 2007<\/a>).\u00a0 Beth went on to demonstrate that while APOBEC3 deaminase activity remains low in primary human T cells despite treatment with a variety of cytokines, APOBEC3 enzymatic activity in primary human monocytes and macrophages undergoes a dramatic increase upon treatment with interferon alpha or interferon-alpha-inducing TLR ligands\u00a0 (Journal of Biological Chemistry 285(36): 27753-27766, 2010<\/a>).\u00a0 In this paper, Beth used knockdown studies to demonstrate that the increase in deaminase activity in interferon alpha treated primary human monocytes is due entirely to induction of APOBEC3A, another member of the APOBEC3 family.\u00a0 She also showed that APOBEC3A enzymatic activity in primary human monocytes could be modulated by a variety of other signaling pathways.\u00a0 In contrast, APOBEC3A enzymatic activity could not be induced by interferon alpha in two different monocytic cell lines.\u00a0 Thus, in primary human T cells, monocytes, and macrophages, the enzymatic activity of APOBEC3 proteins is under the control of regulatory pathways that are not present in transformed cell lines typically used to study APOBEC3 proteins.<\/p>\n <\/p>\n Search PubMed<\/a><\/p>\n Julia Dooher grew up in Melrose, MA and received her BS in Biology, Magna cum Laude, from Tufts University. As an undergraduate, she worked in the laboratory of Tom Gilmore at Boston University, and was a recipient of an NSF undergraduate summer research award and Lemelson Fellowship from Hampshire College. As a PhD student in the Lingappa Lab, Julia received an\u00a0NSF graduate fellowship<\/a>, an ARCS Foundation award, a UW Magnuson Scholarship, and the 2005 UW Pathobiology Dept. Outstanding Student Award. Julia was awarded her PhD in Pathobiology in December 2005.<\/p>\n After leaving the Lingappa lab, Julia pursued postdoctoral research in\u00a0Alan Friedman\u2019s lab\u00a0at Johns Hopkins University (JHU) in Baltimore, MD.\u00a0 In 2011, she was awarded a\u00a0Christine Mirzayan Science & Technology Policy Fellowship<\/a>\u00a0from the Institute of Medicine at\u00a0The National Academies.\u00a0 In 2012, she was awarded an\u00a0American Association for the Advancement of Science (AAAS) Science & Technology Policy Fellowship<\/a>\u00a0in Washington, DC, where she focused on biosecurity.\u00a0 Julia is currently Senior Professional Staff at the Applied Biological Sciences Group in the Johns Hopkins University Applied Physics Lab.<\/p>\n While in the Lingappa lab, Julia spearheaded a number of new research directions.\u00a0 She demonstrated that Gag polypeptides of diverse primate lentiviruses associate with ABCE1<\/a> in primate cells, even though these Gag proteins have limited sequence homology. Julia also used apyrase, an enzyme that hydrolyzes ATP, to trap elusive capsid assembly intermediates, allowing such intermediates to be isolated from cells for the first time (Dooher and Lingappa 2004).<\/p>\n Julia also used pulse-chase labeling approach to demonstrate that in primate cells HIV-1 Gag enters ABCE1-containing assembly intermediates within minutes after synthesis and exits the assembly pathway just before virus maturation and release begins. Julia\u2019s pulse-chase studies of HIV-1 mutants suggest that when progression of Gag through ABCE1-containing assembly intermediates is slowed, virus release is also delayed; thus, viral-host interactions can affect overall virus production.\u00a0 Along with Bobbie Schneider at the FHCRC Electron Microscopy lab, Julia piloted immunogold labeling electron microscopy double labeling techniques to demonstrate that ABCE1-containing assembly intermediates can be found at the plasma membrane (Dooher, Schneider, Reed and Lingappa 2007).\u00a0 Subsequently, the Lingappa lab extended the immunoelectron microscopy techniques Julia initially developed to demonstrate that ABCE1-containing assembly intermediates are also present in the cytoplasm (Klein, Reed et al. 2011) and that assembly intermediates also contain the DEAD-box RNA helicase DDX6 and the Argonaute protein AGO2 (Reed et al. 2012).<\/p>\n At the end of her time in the Lingappa lab, Julia initiated a project in which she used Gag derived from a highly pathogenic HIV-2 isolate to demonstrate that polymorphisms in Gag that arise in vivo can lead to more rapid progression through the assembly pathway.<\/p>\n <\/p>\nResearch:<\/h5>\n
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Go to top<\/a><\/div>\nKevin Klein<\/h3>\n
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Go to top<\/a><\/div>\nBeth Thielen<\/h3>\n
![\"Beth](\"https:\/\/depts.washington.edu\/jaisril\/wordpress\/wp-content\/uploads\/2015\/09\/beth.img_assist_custom-180x205.png\")
Beth Thielen grew up in New Brighton, MN and received her B.S. in Microbiology, summa cum laude<\/em>, from the University of Minnesota.\u00a0 While at the University of Minnesota, she also studied health care policy at Denmark\u2019s International Study Program in Copenhagen and worked for 2 years as a certified emergency medical technician (EMT) for the University of Minnesota Emergency Medical Response Team.\u00a0 Beth joined the\u00a0Medical Scientist Training Program (MD\/PhD program) at the University of Washington\u00a0(UW) in 2003.\u00a0 After finishing her first two years of medical school, Beth pursued her Ph.D. studies in the Lingappa lab from 2005 – 2009. \u00a0During that time, she received support from the\u00a0CFAR STD\/AIDS training grant, a Poncin scholarship, and an ARCS (Achievement Rewards for College Scientists) award.\u00a0 She was awarded the Gilbert S. Omenn Award for Academic Excellence at UW in 2008.\u00a0 She defended her Ph.D. in 2009, obtained her M.D. in 2011, and is currently completing a Medicine Pediatric Residency at the University of Minnesota. \u00a0In July 2016, Beth will begin an Infectious Disease Fellowship at the University of Minnesota.<\/p>\nResearch:<\/h5>\n
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Go to top<\/a><\/div>\nJulia Dooher<\/h3>\n
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