Sara Cherry - US grants

The ability to clear pathogens is critical to survival. During evolution, two defense strategies to deal with microorganisms have developed: the innate and the aquired immune systems. Drosophila is a perfect model system to study innate immunity. It is genetically manipulable and several pathways involved in mammalian immune functions are highly conserved and activated in response to immune challenge. The immune response to pathogens in Drosophila leads to the appearance of "melanotic tumors" that are the result of blood cell encapsulation of pathogens. Melanotic tumors can also be caused by an autoimmune response or hyperproliferation of blood cells. Therefore, the goals of this proposal are. 1) to conduct a genetic screen for mutations associated with melanotic tumors. 2) characterize the mutants phenotypically and genetically. In particular their effects on NFkB, Jak/Stat, and Jnk signalling will be analyzed. 3) to clone mutants isolated in the screen. We will identify genes involved in both immunity and growth control.

DESCRIPTION (provided by applicant): Innate immunity is the first line of defense against pathogens and is highly conserved from insects to humans. While many key facets of the innate immune system have been elucidated, there is a fundamental gap in our knowledge of the pathways that restrict arthropod-borne viral infections both in the insect vector and the mammalian host. Importantly, a molecular understanding of these mechanisms is essential to overcome the lack of effective antiviral therapeutics and combat human disease. The Drosophila immune response is highly homologous to that of vector insects and additionally shares striking similarities with mammalian innate immunity. Thus, discoveries in fruit flies may have profound impacts on human health by identifying novel approaches to interfere with disease transmission in vector species and revealing conserved immune pathways in mammals. One essential pathway that restricts the human arbovirus Vesicular Stomatitis virus (VSV) in Drosophila is autophagy, an ancient intracellular degradative program evolutionarily conserved from yeast to humans. Based on preliminary data, this response depends on the interaction between VSV and a previously uncharacterized Toll receptor, Toll-7; however, the downstream pathways and global significance of Toll receptors remain unknown. The long-term objective of this application is to define the molecular mechanisms by which Drosophila recognizes viral infections to induce protective antiviral autophagy. Because these pathways share a number of molecular and functional similarities to mammalian TLR pathways, and increasing in vitro data suggest that TLRs activate autophagy during a variety of infections, the proposed research also has the potential to extend our understanding of the mechanisms that link innate immunity to autophagy in mammals. As such, this application proposes three specific aims: (1) elucidate the mechanism by which virus is targeted by autophagy; (2) identify the role of Toll receptors in antiviral autophagy and defense; and (3) explore the signaling pathways that link virus recognition to autophagy in flies and mammals. These studies will expand our understanding of the molecular mechanisms underlying antiviral autophagy in flies, as well as conserved autophagy-inducing pathways likely downstream of TLR signaling in mammals. Moreover, this research opens a previously unappreciated and unexplored realm of Toll receptors in immunity, which may serve as effective targets for novel vector-based antiviral therapeutics given their conservation in other insects.

Animals, Domestic; Antiviral Agents; Antiviral Drugs; Antivirals; Applications Grants; Bunyavirus; Categories; Cells; Development; Domestic Animals; Drosophila; Drosophila genus; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Epidemic; Fruit Fly, Drosophila; Generalized Growth; Genes; Genetic Screening; Genome; Goals; Grant Proposals; Grants, Applications; Growth; High Throughput Assay; Host Factor; Host Factor Protein; Human; Human, General; Immune; Immunity; Individual; Infection; Integration Host Factors; Man (Taxonomy); Man, Modern; Methods and Techniques; Methods, Other; Microscopy; Modeling; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Orthobunyavirus; Pathogenesis; Pathway interactions; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNA, Small Interfering; RNAi; Receptors, Virus; Research; Resistance; Rift Valley fever virus; Screening procedure; Sequence-Specific Posttranscriptional Gene Silencing; Small Interfering RNA; System; System, LOINC Axis 4; Techniques; Technology; Testing; Therapeutic; Tissue Growth; Viral; Viral Receptor; Virus; Virus Receptors; Virus Replication; Viruses, General; arboviral disease; arbovirus disease; cohort; domesticated animal; fruit fly; high throughput screening; novel; ontogeny; pathogen; pathway; resistant; screening; screenings; siRNA; virus multiplication; virus pathogenesis

The emergence of epidemic arboviral diseases infecting both humans and domestic animals has led to significant world-wide morbidity and mortality. Little is known about the host factors required for the replication cycles of these viruses, and less about the innate immune pathways that restrict pathogenesis, impeding the development of antiviral treatments. The identification of cellular factors involved in viral replication and pathogenesis has been difficult due to the lack of virus-host systems amenable to genetic screening. Our primary research goal is to use a high-throughput RNAi and small molecule screens in Drosophila and mammalian cells to identify inhibitors of both cellular and viral factors that regulate pathogenesis, including both those factors hijacked by the virus for replication, and those systems used by the host to combat the viral invader. Our studies will be focused on Rift Valley Fever Virus (RVFV, a Category A bunyavirus), but will be extended via collaborations within MARGE to other bunyaviruses of medical significance. RVFV is of particular interest due to its unmet medical and agricultural need, its identification by the USDA as a likely agroterrorism agent, the availability of reagents and experimental systems, and because of the opportunity to collaborate with members of the MARCE. Our cell-based screening approach using replication competent virus enables the identification of compounds or genes that target any factor¿viral or cellular¿that is required for viral replication. Furthermore, our method has the ability to elucidate the mechanism of this requirement by dissecting viral replication with secondary assays against each step in the lifecycle. The simultaneous application of genome-scale RNAi screens against RVFV and small molecule screens, both using a 384 well plate-based high throughput screening assay (HTS) that we have developed, should lead to more rapid identification of cellular targets for antiviral drug development for this medically important virus, as well as for other members of this under-studied virus family.

DESCRIPTION (provided by applicant): Alphavirus interactions with cellular receptors are likely to play a major role determining viral tropism and driving virus-induced disease. However, the viral receptors that mediate alphavirus entry are poorly understood. A genome wide screen using Sindbis virus identified NRAMP as a potential alphavirus entry receptor in insect cells. Additional studies suggest that NRAMP is also an entry receptor for Venezuelan Equine Encephalitis virus. Furthermore, in mammalian cells the ubiquitously expressed homolog, NRAMP2, can mediate infection of these alphaviruses. Our long-term goal is to dissect the role of NRAMPs in infectivity and pathogenesis of alphaviruses with the goal of developing strategies to combat these pathogens. We will study this important interaction from both the perspective of the viruses and their varied hosts, including studies to determine whether additional alphaviruses use NRAMP as a receptor and to define the regions of the viral glycoproteins that mediate NRAMP binding. Since mammals have two homologous genes, NRAMP1 and NRAMP2, we will also test whether NRAMP1 and NRAMP2 can functionally substitute for one another as alphavirus entry receptors and test whether one or both of these molecules is required for alphavirus infection in primary cells from diverse lineages and in vivo. We will take advantage of powerful assays that we have developed to study alphavirus entry and infection both in insect and mammalian cells and extend these studies to mice and adult flies to explore the role of NRAMPs in viral pathogenesis and spread. Our central hypothesis is that dissecting the interactions of these medically important arboviruses with their receptor will reveal mechanisms that will aid in the development of antiviral treatments against these understudied pathogens for which there are no vaccines or therapeutics.

DESCRIPTION (provided by applicant): Alphavirus interactions with cellular receptors are likely to play a major role determining viral tropism and driving virus-induced disease. However, the viral receptors that mediate alphavirus entry are poorly understood. A genome wide screen using Sindbis virus identified NRAMP as a potential alphavirus entry receptor in insect cells. Additional studies suggest that NRAMP is also an entry receptor for Venezuelan Equine Encephalitis virus. Furthermore, in mammalian cells the ubiquitously expressed homolog, NRAMP2, can mediate infection of these alphaviruses. Our long-term goal is to dissect the role of NRAMPs in infectivity and pathogenesis of alphaviruses with the goal of developing strategies to combat these pathogens. We will study this important interaction by dissecting the determinants on NRAMPs that mediate binding and entry. Furthermore, we will sequence NRAMP from different enzootic and epizootic hosts to determine whether polymorphisms impact virus infection. We will take advantage of powerful assays that we have developed to study alphavirus entry and infection both in insect and mammalian cells to explore the role of NRAMPs in viral infection. Our central hypothesis is that dissecting the interactions of these medically important arboviruses with their receptor will reveal mechanisms that will aid in the development of antiviral treatments against these understudied pathogens for which there are no vaccines or therapeutics.

? DESCRIPTION (provided by applicant): Abstract In response to a ground-roots faculty effort, the University of Pennsylvania School of Medicine (Penn) is establishing a new Screening Core in 2014. The mission of the Core is to facilitate broadly the use of high-throughput screening approaches as a mechanism to drive innovative research for Penn investigators. The ability to efficiently and economically screen small molecules and genetic libraries including siRNAs, shRNAs, miRNAs and cDNA libraries will undoubtedly reveal new targets for interventions. A cornerstone of this Core is the Janus Workstation liquid handling delivery robot, which is essential to the Core function and mission. The Core is overseen by Dr. Sara Cherry (Scientific Director) and will be managed by a Technical Director (TBN). Penn will provide all other necessary resources for start-up and operation. Users will be charged a fee for instrument use and funds generated will be cycled back into the Core operations. The Core will thus coalesce all of the equipment required for high throughput screening and will contribute to the research of the 4 major and 15 minor users, each of whom are NIH-funded - with a total of 55 grants and $21M in support overall. Use of this equipment will reveal new insights into biology, generate new biological tool compounds, and directly advance translational medicine at Penn.

This application represents the third competing renewal for our Training in Emerging Infectious Disease (EID) Training program based at the University of Pennsylvania that supports 2 predoctoral and 2 postdoctoral trainees per year. Of the more than 70 faculty who are affiliated with the Penn microbiology program, a select group of 14 faculty are trainers with this EID T32. All of the trainers have significant EID research programs involving parasites, bacteria, and viruses. An active Executive Committee coupled with an experienced Internal Advisory Committee insures that this program retains a very tight focus on the study of emerging and re-emerging pathogens. As a result, trainers have been dropped from the program when their EID programs have faded while others have been added. This program has served to coalesce EID research training on our campus, with our trainers instituting a popular EID lecture course, a rigorous BSL3 training program, and a Certificate in Public Health Program. We now use Individual Development Plans for our students and postdocs which makes it possible for us to tailor our training activities to best meet the needs of our trainees. Importantly, Penn continues to provide significant, direct support to training activities, including $7 million for our ABSL3/BSL3 and via supporting predoctoral trainees for their first 21 months of graduate school. Thus, our T32 supports trainees only after they have completed all coursework and their prelim exams. Thus far, this T32 has supported 39 trainees, almost all for 2 years each, including 19 Ph.D. students, 4 M.D./Ph.D. students, and 16 Ph.D. postdoctoral fellows. Our 39 trainees have worked in the labs of 18 different trainers. Of the 39 current and past trainees, 20 are women, 19 are men, and 7 are minorities/disadvantaged (18%). Our retention rate is 92%, and those who have completed training and left Penn have obtained good positions (for the postdocs) and excellent postdoctoral positions at leading institutions, with most continuing to study emerging infectious agents. The accomplishments of our trainees coupled with their career progress since leaving Penn shows that our T32 program is indeed training promising young scientists to enter careers in EIDs.

? DESCRIPTION (provided by applicant) Enteric pathogens, which represent a major group of disease-causing agents, must overcome barrier immunity within the intestinal environment to infect a host. To counter this, the gastrointestinal tract has evolved as a physical and immunological barrier. Moreover, many enteric viruses infect intestinal epithelial cells which are both targets of and act as sentinels for infection. Increasing evidence suggests that epithelial cells sense infection directly to induce antimicrobial pathways. It is also clear that the microfloa within the intestinal tract plays a fundamental role in immunity and that imbalanced bacterial communities have detrimental consequences to immune defense. Aging animals present with immune deficiencies and an altered microbiome. Mechanistically, which microbes or microbial ligands, mediate these effects and how they impact antiviral immunity is unclear. To overcome our gap in knowledge of the molecular mechanisms that control enteric viral infection, we developed an oral model of arboviral infection using the powerful genetic model organism, Drosophila melanogaster. We found that the gut presents a high barrier to infection: young wild type flies are refractory to oral challenge with enteric viruses, while inoculation into the body cavity, which bypasses the gut, results in robust infection. Importantly, we found that the ERK pathway is activated in the intestinal epithelium by infection, and that genetic depletion of the ERK pathway only in the intestinal epithelium leads to increased infection. Furthermore, we found that the microbiota plays an important role in susceptibility to infection. In young flies, te loss of commensals in the gut led to increased permissivity to viral infection, suggesting that signals from the microbiota impact innate signaling. However, we found that older flies that present with dysbiosis are more susceptible to oral viral infection. Under these conditions ablation of the commensals protects the animals from enteric viral infection. Therefore, the microbiota, depending on its composition, can either be protective or detrimental for antiviral defenses. In this proposal we will address fundamental questions in antiviral immunity by determining the mechanisms by which the microbiota and epithelial cells control immunity in the intestine against human viruses, using a genetically tractable organism with a highly manipulable microbiome and short lifespan.

Flaviviruses are a genus of related positive-stranded enveloped RNA viruses that significantly impact human health, including dengue (DENV), Zika (ZIKV). West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. The discovery of host factors critical for viral infection reveals new aspects of cell biology, intricate virus-host relationships, and potential targets for antiviral therapeutics. After entering cells and fusing in the acidified endosome, the flavivirus RNA genome penetrates into the cytoplasm and is then translated into a polyprotein and processed at the endoplasmic reticulum (ER). In addition to utilizing many functions of the ER for protein production, flaviviruses extensively remodel ER membranes to create a niche for RNA- dependent RNA replication. The ER also is the site for flavivirus assembly, which enables the production and secretion of new infectious viruses. Thus, the ER serves as a central point for orchestrating many of the essential steps in the flavivirus infection life cycle. Despite this, little is known about the host factors and molecular mechanisms at the ER that are required for optimal translation and processing of the viral proteins or for the assembly of the replication niche. We recently have performed several genetic screens to identify important components in this process. Our genome-wide RNAi screen with WNV in insect cells validated 18 genes associated with ER biology that promote infection. Our CRISPR/Cas9 gene-editing screen in human cells with WNV also identified 12 ER-associated genes. These screens converged on ER-resident proteins as being critical for WNV infection and included genes associated with ER-translocation and signal peptide processing, ER-associated degradation (ERAD), protein glycosylation, protein folding and lipid metabolism. Indeed, infection of WNV, ZIKV, JEV, DENV, and YFV all required specific subunit components of the host signal peptidase complex (SPCS) for processing of the viral polyprotein, the production of viral glycoproteins and thus generation of nascent virions. The objective of this proposal is to define the molecular mechanisms by which flaviviruses use specific ER-associated host proteins to promote viral translation, polyprotein processing, RNA replication, and/or assembly. Aim 1 will define the mechanism by which the ER translocon promotes polyprotein translation and processing while Aim 2 will dissect the role of ER-associated decay (ERAD) in promoting flavivirus replication. Our long-term goal is to determine the mechanisms by which flaviviruses exploit the ER for their replication, as this will reveal both fundamental aspects of virology as well as new avenues for antiviral therapeutics.

Enteric pathogens represent a major group of disease-causing agents, and must overcome the physical and immunological barrier of the gastrointestinal tract. The resident microbiota presents with a large array of ligands and pathogen-associated molecular patterns (PAMPs) which can prime immune defenses, through pattern recognition receptors (PRRs), both on enterocytes and immune resident cells. Indeed, microbial-derived TLR ligands are necessary for the development and maintenance of the intestinal barrier and immune homeostasis. Moreover, the microbiota is not static and imbalanced bacterial communities, termed dysbiosis, impact immunity, in particular during aging. Aging is associated with increased susceptibility to enteric pathogens, and how the dysbiotic microbiota alters susceptibility is largely unknown. The complement of microbial-derived ligands that are sensed and that can prime antiviral immunity is incomplete. A better understanding of the molecular mechanisms by which immunity is maintained, how the microbiota and epithelia interact, and how this impacts infection and pathogenesis has the potential to reveal novel strategies to treat enteric viral infections. Studies exploring the role of the microbiota and host genes in the context of aging in enteric infections are challenging in small animal models due to costs and technical hurdles. To overcome our gap in knowledge of the molecular mechanisms that control enteric viral infection, we developed an oral model of infection using the powerful genetic model organism, Drosophila. We found that the gut presents a high barrier to infection: young wild type flies are refractory to oral challenge with human viruses, while inoculation into the body cavity, which bypasses the gut, results in robust infection. The spectrum of antiviral pathways engaged in the gut, and how the microbiota shapes immunity in the intestine is incompletely understood. Preliminarily, we found that Drosophila STING controls infection in the intestine; dSTING mutants are more susceptible to enteric viral infection. STING is known to be activated by cyclic dinucleotides (CDNs). While cGAS can produce CDNs endogenously, STING can also be activated by bacterially derived CDNs. This led us to explore the possibility that commensal bacteria-derived CDNs may impact innate defenses in the gut through STING, as it is known that microbiota-derived CDNs are present in the gut. Our new data identifies a role for microbiota-derived CDNs in antiviral defense. Ablation of the microbiota in young animals leads to increased infection, and feeding these microbiota-deficient flies CDNs was protective. In Aim 1 we will define the role of dSTING in antiviral defense and in Aim 2 we will define the role of commensal-derived CDNs in antiviral defense in young and old animals.

Summary Chikungunya virus (CHIKV) is an emerging arthropod-borne virus for which there are no vaccines or therapeutics. Infection with CHIKV can lead to severe and chronic arthralgia. CHIKV displays wide tropism targeting the epithelial, endothelial, and myeloid compartments. The innate immune system is the first line of defense. Viral nucleic acids are sensed, leading to the activation of classical type I interferons (IFN) which induces hundreds of interferon stimulate genes (ISGs). In response, viruses including CHIKV, encode antagonists of interferon signaling to attenuate these antiviral activities. In addition, there is a growing list of basally expressed, effector proteins known as intrinsic factors, which play roles in controlling infection. Altogether, it is clear that there are additional antiviral regulators that play important roles in controlling infection, and in particular, there is less known about antiviral responses in non-hematopoietic cells which are the major targets of CHIKV. Moreover, while much of our understanding of antiviral effectors has been focused on antiviral proteins, there is an emerging literature that suggests that noncoding (nc)RNAs can also play an important role in controlling infection. Long noncoding RNAs (lncRNAs) are an emerging and abundant subclass of ncRNAs that are known to dynamically regulate transcription and translation and can control innate immune responses. The contribution of lncRNAs to innate defense downstream of viral infection is poorly understood, and there is nothing known during CHIKV infection. We hypothesize that lncRNAs play a regulatory role in the control of innate antiviral immunity against CHIKV. Therefore, we will characterize the role of lncRNAs in the innate immune response to CHIKV infection. We are beginning our studies in endothelial cells, as these are a target during infection and little is known about antiviral defenses in this tissue. In addition, we are particularly interested in cytoplasmic lncRNAs as there is less known about their functions, and CHIKV replication occurs exclusively in the cytoplasm. To achieve these goals, our specific aims will (1) Characterize lncRNAs that control CHIKV infection and (2) define the mechanism of action of one antiviral lncRNA we have validated (chikvLNC)

Summary The current outbreak of the coronavirus in China (SARS-CoV-2) has spread rapidly. There are experimental drugs which will be tested; however, there are no approved therapeutics or vaccines. Indeed, there are tests underway to determine whether remdesivir, which was developed against filoviruses, can be repurposed against SARS-CoV-2 infection. It would be transformative if we could identify additional small molecules that could be repurposed to treat the outbreak of SARS-CoV-2 infection. Given that the goal of the parent grant (R01AI150246) is to discover antivirals active against bunyaviruses, based on findings from cell based screening, and that we have broad expertise in diverse viruses, we are applying for Supplemental funding (notice number NOT-AI-20-030, PA-18-035) to expand the scope of the existing grant to use the same methods (small molecule screening) to identify antiviral therapeutics active against SARS-CoV-2 infection. We will screen two libraries of known bioactives to potentially repurpose existing therapeutics. First, we will test a library of innate immune agonists (~100 PAMPs) for their ability to block SARS-CoV-2 infection in human cells including airway cells. Second, we will screen another ?actionable? library that I have created as the Director of the High-throughput Screening core at UPENN. We created a library of ~3000 drugs that includes ~1500 FDA approved compounds, ~1000 drugs in clinical trials and the remaining drugs have known targets. This library has been used for repurposing (as is being done with the Gilead drug remdesivir that was originally developed against filoviruses) to more rapidly identify active therapeutics for future testing in humans. We will also determine if any of our active antivirals act synergistically with remdesivir since this drug is currently under development for use against COVID-19. We expect to identify additional drugs with activity against SARS-CoV-2.

Summary Innate immunity is the first line of defense against pathogens and is highly conserved from insects to humans. While many key facets of the innate immune system have been elucidated, there is a fundamental gap in our knowledge of the pathways that restrict arthropod-borne viral infections in the diverse target tissues that are infected during the infection of the mammalian host. Importantly, a molecular understanding of these mechanisms is essential to overcome the lack of effective antiviral therapeutics and combat human disease. The Drosophila immune response is highly homologous to that of vector insects and additionally shares striking similarities with mammalian innate immunity. Using the Drosophila system, we previously found that the emerging bunyavirus Rift Valley Fever virus (RVFV) is sensed by the Drosophila Pattern recognition receptor (PRR), Toll-7, which activates antiviral autophagy and that this is conserved in mammalian cells. We found that TLR2-dependent antiviral autophagy can control RVFV in some cell types while in other cells engagement of TLR2 leads to cell death. Moreover, we found that pharmacological activation of autophagy is restrictive against RVFV in mammalian primary neurons, suggesting that this pathway may be harnessed for antiviral protection. Since tissue-specific signaling of PRR pathways are poorly characterized we screened a panel of PRR agonists for those that could block RVFV infection in neurons and in non-neuronal cells in parallel. We identified two classes of antiviral PAMPs. First, we identified TLR2 agonists as antiviral in both cell types. Since pharmacological activation of autophagy can protect primary mammalian neurons from infection, we suggest that TLR2 activation may be harnessed to defend neurons from encephalitic viruses. Second, we identified Dectin-1 agonists as specifically antiviral in neurons which will be further explored. Therefore, the long-term objective of the proposed research is to understand the molecular mechanisms by which viral infections are sensed and controlled by innate pathways and how this may be harnessed to induce protective defenses in diverse cell types including neurons. To accomplish these goals, this application proposes two specific aims: (1) to identify the mechanism by which RVFV is sensed by TLR2 leading to diverse outcomes, autophagy or cell death; and (2) explore the PRR pathways that can control bunyaviruses in mature mammalian neurons.