Anne E. Simon - US grants

0086952 Anne Simon
Although RNA viruses have been the subjects of intensive investigations for the past 100 years, replication, a fundamental process in the life cycle of a virus, remains poorly understood. The viral-encoded enzyme involved in replication, the RNA-dependent RNA polymerase (RdRp), together with possible proteins from the host, must recognize specific viral RNAs, initiate complementary strand synthesis and then use these complementary strands as templates for synthesis of the viral genome. Promoters for RdRp are poorly understood, and much of the research to date involves viruses terminating with either tRNA-like structures (e.g., Brome mosaic virus) or poly(A) tails (e.g., Poliovirus). Much less is known about viruses whose genomic RNA(s) terminate with a free hydroxyl group such as turnip crinkle virus (TCV). In addition, it is only recently becoming clear that internal sequences, and not just end sequences, are important for initiation of transcription by RdRp. Possible roles for such sequences are (i) RdRp-binding sequences, or (ii) sequences that engage in RNA-RNA interactions with end sequences to bind the RdRp to the viral genome. TCV is used as a model to study sequences and structures involved in replication since it is among the smallest and simplest of the RNA viruses. More importantly, TCV is associated with very small subviral RNAs that can be used in replication studies. Protoplast and in vitro systems have been developed to study replication of TCV genomic and subviral RNAs and several regions involved in replication of the subviral RNA satC have been identified.
The TCV RdRp undergoes a process called abortive initiation when using TCV as template, which produces short RNA sequences able to function as primers for repair of deletions of satC ends. Preliminary results suggest that promoters at the ends of TCV and satC strands may not be functioning as units independent of upstream sequence as previously thought, but rather may be involved in specific interactions with interior sequences. To address this hypothesis, the sequence/structural requirements for abortive initiation from the TCV hairpin promoter will be determined by analyzing the importance of the 3' single-stranded tail and an upstream sequence element in abortive initiation by the TCV RdRp. This objective should permit the delineation of an element required for abortive initiation on the TCV genome. Mutations that are able to restore replication of TCV with the promoter from satC and that are outside the promoter sequence will be mapped by replacing segments of the mutant TCV into TCV with the promoter of satC and assaying for infectivity. This objective should provide another means for elucidating internal cis-acting sequences that interact with the 3' terminal promoter of TCV. In addition, the importance of an interior sequence in satC required for transcription in the absence of the 3' end bases will be investigated. Successful completion of these experiments should provide significant advances in our understanding of the function of RdRp's and their ability to recognize a wide variety of linear and structural elements as promoters for complementary strand synthesis.

DESCRIPTION provided by applicant): The long-term goal of this research is to determine the molecular mechanisms of replication, a process fundamental to pathogenicity arid virulence, in (+)-strand RNA viruses that include many human, animal and plant pathogens. The use of powerful model eukaryotic virus systems, such as the turnip crinkle virus system, is necessary to address many important questions in RNA virus replication, and results have already been shown to be applicable to human viruses. TCV is associated with small, non-coding parasitic RNAs like satC that contain cis-acting elements recognized by the TCV RdRp. Already identified are: a 3-terminal carmovirus consensus sequence (CCS); 3PE also containing a CCS; the motif1-hairpin (M1 H) that is a recombination hot-spot, enhancer of transcription, and also contains a CCS; and 5 PE required for (+)-strand synthesis. The similar sequences that comprise the elements and the ability of the 3PE and 5PE to serve as independent promoters in vitro, suggests that they all attract the RdRp. To address the function and relationship among the elements, Specific Aim 1 will test the hypothesis that the in vivo and in vitro structures of satC (-)strands differ in the accessibility of the 3 end to the RdRp, with the in vitro structure promoting internal primer extension and the in vivo structure promoting full-length complementary strand synthesis. We will also convert the in vitro structure into the more active in vivo structure using mutations to stabilize the in vivo structure and RNA chaperones. In Specific Aim 2, an analysis of the M1 H of satC and theanalogous motif-3 hairpin of TCV will be conducted. We will test the hypothesis that functional replacement sequences can enhance either replication of satC or systemic movement of TCV. We will also test why the most common Ml H replacement motif CAACCCC, also found in the 5PE of the related virus Cardamine chlorotic fleck (CCFV), inhibits replication in certain sequence contexts. Loop-out and scanning models will be tested for how the RdRp finds the 3 end promoter after M1H binding. In Specific Aim 3, a functional analysis of the 5PE of satC, TCV and CCFV will be conducted, addressing the sequence specific nature of the 5 PE and determining whether the element, which can serve as an independent promoter for the TCV RdRp in vitro, plays an enhancer or a structural role in vivo.

DESCRIPTION (provided by applicant): Research in Virology requires more than a focused understanding of human pathogens. It requires comprehensive knowledge of viruses that infect hosts ranging from simple eukaryotes to complex multicellular organisms. A more comprehensive understanding of the breadth of Virology promises to provide the tools needed to control perplexing and devastating diseases in humans, as well as animals and plants. Substantive steps in knowledge acquisition require programs that prepare students to synthesize data from diverse systems, including model systems. The systems studied by University of Maryland Virology Program members include such important human pathogens as HIV, influenza, papillomavirus, poxvirus. West Nile, caliciviruses, as well as prions, viroids, fungal hypoviruses, and the model plant viruses Tobacco mosaic virus and Turnip crinkle virus. At the core of our training program are 14 well-funded investigators recognized as leaders in their fields from the Department of Cell Biology and Molecular Genetics, Maryland-Virginia Regional Vet Medicine, University of Maryland Biotechnology Institute Center for Biosystems Research, NIH, NCI, and USDA. Our faculty bring an extraordinarily broad range of expertise, from cellular and molecular biology to genomics and evolution, and from biochemistry to nanotechnology and vaccine development Trainees progress through innovative rotations, advanced courses in molecular and cellular biology, signal transduction, virology, and pathogenesis, journal clubs, attend monthly group meetings and a yearly retreat This training, along with state-of-the-art research projects, prepare trainees for careers centered on combating current and emerging viruses that threaten human health. This renewal application requests financial support for five graduate student traineeships. By combining this support with funds available through grants and intramural funds, over 20 pre-doctoral students will be trained in a five year period. The requested support will focus on attracting incoming students and students in their final years. We are committed to offering this exceptional training experience to highly-motivated students, with a special outreach to minorities, who will greatly enrich the potential for a global public health impact. RELEVANCE: Viruses infect nearly all life forms, are responsible for some human cancers, and are devastative disease agents that can cause world-wide pandemics. When properly harnessed, viruses can also be powerful research tools for dissecting cellular processes. This training proposal seeks continued support for training virologists prepared to meet research challenges common to all viruses- which include developing strategies to protect humans, animals, and plants from viral diseases to improve the quality of people's lives.

To begin to improve biology education in Prince George's County, MD, a pilot workshop will be held in early August 2006, focusing on updating the knowledge and skills of teachers. It is increasingly evident that Biology education in the USA is seriously deficient. Solutions will necessitate partnerships between universities and high schools, tapping into the expertise of professors who not only are experts in the subjects, but experts in teaching the material. If teachers have a better understanding of how chemistry, biochemistry, molecular biology and genetics are integrated, they should be able to better convey the information to their students, who will be better prepared citizens and ready for college level science. The pilot workshop is designed to provide at least one teacher from each high school with a thorough understanding of the material, best ways to present difficult concepts to students, and problem sets designed to engage students in active learning. The proposal is for funds to conduct an intensive four hours/day, two-week workshop for 30 teachers that will cover 75% of the high school biology curriculum (biochemistry, cell biology, molecular biology, genetics), which is viewed as the most problematic by teachers. Three hours each day will be devoted to mastering the material at the AP-biology/Introductory college level with one hour dedicated to strategies for teaching the material at the 10th grade level. Central to this hour will be homework/classroom assignments designed to help students master the fundamental concepts that underlie more complex biological mechanisms. If this pilot program is successful, as judged by improved student performance on state assessment exams, the objective is to apply for a substantial award to expand the program and help additional teachers the following year, with the goal of updating the skills of all biology teachers currently in the district.

One of the most exciting advances in modern molecular biology was the recent discovery that small RNAs regulate numerous cellular activities including development and virus infection through mechanisms referred to as RNA silencing. RNA silencing is an ancient, widespread mechanism for regulating eukaryotic gene expression, and is also used as a means of artificially reducing gene expression in a wide variety of important plants and animals. Based on very limited findings with plant and animal viruses, host small RNAs can be usurped to aid in virus replication or target viruses for destruction. Despite considerable efforts, little is understood about whether small RNAs derived from viral genomes can also control host gene expression, which could have substantial consequences for disease production. This project will combine the best studied viral replication system, Turnip crinkle virus (TCV)and its satellite RNA satC, with the best developed host silencing model, Arabidopsis thaliana. Preliminary experiments have already demonstrated that host enzymes dice TCV and satC into small RNAs that are mainly derived from "hotspot" regions of their genomes. One TCV small RNA tested was able to reduce the expression of at least six host mRNAs, which was related to symptom production and may be providing a more amenable environment for a successful infection. To gain an understanding of (1) the full range of genes targeted by TCV/satC; (2) how the enzymes that generate the viral small RNAs chose their target sequences, and (3) how the TCV coat protein, which naturally suppresses host RNA silencing, contributes to enhancing TCV replication while suppressing satC replication, this research will identify additional cellular targets of TCV and satC vsRNAs, determine the sequence and structural requirements for viral small RNA excision and analyze if the coat protein is responsible for altering the levels of specific small RNAs. This research will help blend together the currently separate fields of RNA virus replication and RNA silencing, dispel incorrect assumptions about RNA virus replication in plants and provide new ideas for the full role of viral RNA silencing suppressors. Successful completion of these experiments should significantly advance our understanding of the interplay between the host RNA silencing pathway and invading viruses with applications to both animal and plant viral pathogenesis, and will likely provide fundamental new information in the broad area of RNA silencing in plants.

Broader Impact of the Project (education and outreach):
Research and knowledge acquisition is being paired with promotion of teacher training in Prince George's County, a substantially minority school district adjacent to Washington DC. An effort was initiated by the PI in 2005 to improve teacher understanding of cutting edge Biology/Virology at DuVal High School. Teachers reported that their students improved both their understanding and enthusiasm for biology and midyear biology assessments improved by 100%. By also working weekly with AP Biology students, all students took the AP exam (compared with only 2 last year) and all applied to and were admitted to research-oriented universities, which were not their original choices (including two students who will join us at the University of Maryland- which is rare for this school district). This pilot program has led to the development of a two week summer workshop to work with 30 high school teachers selected from throughout the county (50 applications were received). The final lecture of the workshop will incorporate the development of the ideas that led to this research project and progress will also be presented at future planned summer workshops. Continued interaction is planned with AP biology students in the Fall, with each session leading off with the "results of the week", which will allow students to follow how this research is planned and how new ideas are generated. By pairing up all 30 teachers at the workshop with other investigators in the College of Chemical and Life Sciences, many with NSF funded research, the aspiration is to expand this model and excite AP-biology students throughout the county by describing how cutting-edge research is conducted and the role that NSF plays in advancing knowledge acquisition and science education.

DESCRIPTION (provided by applicant): Positive-strand RNA viruses are serious pathogens causing encephalitis, hemorrhagic fever and hepatitis in humans and animals and devastating crop losses in plants. Despite extensive studies, replication of these viruses remains poorly understood. A major stumbling block in efforts to fully understand virus replication is the large size of viral genomes, which complicates efforts to link RNA structure with RNA function. We have discovered that a novel conformational switch activates (-)-strand synthesis in satC (356 nt) associated with the model virus Turnip crinkle (TCV;4054 nt). Since satC contains all sequences and structures necessary for replication by the TCV RdRp, studying its replication in the past has provided significant information subsequently found to be applicable to much larger viral genomes, including those that cause significant diseases in humans and animals. Analyses of satC and TCV replication elements has revealed astonishing complexities and differences in how satC and TCV use nearly identical sequences to replicate their genomes, which has important implications for interpretation of results using subviral RNA replicons. In this proposal, we will use biophysical and genetic approaches to define secondary and tertiary interactions that characterize the satC pre-active structure and structural transitions of wt and mutant satC. Full length and selected satC fragments will be analyzed by temperature gradient gel electrophoresis, UV melting curves, oligonucleotide accessibility and UV cross-linking. Site-specific mutagenesis and in vivo genetic selection (selex) will help define individual elements and the relationship between elements. We will also use mutagenesis approaches combined with RdRp binding analyses to explore TCV sequences that are uniquely important for (-)-strand synthesis although also found in satC. Finally, we will examine RdRp binding to specific satC and TCV hairpins and determine if elements that flank one satC hairpin affect satC replication while simultaneously interfering with TCV replication and repressing virion accumulation function through an interaction between the two viral RNAs. Successful completion of these experiments will provide new paradigms for virus replication and interactions between helper viruses and associated subviral RNAs and provide the most detailed understanding of RNA conformational switches and the steps that lead to initiation of (-)-strand synthesis for any RNA virus.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
In the United States, crop loss due to infection by plant pathogens, including plant viruses, has been estimated to exceed thirty billion dollars per year. In the environment, the genomes of plant viruses, as well as small, virus-associated (subviral) RNAs that often augment the severity of symptoms in infected plants, can exchange genetic information by recombination and/or rapidly self-evolve, which can further enhance infectiousness of these pathogens. A major question in the field of RNA virology remains "What makes an RNA infectious?" One thought is that the viral RNA sequence and/or structure that the RNA forms relates to its ability to replicate its genetic material, accumulate in its host, and cause disease. Due to its small size, the subviral RNA satellite C (satC) of Turnip crinkle virus (TCV) is an excellent model for identification and characterization of RNA sequences and/or structures involved in replication and pathogenesis. In vivo SELEX (systematic evolution of ligands by exponential enrichment) is a method in which a portion of the satC RNA is randomized, and then pools of these randomized satC RNAs are used to infect plants in the presence of TCV. Only functional satC molecules move through the plants and can be recovered in new leaves; therefore, required RNA sequences and/or structures in these satC can be identified. Through this approach of de-evolving and re-evolving different elements within the 5´ portion of satC, signals that control (i) satC (+)-strand synthesis from (-)-strand replication intermediates and (ii) the ratio of satC monomers to dimers will be defined. Defining sequence/structure requirements in the satC 5´ end will enhance our current understanding of how satC functions in TCV infection.
Broader impacts. About 30 undergraduates will contribute to the research most via initiating the research projects during semester-long laboratory exercises in two offerings of an RNA Biology course at Dickinson College; others will complete the studies in the principal investigator's research lab. The students will answer state-of-the-art questions in RNA biology and virology by learning molecular biology techniques and performing in-depth data analysis, therefore partaking in a true research experience (as recommended in the BIO2010 guidelines). Furthermore, this research will engage women and underrepresented minorities based on current demographics of student majors in Biology and Biochemistry & Molecular Biology (likely participants in the course) as well as new (Dickinson science posse; NSF DUE-0856704) efforts to enhance recruitment and retention of minorities with interests in the Natural Sciences. The undergraduates who work on this project in the principal investigator's research lab will present at national meetings so that they can contribute to the research culture while enhancing their research awareness. The students also will contribute to the writing of research publications. Additionally, the principal investigators will write an education paper in order to disseminate the concept of in vivo SELEX experiments as a pedagogical tool for open-ended, molecular biology research projects for undergraduates.

Intellectual Merit. All organisms, from viruses to man, must synthesize proteins from their genomes by the process of protein synthesis (translation). Plant viruses are especially intriguing, as they must compete with their host cells for limited translation factors without killing the cell. Research on plant viruses has revealed that the end of the RNA genome most distal to where translation begins (the 3' end) is a critical region for attracting components of translation to the genome, leading to substantially enhanced translation. The 3' region of one virus with an RNA genome, Pea enation mosaic virus, was recently found to contain a translational enhancer that attracts ribosomes, the central protein synthesizing machines, and may function by helping ribosomes recycle following genome translation. In addition, this translational enhancer also participates in a long-distance joining with another element near the other end (the 5' end) of the virus. This project will examine how this translational enhancer interacts with ribosomes and whether ribosomes can "bridge" the ends of the viral genome by interacting simultaneously with both 3' and 5' RNA elements. Since viral genomes cannot be replicated and translated at the same time, this research will also investigate how the virus switches between these incompatible activities. Finally, the research will explore the possibility that the host plant's genome contains similar ribosome-binding elements that enhance translation of host RNAs. Developing new paradigms for how the 3' region participates in translation will prompt other investigators to correctly examine their genes or viruses for 3' translational enhancers, which will be transformative in the translation field. By understanding how these translational enhancers function, it should be possible to include them when designing new ways to produce higher levels of products in plants for improvement of agricultural traits.

Broader Impacts. This research will be conducted by graduate and postdoctoral researchers, who will learn state-of-the art techniques and model-building to enhance their development as scientists. Undergraduate researchers will also participate in all aspects of this project, learning about the excitement of scientific discoveries. Enthusiasm for research-based discovery will continue to be conveyed weekly to Advanced Placement high school students and teachers at DuVal High School (98% minority) in Prince George's County, Maryland, with several students already benefiting by being accepted at top colleges/universities and majoring in STEM subjects. The principal investigator will also continue to effectively represent science and scientists to the public, most recently as a consultant for a Discovery TV Series and participation in activities such as the Saturday morning science program at the University of Toledo. Specific examples from this research will also be used to excite and enhance the education experience of students in their first year as science majors at the University of Maryland through an introductory biology course taught by the principal investigator.

Intellectual Merit: This project is to help support the Plant Virology Satellite Saturday Symposium at the 31st Annual Meeting of the American Society of Virology, which will take place from July 21-25, 2012 at the Monona Terrace Convention Center, Madison, Wisconsin. The Symposium is sponsored by the Plant Virology Club (PVC), an organization within the American Society of Virology that was initiated to support research and speakers at the annual meeting who focus on plant viruses. The PVC sponsors a one day Symposium every other year at the Annual Meeting of the ASV. The Symposium this year was organized by Dr. Anne Simon, the elected Plant Virus Councilor and the PI on this project. Dr. Simon has worked throughout her career to bring together researchers working on plant and animal viruses and to make animal virus researchers more aware of the exciting work that uses plant viruses as models. Most virologists receive their graduate education in Medical Schools, which nearly uniformly ignore research that pertains to plant viruses. Similarly, most students studying plant viruses are educated in plant pathology departments that likewise spend little time on animal viruses. This separation has created a gap in the appreciation of the research currently being conducted using plant viruses by animal virologists working in the same area. This lack of appreciation reduces the impact of the work on model plant viruses and also has resulted in the loss of support (from NIH and USDA) for research that uses model plant viruses. The low level of 'cross-talk' between animal and plant virologists has also retarded research in the broad areas of virus replication/gene expression and RNA structure/function as well as innate immunity. The most prominent example of the negative impact on research that accompanies segregation of viruses according to host is the field of RNA silencing, which was well known to exist as a protective mechanism against plant viruses before its belated discovery in nematodes. Conferences that intermix talks on animal and plant viruses are rare (e.g., the Plus-strand RNA Virus meetings every three years), and the vast majority of talks even at these meetings continue to focus on animal viruses. The idea for this year's Satellite Symposium, entitled: Virus Replication and Movement: Building Bridges Between Plant and Animal Viruses came from an issue of Current Opinion in Virology (Nov, 2011), which was edited by Anne Simon and Grant McFadden. This issue was designed around a series of topics that were relevant to both plant and animal virologists, with each topic covered by an animal and a plant virologist. These topics are: Virus replication; Virus translation; Virus origins; Virus reorganization of intracellular membranes; and Virus cell-to-cell movement. The authors, selected as being world leaders in their fields, incorporated information from their counterpart's chapter into their submission, to better tie together the intersecting fields. Dr. Simon's idea was to organize a novel Plant Virology Symposium around each of these topics and invite the authors to speak. All authors gave enthusiastic agreement to come and present their current research, and place this research within the broader disciplines of plant and animal virus research. This should result in a broadening of the appreciation of research fields regardless of the host by audience members and hopefully foster new areas of communication within the broad field of Virology.

Broader Impacts: Graduate Students/Postdocs who work on plant/insect/fungal viruses will be eligible for these NSF-sponsored awards. This will assure that the students benefiting are not engaged in human health/related research, which is less appropriate for NSF support. Attention will be paid to distribution of the travel awards to labs in all regions of the US, and preference will be given to students/postdocs who are underrepresented minorities, women and persons with disabilities. We have also arranged with Elsevier to present free copies of the Current Opinion in Virology that this symposium is based on, which should allow participants to immediately access review articles that span animal and plant virology, thus further broadening the educational opportunity. Dr. Simon has spent her career advocating for the broadening of the education of virologists in this country and this symposium is the culmination of these efforts. These travel awards will allow students who might not have been able to attend for financial reasons due to the current funding climate, to participate in this exciting intersection among many fields of virology.

Climate changes leading to new migrations of hosts that viruses require for transmission is causing serious new outbreaks of viruses that threaten agriculture throughout the world. How viruses naturally cross species barriers and evolve to populate new hosts is a key theme of the Plant Virology Satellite Saturday Symposium. This grant is to help support travel for 10 graduate students/postdoctoral fellows to attend the Plant Virology Satellite Saturday Symposium at the 33rd Annual Meeting of the American Society of Virology (ASV), which will take place from June 21-25, 2014 at Colorado State University. Attending top meetings in their fields, such as the annual meeting of ASV, is critical for students as they learn new research, expand their scientific thinking, network with peers and meet top researchers in the field of virology. The Satellite Symposium, entitled: Viruses in a Transitioning World: Species Jumping and Emerging Plant and Animal Viruses is sponsored by the Plant Virology Club, an organization within the American Society of Virology that was initiated to support research and speakers at the annual meeting who focus on plant viruses. Because of the world class reputations of both the animal and plant virologists who will be presenting at the Symposium, a large audience of students and researchers in both animal and plant virology is expected. This should result in a broadening of the appreciation of research fields regardless of the host and hopefully foster new areas of communication within the broad field of virology.

This request is for funds to help provide for travel fellowships for graduate students/post-doctoral fellows who are currently conducting research in plant/insect/fungal viruses that will support their attendance at the conference. Attention will be paid to distribution of the travel awards to labs in all regions of the US, and preference will be given to students/postdocs who are underrepresented minorities, women and persons with disabilities. Providing support to allow these young investigators to attend the satellite meeting and the larger ASV meeting should encourage them to have excellent interactions with the larger viral scientific community and make connections that will be useful for their future work.

? DESCRIPTION (provided by applicant): Ribosome recoding (frameshifting, stop-codon readthrough) is used by many plant-, fungal-, animal- and human-infecting viruses to produce two proteins from a single 5'-end translation initiation site. Most recoding is dependent on a pseudoknot-containing Recoding Structured Element (RSE) positioned just downstream from the stop codon/frameshift site. This exploratory proposal is designed to test the overarching hypothesis that similar recoding events necessary to generate the RdRp by ribosome recoding in Turnip crinkle carmovirus (TCV) and SARS-coV involve similar overall mechanisms. We posit that both viruses require similar alternative basal conformations requiring an additional hairpin and sequences upstream of their RSE and similar long-distance interactions between their RSEs and the 3' and 5' ends of their genomes. This hypothesis is based on finding that TCV contains a stable alternative (basal) structure that disrupts its RSE, and that similar alternative structures are predicted for all coronavirus RSEs examined. In addition, a long-distance RNA-RNA interaction that connects RSEs in plant viruses with their 3' terminus is also conserved in coronavirus RSEs. Furthermore, the RSE sequence in carmoviruses and coronaviruses that has the pairing partner near the 3' end also has a possible pairing partner (7-8 nt for coronaviruses) near their 5' ends. We propose that the 3' end interaction stabilizes the active RSE structure allowing ribosomes to readthrough /frameshift, and that the RSE-5' end interaction occurs when the RSE is in the basal conformation to aid in ribosome recycling following translation termination. In Specific Aim 1, we will investigate the importance of a conformational switch between basal and active structures in the RSE region of TCV using SHAPE RNA structure probing of full-length virus combined with selective mutations. We will also determine if the known long-distance interaction between the TCV RSE bulge loop and 3' terminal sequences stabilizes the active RSE structure. Using single and compensatory mutagenesis, we will also test (with collaborator Dr. Ralph Baric) if a basal conformation exists for the RSE of SARS-coV and if a phylogenetically conserved hairpin loop in the RSE of SARS-coV is involved in a very similar interaction with a 3' sequence. In Specific Aim 2, we will investigate if the RSEs in TCV and SARS-coV play an additional role in ribosome recycling by engaging in a predicted long-distance interaction with the 5' end. We will also investigate if release of this interaction in TC promotes a conformational switch to the RSE active structure for ribosome readthrough. In Specific Aim 3, we will use in-line RNA structure probing of isolated TCV and SARS-coV fragments to investigate structural requirements for the basal and active RSE conformations. The results of these experiments will likely transform current models on ribosome recoding and provide evidence for the importance of ribosome recycling, which has long been lacking in the translation field. It should also, importantly, open up new targets for antiviral agents against viruses that are important human pathogens.

This project addresses the question of how viruses, which depend on the infected host cells for making virus proteins, use the host cell protein-producing machinery, the ribosome, in a manner that is highly efficient. The research will exploit and extend the novel discovery that the genetic material of a virus contains specific structures that can bind to the ribosomes and that thereby allow them to be quickly and efficiently reused. Key outcomes of this project will be broadly applicable in many fields, including in biotechnology to enhance the efficiency of protein production. The outcomes may also lead to novel approaches to control a variety of viruses that infect plants and animals. This project will provide educational and research training opportunities for high school students, college undergraduates, graduate students and postdoctoral researchers. The outcomes of the research will be presented at scientific conferences, taught in introductory courses to biology students, and used to introduce undergraduates and high school students to the life cycle of viruses. The principal investigator will work with Advanced Placement students at DuVal High School (98% under-represented minority students) to prepare them for college, serve on the board of the National Academy of Sciences partnership with the entertainment industry and will also serve as a scientific consultant for programming by the Discovery and National Geographic Channels.

The plant virus "Pea enation mosaic umbravirus" has a bipartite positive-strand RNA genome containing two 3' proximal T-shaped structures (kl-TSS and 3'TSS). These structures are critical for translation of the uncapped viral RNAs into proteins. 3'TSS is an example of the long-known, better-studied tRNA-like structures found in viral RNAs, but the existence of the kl-TSS was unsuspected and came to light during previous research. The newly found, internal kl-TSS, with its three-way branched secondary structure, binds to 40S, 60S and 80S ribosomes and can simultaneously engage in a long-distance interaction with 5H2, a coding region hairpin located near the 5'Untranslated Region (UTR). The 3'TSS is also likely to connect with 5H2, and the whole system will be exploited to learn how different tRNA-mimics interact with ribosomes to enhance translation. Specifically, a novel "pick-up and delivery" hypothesis will be evaluated, i.e., the idea that multiple TSS may be connected via RNA:RNA interactions with the ends of virus-encoded Open Reading Frames (ORF), to gather ribosomal subunits downstream of an ORF at the end of translation and relay them back to the 5'UTR for another round of translation. In addition, it will be tested whether the kl-TSS can enhance translation of capped mRNAs produced from expression constructs. These experiments will inform our understanding of how this novel and possibly widespread use of tRNA mimicry operates to enhance translation and to control plant virus life cycles. The project will also lead to the development of expression cassettes with enhanced translation of encoded proteins with potentially extensive agricultural as well as laboratory applications.

The Virology Training Program at the University of Maryland was founded 13 years ago on the principle that transformative research on host-virus interactions and viral immunology/vaccine design would be facilitated by a comprehensive understanding of virology that extends beyond selective, traditional training in human pathogens. The central location of the University of Maryland in the most significant concentration of virologists in the world (University of Maryland, NIH, NCI, Institute for Biosystems and Biotechnology Research [IBBR], Virginia-Maryland College of Veterinary Medicine [VetMed]), led to the establishment of a program that today brings together 17 outstanding virologists (16 mentors) dedicated to training future leaders in the field. This training program is the first of its kind to include participation by major researchers at the NIH, providing trainees with an exceptional opportunity to explore how research is conducted in both academic settings and at the nation's premiere research institute. The Virology Program also fully supports the NIH directive to offer ?structured, career development advising and learning opportunities for trainees to prepare them for possible careers outside of academia? by partnering with MedImmune, the worldwide biologics research and development arm of AstraZeneca, to offer interested predoctoral and postdoctoral trainees career mentors and 2-month internships. Our affiliated institutions and partnership with MedImmune provides trainees with unmatched opportunities for research and career mentoring, and for exploring how research is conducted in academic settings, at the nation's premiere federal government research institutes, and at one of the world's leading pharmaceutical companies targeting viruses. The University of Maryland Virology Training Program is requesting 3 predoctoral and 2 postdoctoral traineeships in years 1, 2 and 3, and 4 predoctoral and 3 postdoctoral traineeships in years 4 and 5. Through focused didactic training, monthly group meetings and workshops where experts discuss the future of the field, an annual retreat, and individualized career and research mentoring, our training program produces virologists who (1) have exceptional research skills acquired from working in top Virology labs; (2) have gained a comprehensive background in basic virology through didactic training in viruses that infect humans, animals and plants; (3) have an understanding of the impact of their research in the context of broader biomedical and agricultural issues; (4) embrace the value of integrative, collaborative research; (5) have developed a teaching portfolio allowing them to be competitive for positions at traditional academic research intensive institutions and undergraduate research institutions; and (6) can effectively disseminate the knowledge that they have gained. We are committed to offering this exceptional training experience to highly-motivated predoctoral and postdoctoral students, with a special outreach to minorities, who will greatly enrich the potential for a global public health impact.

Understanding how plants respond to pathogens is critical for maintaining a robust food supply. Key to this response is the movement of RNA, a type of genetic information, from one part of the plant to another. However, detailed understanding of how RNA moves in plants is not well understood due to the small amounts of transiting RNAs. In addition, nearly all of our knowledge comes from research on short-lived plants and very little is known about long-lived trees due to the time it takes to grow and study them. The discovery of a novel virus-like RNA (called CYVaV) that infects virtually all citrus varieties provides an unparalleled opportunity to explore the biology of trees and the movement of RNA in plants. CYVaV accumulates to high levels in tree veins and moves in the absence of specific virus-produced "movement" proteins. Since CYVaV RNA must use the trees molecular machinery to move its RNA, it is an ideal system to study RNA movement. This project uses CYVaV to develop technologies to understand the movement of RNAs in plants. In addition, CYVaV may enable researchers to introduce genetic elements in trees. An important broader impact is the possibility of using CYVaV as a vehicle to combat the bacterial disease Citrus Greening by targeting the bacteria and the insect depositing the bacteria. This could help save the US citrus industry from Citrus Greening.

Recent revelations that 50% of the companion cell (CC) transcriptome is moving long distances through sieve elements (SE) have elicited numerous questions concerning how and why this migration of mRNAs is occurring. Answering key questions on intracellular and intercellular movement of mRNAs requires a model system consisting of an abundant mobile RNA, whose location can be traced in living tissue under different cellular conditions. The recently discovered Citrus yellow vein associated virus (CYVaV) is a highly unusual virus-like RNA that traffics bi-directionally through the phloem of all commercial varieties of citrus and laboratory host Nicotiana benthamiana in the absence of encoded movement proteins (MPs), coat protein or silencing suppressors. This project develops CYVaV as a model for transiting mRNAs using MS2 CP and λN22 systems and as a VIGS vector to study effects of altered gene expression in citrus phloem. CYVaV causes few if any symptoms on citrus although it accumulates to levels detectable on ethidium-stained gels in infected plants. This should allow for high level expression of small RNAs, with the initial goal of down regulating CC callose synthase, which causes callose deposition in SE during stress or pathogen invasion. Since CYVaV should have no host-MP specific incompatibility, it will also be examined for an extended host range in other woody plants and vines, which currently have no useable vectors. By studying CYVaV movement and ability to serve as a VIGS vector, one broader impact of this work is using CYVaV to block the devastating effects of Citrus Greening.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The project tackles the ongoing need to develop new technology for rapid, large-scale and cost-effective production of therapeutic peptides/proteins such as vaccines ? now made even more acute by the COVID-19 pandemic. Using plants to produce biological products offers advantages of speed, low cost and low toxicity, but also disadvantages of low yields and complex purification processes. The PI will investigate a novel RNA vector for molecular determinants of stability that can hinder protein expression, and develop pumpkin as a host for producing large amounts of proteins encoded by this RNA vector. Overcoming hurdles to vaccine production in plants has potential for immense broader impact. The project also offers research training and career development opportunities to a post-doctoral scholar.

The research builds on substantive preliminary data indicating that a novel virus-like RNA vector (CYVaV) can be manipulated to control its stability and related properties in host plants. The PI?s team plans to modify CYVaV RNA sequence and secondary structure with the goal of optimizing stable versions of the vector for over-expression of engineered proteins in host plants. Development of a vector platform that can serve to infect a variety of plants for large-scale production of biologics will have broad biotechnological and biomedical applications.

This project is funded by the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.