Peter Sarnow - US grants

An ideal tool to study biosynthesis pathways in mammalian cells is a virus which interferes with these processes. Poliovirus is a positive-strand cytoplasmic RNA virus that undergoes extensive interactions with its host cell, including the inhibition of cellular transcription and translation. The discovery that a cDNA clone of the viral genome gives rise to infectious virus upon introduction into mammalian cells, created the opportunity to construct defined viral mutants by site-directed mutagenesis and to study their biological properties. The aim of this proposal is to apply genetics and biochemistry to identify functions of a viral RNA genome and its interaction with the host cell. Using a temperature-sensitive (ts) poliovirus mutant, 3NC202, which bears an eight nucleotide insertion in the 3' noncoding region and is defective in the initiation of minus-strand RNA, we will identify structural and functional elements in the RNA molecule which control the amplification of negative-strand RNA. Specifically, we are going to analyze the structure of the mutant RNA using chemical probes and reverse transcriptase. In addition, we will search for viral trans-acting factors which might interact with the 3' untranslated region. Therefore, revertants of the ts-mutant will be isolated, and the altered protein factor tested for its ability to bind to the viral "ts-RNA" molecule. In addition, we will study the biological properties of a small plaque mutant, 2B201, mapping in the gene encoding 2B, a protein with unknown function. Genetics (analysis of revertants) and biochemical approaches (isolation and in vitro assays of 2B) will be applied to gain understanding of the role of the 2B polypeptide in the viral life cycle. We will also investigate why the cellular mRNA encoding the glucose regulated protein (GRP80) is translated in mutant-infected cells at a time when the translation of other host mRNA molecules is inhibited. Translatability of hybrid-selected GRP80 mRNA in infected and uninfected cellular extracts will be analyzed. We will construct hybrid genes between GRP80 cDNA sequences and a test gene and examine translation of in vitro made transcripts, to identify sequences which are involved in cap- independent translation. The health relatedness of this project lies in its contribution to the understanding of how animal RNA viruses interact with their host cells. Poliovirus is one of the best studied animal viruses because its genome organization is known and its three dimensional structure has been solved. The recent sequencing of other RNA virus genomes has revealed great homology between the members of the picornaviridae family. Thus, knowledge of the structure and function of the poliovirus genome might be applicable to the genomes of other medically important RNA viruses such as foot and mouth disease virus and human rhinovirus, responsible for the common cold.

Comparison of neurovirulent and attenuated poliovirus genomes has implicated the 5' noncoding region of the viral RNA in a rate limiting step in viral growth and neurovirulence. The poliovirus 5' noncoding region presumably contains signals important for the translation and amplification of the viral RNA genome. The availability of an infectious cDNA clone of the viral genome and the discovery that in vitro synthesized viral RNA is infectious when introduced into mammalian cells, allow the application of genetics to study this RNA virus. The aim of this proposal is to introduce defined mutations into the 5' noncoding region by site- directed mutagenesis of the cDNA and to study their effects on the translation and amplification of the viral RNA genome. Specifically, we will analyze wild type and mutant RNA molecules in in vitro translation and replication systems. Hybrid genes containing the 5' noncoding region of wild type or altered viral genomes and the coding region of a test gene will be constructed and the translatability of the hybrid mRNAs will be tested in cell lines harbouring the hybrid genes stably integrated in the chromosome. In addition, we will search for trans-acting factors which might interact with the 5' noncoding region. These studies will be complemented by the structural analysis of wild type and mutant 5' noncoding regions employing RNA-conformation specific chemical probes and reverse transcriptase. The recent sequencing of other picornavirus RNA genomes has revealed great homology in the 5' noncoding region. Thus, knowledge of the structure and function of this region will increase our understanding of the neurovirulent behaviour of other medically important RNA viruses such as Theiler's virus, a neurotropic mouse RNA virus, which is studied extensively in the laboratories of Drs. Murray and Rotbart at the University of Colorado Health Sciences Center.

A ribosome scanning model has been proposed to explain translation initiation of eukaryotic mRNAs. In this model, binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by a concerted interaction between the m/7GpppN cap-structure at the end of the mRNA and the cap binding protein complex eIF-4F. However, certain viral and cellular mRNAs have been identified that can contain internal ribosome entry site elements that recruit directly 43S subunits onto mRNAs. The goal of this proposal is to study the molecular events leading to 5' cap- dependent and 5' cap-independent translational initiation in mRNA molecules. First, it will be tested whether a local critical concentration of translation initiation factors can mediate the recruitment of 43S subunits onto mRNAs. Specifically, it will be tested whether hybridization of "initiation competent" RNAs to "initiation non- competent" RNAs will result in the translational activation of the latter RNA species. Secondly, the mechanism by which an appropriate AUG codon is selected as start codon for translation will be investigated. Specifically, a putative role of the La autoantigen in AUG start site selection will be tested by monitoring the effects of La and La-associated factors on the translation of mRNAs containing the AUG embedded in different sequence motifs. Third, the mechanism by which a novel "cap- binding" RNA (SELEX RNA) inhibits translation of capped but not of uncapped mRNAs will be examined. Fourth, the SELEX RNA will be used as a tool in the identification and characterization of naturally occurring mRNAs that can be translated cap-independently. Specifically, SELEX RNAs will be added to an in vitro translation system, resulting in the dissociation from polysomes of mRNAs that are translated cap-dependently. RNAs that are still associated with polysomes in the presence of SELEX RNAs will be isolated using differential mRNA method and characterized for their ability to be translated cap-independently. Lastly, the mechanism will be determined by which HAP4 mRNA of Saccharomyces cerevisiae is translationally initiated when the organism encounters nonfermentable carbon sources. Overexpression of the cap binding protein 4E can lead to cell transformation. Thus, regulating the rates of translational initiation of certain mRNAs, such as those encoding growth factors, is essential for normal cell growth.

DESCRIPTION: With the discovery that many cDNA copies of picornaviral RNA genomes result in the synthesis of infectious virus after transfection into mammalian cells, picornaviruses became amenable to genetic manipulations. Because picornaviruses interact extensively with host macromolecular pathways, these viruses have been used as tools to study the mechanisms of host cell translation, transcription and protein secretion. The overall aim of this proposal is to study specific viral-host interactions that occur in infected cells, with the ultimate goal to understand picornavirus-induced pathogenesis. The first aim examines a functional role of the specific interaction of nucleolin with the viral 3' noncoding region. Specifically, the outcome of the relocalization of nucleolin from the nucleolus to the cytoplasm on viral gene expression will be studied in cell-free extracts that mimic many aspects of the infectious cycle. In addition, a virus-encoded function that induces the relocalization of nucleolin will be sought by monitoring the intracellular localization of nucleolin in the presence of individual viral polypeptides. Various forms of the translation initiation factor eIF4G have been implicated to be important in translation initiation by both the conventional 5' end-dependent scanning mechanism and the internal inititation mechanism. In the second aim, the roles of various forms of eIF4G in host cell translation and the poliovirus infectious cycle will be studied in cells in which the normal eIF4G gene is inactivated. In the last aim, genes will be identified that confer susceptibility to picornavirus infections. Random host genes will be disrupted by retroviral insertions and inactivated by expression of antisense RNA. Cells that have functionally inactivated allelic loci and that are resistant to infection by either picornavirus or rhinovirus will be selected and the disrupted genes will be identified and characterized in detail.

It has been estimated that at least 1 percent of the world's population is chronically infected with hepatitis C virus (HCV). Infection by this virus can cause acute or chronic hepatitis, as well as liver cirrhosis which can lead to hepatocellular carcinoma. Only 50 percent of HCV infected patients respond to alpha-interferon and fewer than 25 percent of treated patients will have long term remission. In addition, the lack of protective immunity after HCV infection has impaired the development of effective antiviral therapies and vaccines. Although there is genetic diversity among different HCV isolates, there is a significant restriction on the sequence diversity within the 5' noncoding regions (5'NCR) of these RNA genomes. This conservation can be attributed to the presence of RNA elements that are involved in viral genome replication and translation. Distinct structural elements in the viral 5' NCR, especially a pseudoknot structure located near the start site AUG codon, are required to permit the translation of the viral genome by an unusual mechanism of internal ribosome entry which is used by all picornaviral and certain cellular mRNAs. However, the internal ribosome entry site (IRES) in HCV has functional features that are different from those of any other known IRES element or 5'NCR. All eukaryotic mRNAs studied to date recruit 43S ribosomal subunits, 40S ribosomal subunits that carry factors eIF3 and eIF2-tRNAinit. In contrast, the HCV IRES can bind "empty" 40S subunits directly at the translational initiation start codon, very much like prokaryotic mRNAs which are known to recruit 30S subunits directly. Thus, the HCV IRES is an ideal target for the design of antiviral therapeutics, because such therapeutics may not affect other cellular functions. Molecular genetic approaches are proposed to select and to characterize conformationally constrained peptides, expressed from combinatorial libraries, that selectively inhibit the HCV IRES in living cells. Furthermore, three-dimensional structures of domains of the HCV IRES RNA, including the pseudoknot structure, will be determined by NMR. The use of a combination of molecular, biochemical and structural approachers to study the HCV IRES, will greatly assist in more rational design of antiviral reagents.

DESCRIPTION (provided by applicant): The goal of this application is to investigate the roles of miRNAs in the regulation of gene expression in mammalian cells. Although at least 135 miRNAs have been identified in Caenorhabditis elegans, Drosophila melanogaster and humans, a functional role has been determined for only two: lin-4 and let-7 of C. elegans. Both of these miRNAs interact by incomplete pairing with the 3' noncoding regions of their target mRNAs. It was demonstrated that the C. elegans-specific lin-4 miRNA inhibits translation of its target mRNA, lin-14. Although the expression of the highly conserved let-7 is known to reduce the accumulation of the lin-41 gene product in C. elegans, neither the mechanism of this inhibition nor the target mRNA of let-7 orthologues in other species are known. To investigate the function of let-7 in human cells, we will isolate its human target mRNAs by expressing biotin-tagged let-7 RNA in cells and isolating target mRNAs by affinity chromatography. After obtaining cDNA copies of the target mRNAs, the nucleotides that participate in let-7/miRNA interactions will be determined by psoralen crosslinking and site-directed mutagenesis. To study the physiological roles of let7-mRNA complexes, the stability and translational efficiency of mRNAs containing wildtype and mutated binding sites for let-7 will be examined in cultured cells. To gain an understanding of the prevalence of miRNAs that regulate mRNA translation, we will search for miRNAs that are associated with polysomes in cycling cells and in mitotically arrested cells using cDNA microarrays. Identities of associated miRNAs will be determined by obtaining cDNA copies and analyzing their sequence. Using several different approaches, the experiments described in this proposal will address the function of the highly conserved miRNA let-7 and will identify distinct miRNA/mRNA complexes that are regulated during the cell cycle. If the hypothesis is correct that miRNAs can facilitate a rapid and reversible control of translation, then the proposed experiments should provide a large amount of novel biological information.The health-relatedness of this project lies in the potential roles of this novel mechanism of gene regulation in cell growth or development.

DESCRIPTION (provided by applicant): Hepatitis C remains a serious health threat, with few therapeutic options. Amplification of the positive-stranded viral genome is regulated by highly conserved RNA elements located in the viral 5' and 3' noncoding regions. Translation of the viral proteins is mediated by an unusually divergent internal ribosome entry site (IRES) located in the viral 5' noncoding region. The IRES is a conserved 320 nucleotide RNA that binds directly to ribosomal 40S subunits to direct translation initiation. Initiation of viral minus-stranded RNAs is modulated by a conserved landscape of RNA stem-loop structures located at the very 3' end of the viral RNA genome. Here, a combined genetic, biochemical, biophysical and structural approach is proposed to determine the mechanism by which the viral noncoding regions control HCV translation and replication. In specific aim 1, biochemical and genetic methods will delineate the mechanisms of HCV translation initiation, and will unravel the roles of a cellular microRNA in modulating gene expression of the viral RNA. The mechanistic information, coupled with a large amount of preliminary spectroscopic data, will be used in specific aim 2 to guide NMR structure determinations of IRES domains and the full-length intact IRES. To complement static structural experiments, specific aim 3 explores the conformational dynamics of free and ribosome-bound IRES RNA using single-molecule fluorescence spectroscopy, and mechanistic details will be obtained by direct observation of tRNA binding and release during translation initiation. The role of the 3' noncoding in IRES-mediated function will be also monitored by single-molecule fluorescence to detect transient end-to-end communication in viral RNA. Similarly, the effects of microRNA-viral RNA interactions on conserved sequence elements located at the 3' end of the viral genome will be examined using single-molecule fluorescence. The results of this proposal will have direct impact on our understanding of critical steps in the viral replication cycle, and the possible development of novel antiviral therapies.

It has been long been known that eukaryotic ribosomes are heterogeneous in nature. Ribosome[unreadable] heterogeneity is exemplified by the differential presence of certain ribosomal proteins and ribosomal[unreadable] RNAs, modification of ribosomal proteins and RNA, and association of non-ribosomal proteins with[unreadable] ribosomal particles. However, it remains unknown whether the heterogeneous pools of ribosomes reflect[unreadable] distinct activities in protein biosynthesis. More recently, it has become clear that the internal ribosome[unreadable] entry sites (IRES) located in certain viral mRNAs can bind directly and with high affinity to mammalian[unreadable] 40S subunits. The aims of this proposal are to examine whether functionally heterogeneous populations[unreadable] of ribosomes exist in mammalian cells and whether distinct ribosome populations are recruited to[unreadable] picornaviral and hepatitis C viral IRES elements in infected cells. In the first aim, the composition and[unreadable] activity of total, unbound and polysome-bound ribosomal subunits from uninfected cells will be compared[unreadable] to those isolated from picornavirus infected cells by electrospray mass spectrometry (ES-MS). In the[unreadable] second aim, the presence of altered ribosomes in viral mRNA-complexes will be examined. Specifically,[unreadable] thiouridine-containing viral RNA will be generated in HeLa cells expressing the Toxoplasma gondii uracil[unreadable] phosphoribosyltransferase (UPRT) enzyme. UPRT will convert thiouracil to thiouridine 5' monophosphate[unreadable] nucleotides whose triphosphates can be selectively incorporated into viral RNA by the viral RNAdependent[unreadable] RNA polymerase in the presence of actinomycin D. Polysomal thio-labeled RNA will be[unreadable] biotinylated, isolated by streptavidin chromatography and associated ribosomes characterized by ES-MS.[unreadable] This method will also be used to identify IRES-binding proteins that are tightly linked to the viral RNA in[unreadable] infected cells. Finally, the roles of modified ribosomes in various steps of translation initiation will be[unreadable] examined in reconstituted translation systems. The outcome from these studies will reveal roles of[unreadable] modified ribosomes in mammalian cells and will provide insights by which IRES elements recruit host cell[unreadable] ribosomes during viral infection, revealing new potential targets for antiviral therapeutics.

DESCRIPTION (provided by applicant): An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5' end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3' terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies. PUBLIC HEALTH RELEVANCE: An estimated 170 million people worldwide and 4 million in the United States are infected with hepatitis C virus (HCV). The majority of patients do not resolve the infection and become chronic carriers, ultimately needing expensive liver transplants. There is no vaccine for HCV, and current treatments, which include ribavirin and interferon 1, are expensive and relatively ineffective. It was discovered that HCV binds two molecules of a liver- specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. This proposal explores the mechanisms by which miR-122 protects HCV RNA in the liver. The dependence of HCV on miR-122 presents an Achilles heel of the virus that can be used for antiviral intervention. We will study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules (locked nucleic acids) that can easily bind and inactivate miR-122 in the liver of animals. This is a highly significant approach, because LNA-mediated sequestration of miR-122 in the liver of HCV-infected chimpanzees resulted in a 2.5 fold drop in viral load without any emergence of resistant virus (Lanford et al. 2010. Science: 327:198-201).

DESCRIPTION (provided by applicant): Support is requested for years 21 through 25 of a Program to train graduate students and postdoctoral fellows at Stanford University School of Medicine in the Molecular Basis of Host-Parasite Interaction. This Training Program crosses the disciplines of infectious disease, molecular biology, genetics, cell biology, biochemistry, immunology and microbiology in the area of microbial pathogenesis and the host native and acquired immune responses. The successful training record of our faculty, including the predoctoral and postdoctoral students trained over the past 19 years of this Program, supports the continuation in the next period. The support provided by this training grant has allowed highly qualified individuals to train in the Program, and has precipitated a range of interactions among the various faculty members. In a modestly sized program, highly regarded scientists who investigate innate and acquired immune responses interact daily with experts in the genetics, cell biology and biochemistry of viruses, bacteria and eukaryotic parasites. The Training Program is based in the Department of Microbiology and Immunology but includes faculty members chosen by students in the program from outside the Department. Faculty expertise in immunology and a wide range of microbiological topics are available to graduate students and postdoctoral fellows in the Program. A rigorous mentoring program, interactive seminar series, departmental retreats and journal clubs are in place to assure scientific exchange across traditional disciplines. Trainees from underrepresented minorities have been well represented, as have been female trainees. To accomplish the overall aim to train future experts in infectious disease, eight predoctoral and four postdoctoral training slots are requested for the continuation of this Program.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Although many cases of HCC are induced by chronic alcohol abuse or environmental toxins, approximately 80% are caused by infection with either hepatitis B or hepatitis C virus (HCV). The burden of HCV infection worldwide is very large, with most of the personal and economic burden yet to come, as cirrhosis and HCC take years to develop. Approximately 170 million people are infected worldwide with HCV. Most infected individuals do not clear the disease, but develop chronic infections that often lead to end-stage liver disease and HCC. Current treatment is limited to co-treatment with ribavirin and interferon [unreadable], a therapy that is expensive and ineffective in 50% of infected individuals. Therefore, there is an urgent need to identify new and accessible viral and cellular targets for therapies against HCV and HCC. It has been shown recently that cellular microRNAs, especially those in the liver, can be readily inactivated by direct intravenous delivery of modified oligonucleotides. Thus, our finding that liver-specific microRNA miR-122 interacts directly with the viral 5'noncoding region of HCV and that this interaction is essential to maintain intracellular abundance of the viral RNA, identifies a promising target for antiviral therapy. What are the host proteins required for microRNA expression in the liver? Recently, it has been found that host cell protein RCK/DDX6, a member of the DEAD box helicase family, is important for microRNA-mediated gene regulation in mammalian cells and that both miR-122 and RCK are greatly overexpressed in HCV-induced HCC in patient livers. Our preliminary results have shown that RCK is an essential positive regulator of HCV gene expression;depletion of RCK results in loss of HCV proteins. In this proposal, we propose to examine the mechanism by which RCK upregulates HCV gene expression at an apparently post-transcriptional step and by which RCK affects microRNA abundance and intracellular distribution in uninfected and HCV-infected liver cells. Using novel in situ hybridization technology, we will determine the effect of RCK expression on microRNA localization to specific cytoplasmic compartments known as P-bodies and stress granules. Finally, we propose experiments to identify targets for microRNAs whose abundance and distribution is affected by RCK, These studies will develop novel tools for HCV and HCC and are likely to point to new targets for antiviral therapy.

DESCRIPTION (provided by applicant): Infection by RNA viruses, whose mRNAs are translated by an internal ribosome entry (IRES) mechanism, such as picornaviruses and hepatitis C virus (HCV), remains a significant health threat. For example, rhinovirus causes the common cold and exacerbation of asthma, and enterovirus 71 is currently epidemic in parts of Asia. Notwithstanding the poliovirus vaccines, no effective antiviral reagent exists for picornaviruses. For HCV, several new compounds are being on the market and their efficacies are being monitored. We have been wondering whether the IRES elements in these viral genomes present an Achilles' heel for the viruses, and we have been searching whether specialized ribosome populations might be involved in internal initiation. We discovered that ribosomal protein RPS25, which is modified during infection with poliovirus, is essential for translation of IRES-containing viral RNAs and curiously, also for Dengue virus who's mRNA is translated in a cap-dependent manner. In this application, we propose to study roles for RPS25 in IRES-mediated translation, using a variety of cell-based assays and a large array of single-molecule approaches that are ideally applied to heterogeneous systems such as modified ribosomes. The first specific aim proposes to use a viable haploid cell that lacks RPS25 to study interactions of ribosomes with known IRES-containing RNAs, Dengue viral RNAs and cellular RNAs, that require RPS25, identified in ribosomal profiling experiments. Novel crosslinking assays will be used to map mRNA-RPS25 interactions in living cells. Aim 2 proposes to study steps in translation that are affected by RPS25 using translation competent-extract and a variety of mRNA targets. Specific Aim 3 details a very comprehensive approach to study the dynamics and kinetics of IRES-mediated translation and the role for RPS25 variants, employing state-of-the-art single molecule approaches. The last aim proposes to examine effects of specific identified modifications in RPS25 on IRES-mediated translation. Overall, this application will address fundamental aspects of specialized ribosomes in the translation of distinct mRNAs. The outcomes of these studies are likely to detail novel mechanisms of gene expression mediated by the ribosome in eukaryotic cells and may point to new venues for antiviral and anticancer therapies.

New direct-acting antivirals against hepatitis C virus (HCV) cure the virus in most patients. However, these compounds are expensive and not available to most of the 170 million people that are worldwide infected with HCV. There are also no approved vaccines or anti-viral compounds for other members of the flaviviridae, such as Dengue virus, West Nile virus or Zika virus. Thus, identification of cellular genes that are essential for virus propagation is highly significant, because such targets are not regulated by the error-prone viral RNA polymerase and, therefore, offer a high barrier to resistance. The long-term goal of this application is to explore the mechanism by which certain noncoding RNAs, such as microRNAs and circular RNAs, display pro-viral or anti-viral activities. It is known that microRNA miR-122 attaches as an oligomeric complex to the 5? end of the viral genome and protects it from degradation by host exonucleases. Curiously, escape mutants that harbor a single C3U mutation in the viral genome were detected in the serum of 122-antagomir treated patients. The overall objective of the first aim to is identify interactions of between C3U HCV genome with nucleic acids or proteins that allow the virus to persist when miR-122 abundance is reduced. The central hypothesis is that novel RNA-RNA or protein RNA interactions in the C3U HCV genome allow stabilization and expression of the viral genome in the cells during reduced miR-122 abundances. Novel cell-based protein biotinylation assays and approaches that detect tertiary RNA structures will be used to study RNA-protein and RNA-RNA interactions in wildtype and mutant viral RNAs in cultured liver cells. Steps in the viral life cycle that are modulated by such nucleic acid-protein interactions will be identified. The overall objective of the second aim is based upon the finding that the host cell-derived circular RNA (cRNAs) landscape is altered during HCV infection. The potential mechanisms by which cRNAs exert their pro- and antiviral functions will be explored. First, effects of cRNA-mediated sequestration of proteins and microRNAs on HCV RNA amplification will be examined. Because methylation of specific adenosines in cRNAs has been shown to induce translation initiation in cRNAs, the methylation status in cRNAs will be determined. The properties of methylated cRNAs to synthesize small peptides will be examined using genetic and proteomic approaches. Overall, the application details innovative paradigm-shifting concepts to study roles for noncoding RNAs in virus-host interactions, using novel detection methodologies. The rationale for this proposal is that noncoding RNAs, such as microRNAs and cRNAs, affect HCV pathogenesis. It has been shown that microRNAs can be targeted in HCV patients, resulting in loss of viral RNA abundance. Thus, targeting pro-viral cRNAs in the liver offers a novel antiviral strategy.

While novel direct-acting antivirals (DAA) against hepatitis C virus (HCV), such as anti-polymerase and anti- proteinase compounds are effective in many patients, these treatments are still very expensive and not available to most of the 170 million people world-wide infected with HCV. In addition, it is not known whether every patient population will respond to the current DAA treatments. Thus, it is significant to continue to search for new compounds that target both HCV and viral host susceptibility factors to combat virus infection. An astonishing recent discovery made by our laboratory revealed that the HCV genomic RNA is processed during viral infection to yield hundreds of different virus-derived circular RNAs (circRNAs). These circRNAs are generated from all parts of the 10,000-nucleotide viral RNA genome, including circRNAs that contain the viral internal ribosome entry site (IRES). It is hypothesized that these circRNAs present a novel class of viral RNA species that likely display novel functions in infected and uninfected bystander cells. Because these viral circRNAs are predicted to be long-lived they could also have important roles as biomarkers. Thus, exploring the pro- and anti-viral effects of the HCV-derived circular RNAs is a significant venue of research and will point to new Achilles? heels in RNA viruses. The long-term goals of this application are to explore the roles for the identified viral circRNAs in pro- and anti-viral responses. Two specific aims are proposed to accomplish these goals. First, the mechanism by which the viral circRNAs are generated is being investigated. The hypothesis is that HCV subverts a cellular splicing mechanism that modulates the unfolded protein response in the endoplasmic reticulum. Secondly, it will be examined whether highly abundant circRNAs and IRES-containing circRNAs have functional roles on HCV gene expression in infected cells. The functions of IRES-circRNA translation products in the viral infectious cycle will be examined both in cultured liver cells and human liver organoids that can be infected with autologous HCV. The expected outcomes of this application will address fundamental aspects about the functions of novel circular HCV-derived RNAs in the viral infectious cycle. This proposed research is innovative because it will examine whether novel virus-derived circRNAs can be targeted in antiviral approaches in RNA virus-infected cells.