archives@tulane.edu / A-to-I RNA editing, catalyzed by a family of enzymes called adenosine deaminases acting on RNA (ADARs), brings broad significance in various biological processes. To date, the roles of ADARs and its associated RNA editing in Epstein-Barr virus (EBV)’s life cycle and pathogenesis are still largely unknown. To fill this significant knowledge gap, we utilized our well-established next-generation RNA sequencing-based computational approaches and traditional molecular biology methodologies to elucidate the triangle relationship between ADARs, RNA-editing, and EBV infection. The expression of ADARs was first evaluated in a cohort of EBV-associated lymphoma cells. A constitutive expression of ADAR1, the predominant form of ADARs, was observed in the examined cells. In synchronous EBV reactivation cell models, we found that EBV reactivation led to a decreased expression of ADAR1 as well as a global suppression of A-to-I RNA editing. Further, we found that expression of the key viral trans-activator Zta inhibited ADAR1 expression in EBV-associated lymphoma cells. Analyses of the ADARs-mediated RNA editing events revealed novel editing sites on viral lytic transcripts. Knockdown of ADAR1 led to a global suppression of RNA-editing accompanied by a more robust EBV reactivation. Meanwhile, the enhanced expression of ADAR1 inhibited Zta’s expression and transactivation function. Together, our findings reveal a novel mechanism controlling the balance of EBV life cycle, in which ADAR1 and associated RNA editing events help maintain the viral latency by silencing Zta; whereas a bona fide lytic signal leads to high-level Zta expression by inhibiting ADAR1 and ADARs-mediated RNA editing. / 1 / Yi Yu

Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from ​Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...

Viruses are obligate intracellular parasites that replicate utilizing the resources of host cells. The replication of positive sense RNA viruses is coupled with alterations to host cell membranes. These viruses are believed to replicate efficiently by using co-opted membrane structures assembled from viral and host cell proteins and lipids. Poliovirus is a prototypical positive-sense RNA virus, however the topological details of viral RNA replication are not well understood. In this work we use electron cryotomography, among other methods, to examine the ultrastructure of fractionated poliovirus RNA replication factories that were formed within infected cells, and to investigate oligomeric interactions within a three dimensional crystal formed by a poliovirus polymerase point mutant. Investigation of the ultrastructure of isolated viral RNA replication factories shows that the low resolution features of cryopreserved membrane structures are essentially identical to previously observed structures within plastic sections of infected cells. Furthermore, greater detail visible using electron cryotomography reveals pore-like structures and other high energy membrane conformations within the replication factories. We see a mix of single, double, and multi-membrane structures that are arranged with openings that connect their interior lumenal space to the exterior environment. The lumen of some of these membranous structures contains a linear polymeric density thought to be RNA. We conclude that the RNA replication of poliovirus may occur on the lumenal surface of vesicular membranes with an opening to the cytoplasm for metabolite and product exchange. Within the poliovirus replication machinery, the principal component is the RNA polymerase 3Dpol. This prototypical RNA-dependent RNA-polymerase forms homo-oligomeric interactions that are key to its functions. To investigate these interactions, previous studies focused on hollow helical structures formed by wild-type polymerase. Here, we investigate the structure of small three-dimensional crystals formed by 3Dpol with a mutation of a single residue, lysine 314, to alanine. Using electron cryotomography and volume averaging, we demonstrate that the crystal packing within this point mutant does not include physiological polymerase-polymerase interactions. Elucidation of the topology of poliovirus replication machinery provides a basis for future development of antiviral therapeutics.

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Desulfovibrio vulgaris strain Hildenborough is a key organism in the bioremediation of environments contaminated with anthropogenic sources of heavy metals and radionuclides. In order for D. vulgaris to be optimized for remediation of such sites in the environment, it is necessary to understand the organism's response to key stressors that are found in conjunction with both heavy metals and radionuclides. Few regulators have been discovered in this bacterium and even less is known about its genetic regulatory response to changing conditions in its environment. This dissertation project aimed to use computational, molecular, and culture-based methods to identify novel, small, regulatory RNAs (sRNAs)--an important class of cellular regulators that have yet to be studied in D. vulgaris--and to determine their effect on cellular processes within the organism. Computational and transcriptomic high-throughput sequencing resulted in over 200 high-quality putative sRNAs with the expression of sixteen of these sRNAs verified by Northern blot analysis. Eight of the confirmed sRNAs were selected and further characterized within this study resulting in three trans--or intergenic--encoded sRNA, three cis--or antisense--encoded sRNAs, and two sRNAs that may encode small proteins. These eight sRNAs included growth phase-specific sRNAs, differentially expressed sRNAs, as well as sRNAs processed into multiple forms. Molecular techniques such as quantitative reverse-transcriptase PCR (qRT-PCR) indicated differential expression of the confirmed sRNAs under a variety of stress conditions. Culture-based methods confirmed growth deficiencies in deletion and over-expression mutants of the sRNAs tested under stressors suggested during qRT-PCR analysis. Both of these tests suggested that the sRNAs discovered in this study participate within the cellular response of the bacterium to environmental stress. Computational methods were employed to reveal likely putative mRNA targets for the confirmed sRNAs in order to determine interactions and function within the regulatory networks. This study presents the first evidence of the existence and expression of sRNAs within D. vulgaris Hildenborough. Furthermore, the information gathered about the sRNAs analyzed in this work indicates that sRNAs are an important part of the regulatory response mechanism of D. vulgaris. Further research into target identification and interaction can close the loop of these regulatory networks and solidify the role of sRNAs in D. vulgaris.

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