RNA Biology of Host-Pathogen Interactions

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RNA Biology of Host-Pathogen Interactions

Friday, March 26, 2010

The New York Academy of Sciences

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Our improved understanding of the intimate details of the functional and molecular interface between microbial pathogens and their hosts is emerging as a crucial area of infectious disease research. This 1-day symposium will explore the role of small non-coding RNAs in host-pathogen interactions from the perspectives of both Pathogenesis and Immunobiology.

Presentations will discuss the RNA biology underlying 1) mechanisms utilized by pathogens of viral, bacterial and parasitic origin to subvert the host and secure their own replication and survival, and 2) the highly regulated response to foreign pathogens by the immune system.

Agenda


8:30 AM

Registration and Continental Breakfast

9:15 AM

Opening Remarks

SESSION I: RNAi in Hosts and Pathogens

Session Chair: Alla Grishok, PhD Columbia University

9:30 AM

siRNAs and piRNAs in Antiviral Immunity
Shou-Wei Ding, PhD, University of California Riverside

10:15 AM

Disruption of the RNAi Pathway Abolishes Antigenic Variation in the Intestinal Parasite Giardia lamblia
Hugo D. Luján, PhD, School of Medicine, Catholic University of Cordoba, Argentina

11:00 AM

Coffee Break

SESSION II: Virus/Host Interaction

Session Chair: Peter Palese, Mount Sinai School of Medicine

11:30 AM

Antiviral RNA Silencing in Drosophila
Sara Cherry, PhD, University of Pennsylvania

12:15 PM

Herpesviruses and microRNAs
Jennifer Umbach, Duke University

1:00 PM

Lunch

2:00 PM

Engineering Virus Vaccines through the Exploitation of microRNAs
Benjamin R. tenOever, PhD, Mount Sinai School of Medicine

2:45 PM

Epstein Barr Virus Modulation of Host MicroRNAs in B Cell Lymphomas
Olivia M. Martinez, PhD, Stanford University School of Medicine

3:30 PM

Coffee Break

Session III: Novel RNA-Based Regulation in Bacteria

Session Chair: Thomas Tuschl, Rockefeller University

4:00 PM

Small RNAs: Mediators of Gene Regulation and Silencing
Dinshaw Patel, Memorial Sloan-Kettering Cancer Center

4:45 PM

RNA Silencing in Prokaryotes: The CRISPR-Cas System
Michael P. Terns, University of Georgia

5:30 PM

Networking Reception

Speakers

Organizers:

Alla Grishok, PhD

Columbia University

Alla Grishok received her BSc degree from the Kiev State University in Ukraine. She conducted her Ph.D. research in the laboratory of Dr. Craig Mello where she studied the genetic pathway of RNA interference, RNAi inheritance and the connection between the RNAi and miRNA pathways in C. elegans. Alla completed her postdoctoral training with Dr. Phillip Sharp at MIT studying RNAi-induced transcriptional gene silencing and biological functions of RNAi in C. elegans. In July 2007 Alla became an Assistant Professor at the Department of Biochemistry and Molecular Bioshysics where her lab is investigating connections between RNAi and chromatin and biological roles of endogenous RNAi processes.

Thomas Tuschl, PhD

The Rockefeller University

Dr. Tuschl received his PhD in chemistry from the University of Regensburg, in Germany, in 1995. He went to the Max Planck Institute for Experimental Medicine in Goettingen, Germany, pursuing research with Fritz Eckstein. He next joined the biology department at the Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research, where he worked with Phillip A. Sharp and David P. Bartel. Dr. Tuschl was a junior investigator at the Max Planck Institute for Biophysical Chemistry before coming to Rockefeller in 2003 as associate professor. He was named professor in 2009.

Dr. Tuschl’s most recent honors include the Ernst Jung Prize for Medicine in 2008 and the Max Delbrück Medal and the Karl Heinz Beckurtz Award in 2007. In 2006 he received the Molecular Bioanalytics Prize from Roche Diagnostics. In 2005 he was named a fellow of the New York Academy of Sciences and received the Meyenburg Prize, the Irma T. Hirschl Trust Career Scientist Award and the Ernst Schering Award. In 2003 he received the Wiley Prize in Biomedical Sciences, the New York City Mayor’s Award for Excellence in Science and Technology and the Newcomb Cleveland Prize from the American Association for the Advancement of Science. Dr. Tuschl was the recipient of the European Molecular Biology Organization Young Investigator Award in 2001 and the Biofuture Award from the German government in 1999. He is also a Howard Hughes Medical Institute investigator.

Speakers

Sara Cherry, PhD

University of Pennsylvania

Dr. Cherry obtained her PhD with Dr. David Baltimore, where she studied VDJ recombination. She did her postdoctoral fellowship with Dr. Norbert Perrimon at Harvard Medical School, where she developed a new approach to study host factors that regulate viral pathogenesis. Specifically, Dr. Cherry coupled the powerful genetics of Drospohila with siRNA technology to identify cellular factors involved in viral replication. She started as an Assistant Professor at the University of Pennsylvania School of Medicine in 2006 where she also is the Associate Director of the Cell Based Screening Core. Since she began there she has continued to apply functional genomic approaches to the study of viral host interactions. In particular, her lab has identified new components of RNA silencing in Drosophila and mammals.

Shou-Wei Ding, PhD

University of California, Riverside

Dr. Ding is a Professor in the Department of Plant Pathology & Microbiology and the Institute for Integrative Genome Biology, University of California, Riverside. He received his PhD from the Australian National University, and subsequently completed postdoctoral training in University of Adelaide, Australia. He has previously served on the faculty of the Institute of Molecular Agrobiology at the National University of Singapore. Dr. Ding has active research programs funded by the NIH, USDA, and California Citrus Research Board to investigate viral immunity mechanisms of plants and animals and viral counter-defense strategies. His lab provided the first evidence for a natural antiviral role of RNAi in the animal kingdom in 2002 and participated in the identification of the first viral suppressors of RNA silencing. His teaching responsibilities at UC-Riverside include undergraduate and graduate courses in virology and RNAi. He is a fellow of the American Association for the Advancement of Science and has served on the Scientific Advisory Panel of Environmental Protection Agency. Dr. Ding is an editor of PLoS Pathogens and serves on the editorial boards of Virology and Journal of Virology.

Hugo D. Lujan, PhD

School of Medicine, Catholic University of Cordoba, Argentina

Dr. Lujan received his PhD in Biochemistry from the School of Chemistry, National University of Cordoba, in 1991. He did postdoctoral work in the United States at the National Institute of Allergy and Infectious Diseases, National Institutes of Health, until 1997. He is Full Professor in the Department of Biochemistry and Molecular Biology of the School of Medicine, Catholic University of Córdoba in Argentina. He also holds a position of Principal Investigator at National Council for Science and Technology of Argentina. He has won numerous awards, including the NIH Inventor Award in 1996 and 1999, and the National Ministries of Health and Education Awards in Argentina. Dr. Lujan is, for a second period, a Howard Hughes Medical Institute International Research Scholar. His work has focused in the last years on the molecular mechanisms of gene expression and on intracellular protein trafficking in the early-branching, intestinal parasite Giardia lamblia.

Olivia Martinez, PhD

Stanford University School of Medicine

Dr. Martinez received her PhD in Immunology from UC Berkeley. She did post-doctoral training at UC Berkeley and UCSF before joining the Faculty at UCSF. Dr. Martinez has been at Stanford University School of Medicine for 15 years and is currently Professor in the Department of Surgery/Division of Transplantation, a member of the Program in Immunology, and Chair of Immunology’s Pre-Doctoral Program. She serves on the Steering Committee of the Immunity, Transplantation, and Infectious Disease Institute at Stanford University School of Medicine. The research in Dr. Martinez’ lab focuses on 1) elucidating mechanisms of pathogenesis and immune evasion in Epstein Barr virus B cell lymphomas and 2) identifying strategies to induce transplant tolerance in solid organ and stem cell graft recipients. Dr. Martinez is author of over 75 original research publications and numerous reviews and chapters in the area of transplant immunology and B cell lymphomas. She serves on the editorial board of Transplantation, Pediatric Transplantation, Transplant Immunology and Virology: Research and Treatment. She is the Associate Editor of Digestive Diseases and Sciences journal. Dr. Martinez has served on numerous NIH advisory and study section committees. She is active in several professional organizations including the American Association of Immunologists where she has held many leadership positions and currently serves on the Public Policy Committee, and the American Society of Transplantation. She served as Director of the AAI Course in Advanced Immunology. In recent years she has been a recipient of the Distinguished Service Award from American Association of Immunologists and the Fujisawa Basic Science Award from the American Society of Transplantation.

Benjamin tenOever, PhD

Mount Sinai School of Medicine

Dr. tenOever completed his postdoctoral formation in Molecular Biology from Harvard University in 2007 after receiving his PhD in Virology from McGill University in 2004. In August of 2007, Dr. tenOever joined Mount Sinai School of Medicine as an Assistant Professor of Microbiology. His work focuses on the intricacies that govern a cell’s response to infection and the subsequent exploitation of that knowledge to generate novel strategies for vaccine and antiviral drug design. His research has been published in more than a dozen high impact journals including Science, Immunity, Nature Biotechnology and Cell. Dr. tenOever’s exceptional promise as a microbiologist has been acknowledged with a number of prestigious honors including becoming a Pew Scholar and receiving Presidential recognition (PECASE), the highest honor bestowed by the United States government on outstanding scientists beginning their independent careers.

Michael Terns, PhD

University of Georgia

Dr. Michael Terns received his BS degree in Biochemistry from The University of Michigan. He obtained his PhD degree in Pharmacology from the Pennsylvania State University, where he studied the mechanism of mRNA 3’ end formation and polyadenylation in Dr. Samson Jacob’s group. He did his postdoctoral training at the University of Wisconsin, where he studied small RNA trafficking and RNP assembly in the laboratory of Drs. James Dahlberg and Elsebet Lund. He is a full Professor in the Departments of Biochemistry and Molecular Biology, and Genetics at the University of Georgia. Together with his wife Dr. Rebecca Terns, he has established a dynamic and productive research program that has made important contributions to our understanding of the role of various non-coding RNPs in gene expression and human disease. In the area of cancer research, his work revealed that the function of telomerase (an enzyme that is key to the development of nearly all cancers) is controlled by regulated intracellular trafficking of the enzyme in cancer cells. In other studies, his group delineated the essential components and architecture of the H/ACA RNP, a complex enzyme that functions in RNA-guided RNA modification. This work led to identification of a structural domain associated with development of dyskeratosis congenita in humans. Most recently, the Terns group identified an effector complex of the emerging CRISPR-Cas system (a prokaryotic RNA silencing pathway that provides protection against viruses and other invaders) and demonstrated that it functions by RNA-guided cleavage of target RNAs.

Jennifer Umbach, PhD

Duke University

Jennifer L. Umbach graduated with a BS from Carnegie Mellon University. She completed her PhD in the Cullen lab at Duke University where she continues to work as a post-doctoral fellow.


*Additional speaker biographies coming soon.

 

Abstracts

SESSION I: RNAi in Hosts and Pathogens

siRNAs and piRNAs in Antiviral Immunity

Shou-Wei Ding, PhD, University of California Riverside

Drosophila melanogaster produce siRNAs, miRNAs and PIWI-interacting RNAs (piRNAs) in three distinct genetic pathways to regulate gene expression and development. Infection of D. melanogaster with diverse positive-strand (+) RNAviruses induces Dicer2-deppendent production of virus-derived siRNAs predominantly 21 nucleotides in length, which are loaded in Argonaute-2 and methylated at the 3’-ends. Mutant fruit flies carrying loss-of-function mutations in any of the key siRNA pathway genes such as Dicer-2, R2D2 and Argonaute-2 display enhanced disease susceptibility to distinct +RNA viruses. We have recently developed a culture-independent approach for virus discovery and shown that infection of three distinct dsRNA viruses in Drosophila cells all triggered production of viral siRNAs with features similar to siRNAs derived from +RNA viruses. Notably, we found that after infection with +RNA or dsRNA viruses, Drosophila ovary somatic sheet cells which express AGO1 and AGO2 as well as PIWI, produced an additional population of virus-derived small RNAs that exhibit features characteristic of primary piRNAs, suggesting a novel function of piRNAs in viral immunity.

Disruption of the RNAi Pathway Abolishes Antigenic Variation in the Intestinal Parasite Giardia lamblia

Hugo D. Lujan, PhD, School of Medicine, Catholic University of Cordoba, Argentina

Giardia, a parasitic protozoan of humans, is a major source of waterborne diarrheal disease worldwide. Giardia is also an excellent system to study the evolution of cellular processes since it belongs to the earliest branch of the eukaryotic lineage. Giardia trophozoites undergo fundamental changes to survive outside the intestine of their host by differentiating into infective cysts. Encystation entails the synthesis, processing, transport, secretion and extracellular assembly of cyst wall components. To survive within the intestine, Giardia go through antigenic variation, a process by which the parasite continuously switches its major surface molecules, allowing the parasite to evade the host’s immune response and produce chronic and recurrent infections. The main goal of our laboratory is to better understand the basic molecular mechanisms involved in Giardia adaptation and differentiation. Regarding antigenic variation, one variant-specific surface protein (VSP) is expressed on the surface of each trophozoite at a particular point in time, from a repertoire of approximately 190 vsp genes present in the parasite’s genome. Here, we show that vsp expression is regulated by a mechanism that involves both RNA-dependent RNA polymerase (RdRP) and Dicer homologs, known components of the RNA interference (RNAi) pathway. Unlike Plasmodium falciparum and Trypanosoma brucei, where transcriptional silencing of non-expressed VAR and VSG genes occurs, Giardia clones transcribe many vsp mRNAs but only accumulate transcripts encoding the single surface antigen that is expressed on the parasite’s surface. Detection of antisense RNAs corresponding to the silenced vsps and small RNAs from the silenced but not for the expressed vsp implicate the RNAi pathway in antigenic variation. Additional findings show that an epigenetic process, involving particular histone modifications, play a role in the variable expression and, therefore, on the concentration of individual VSP transcripts, which, in turn, influence the selection of the VSP that can circumvent the silencing process and be expressed on the trophozoites surface. Remarkably, knockdown of Dicer or RdRP leads to a change from single to multiple VSP expression in individual trophozoites. These results demonstrate the involvement of a post-transcriptional gene silencing (PTGS) mechanism in regulating the expression of surface antigens in Giardia. Infection of experimental animals with these multiple VSPs-expressing trophozoites protect them to subsequent infections with individual clones, providing direct evidence regarding the importance of variable surface antigens of pathogens in the establishment of the infection and on evading the host immune response. Our results also indicate that the manipulation of the mechanisms of antigenic variation in Giardia, and potentially in other human parasites, could facilitate the development of vaccines against important pathogens.

SESSION II: Virus/Host Interaction

Antiviral RNA Silencing in Drosophila

Sara Cherry, PhD, University of Pennsylania

Innate immunity is the most ancient line of defense against pathogens. Recent studies have identified RNA interference (RNAi) as an ancient, cell-intrinsic immune mechanism that restricts RNA viruses in plants and insects. Using high-throughput RNAi screening in Drosophila cells for factors that when lost, lead to increased replication of Vesicular Stomatitis Virus. Using this approach we found that in addition to the canonical components of the siRNA pathway (Dcr-2, Ago2), we identified Ars2 and its binding partners Cbp20 and Cbp80 as novel proteins having antiviral activity against a battery of RNA viruses in both Drosophila cells and adult flies. Mechanistically, we found that Ars2 interacts with Dcr-2 to facilitate cleavage of dsRNA precursors. Previous studies have suggested that viral dsRNA intermediates are recognized and cleaved by Dcr-2 to generate viral siRNAs. This viral RNA represents the Pathogen Associated Molecular Pattern (PAMP) while Dcr-2 serves as the Pattern Recognition Receptor (PRR). Whether the viral PAMP is the same across diverse viruses and whether factors such as Ars2 modulate the specificity of targeting remains unclear. Therefore, to determine the viral PAMPs targeted by Dcr-2 and whether Ars2 plays a role in this we have embarked on the deep sequencing of virus-derived small RNAs generated during infection by a battery of human arboviruses. Our data show that different viruses can be targeted by disparate mechanisms suggesting that Dcr-2 interacts with distinct viral PAMPs perhaps through a variety of specificity factors. Lastly, by continuing to use RNAi screening approaches we have identified additional genes that are both antiviral and involved in RNA silencing. These findings will be discussed.

Herpesviruses and microRNAs

Jennifer Umbach, PhD, Duke University

Herpesviruses encode far more microRNAs (miRNAs) than any other virus family. As a result, there has been great interest in elucidating the roles these miRNAs play in the virus life cycle, particularly given the long-term, latent infections that these viruses establish with their hosts. The diverse roles that KSHV, EBV and HSV miRNAs play in regulating oncogenesis, apoptosis and the expression of viral genes will be discussed.

Engineering Virus Vaccines through the Exploitation of microRNAs

Benjamin R. tenOever, PhD, Mount Sinai School of Medicine

MicroRNAs (miRNAs) are endogenously expressed, short non-coding RNAs that directly inhibit translation of complimentary mRNA targets, and function in concert to globally modulate cellular protein levels. The expression pattern of miRNAs, like all cellular transcripts, can be ubiquitous or demonstrate tissue, lineage, and even species-specificity. By exploiting the diverse expression patterns of miRNAs, we have developed a novel attenuation strategy that harnesses the host cellular small RNA machinery, and utilizes it in an antiviral capacity. Incorporation of perfect, or near-perfect, miRNA-complementary sites into viral mRNA confers antiviral activity upon encountering endogenously expressed miRNAs, effectively restricting viral growth in a miRNA-specific manner. We have successfully generated various influenza viruses encoding diverse miRNA targets, allowing us to modulate both the degree of attenuation as well as the tropism of the virus. We have utilized this technology to study the in vivo function of individual influenza virus transcripts to ascertain the roles these proteins perform in specific cell populations and to extrapolate how these functions relate to viral pathogenicity. Similarly, we have expanded these studies to include the development of high yield egg-grown vaccines through the incorporation of mammalian-specific, ubiquitous miRNA targets. These studies demonstrate that viral manipulation, through the exploitation of miRNAs, has significant potential in controlling and understanding influenza virus biology.

Epstein Barr Virus Modulation of Host MicroRNAs in B Cell Lymphomas

Olivia M. Martinez, PhD, Stanford University School of Medicine

Epstein Barr Virus (EBV) is a B lymphotrophic gammaherpes virus that has infected over 90% of the population. In most cases, infection with EBV is asymptomatic, however, EBV is also associated with a variety of human malignancies including nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s disease, and B cell lymphomas in AIDS patients or immunosuppressed transplant recipients. Our lab has studied the mechanisms by which EBV promotes survival of infected B cells and evasion of the host immune response. Here we examined the possibility that modulation of host cell microRNA by EBV may play a role in these processes. Comparison of microRNA expression in a panel of EBV-negative human B cell lymphomas, and their EBV-positive counterpart, revealed that latent EBV infection modulates cellular microRNA involved in cell cycle regulation. A similar pattern of microRNA expression was observed in EBV+ B cell lines from patients with post-transplant lymphoma. Further, signaling by the EBV-encoded protein, latent membrane protein 1 (LMP1) was sufficient to induce host cell microRNA expression. Ongoing studies are examining the regulation of microRNA expression by LMP1 and the function of specific host cell microRNA in B cell lymphomas.

Session III: Novel RNA-Based Regulation in Bacteria

RNA Silencing in Prokaryotes: The CRISPR-Cas System

Michael P. Terns, University of Georgia

The CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive RNA-based prokaryotic immune system arises from clustered regularly interspaced short palindromic repeats (CRISPRs), which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes found in prokaryotic genomes. Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in the CRISPR-Cas genome defense pathway. We describe the biogenesis of small RNAs from the CRISPR loci. The Cas6 protein “dices” CRISPR transcripts to generate individual invader-targeting RNAs. The mature RNAs include a signature sequence element called the “psi-tag”. We have also identified a CRISPR-Cas effector complex comprised of mature CRISPR RNAs and the RAMP module (or Cmr) Cas proteins. The complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral guide RNAs. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.

*Additional abstracts coming soon.

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