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Humane Coronaviren sind wichtige Pathogene, die vor allem mit respiratorischen (z.B. SARS) und enteralen Erkrankungen assoziiert sind. Coronaviren besitzen das größte gegenwärtig bekannte RNA-Genom aller Viren (ca. 30 Kilobasen). Die Replikation des Genoms und die Synthese zahlreicher subgenomischer RNAs, die die viralen Strukturproteine und einige akzessorische, vermutlich virulenzassoziierte, Proteine kodieren, erfolgt durch die virale Replikase. Die coronavirale Replikase ist ein Multienzym-Komplex, der durch die proteolytische Prozessierung großer Vorläuferproteine (Polyproteine pp1a und pp1ab) entsteht und 16 virale Nichtstrukturproteine (nsp), aber auch einige zelluläre Proteine, beinhaltet. Obwohl die Charakterisierung der Funktionen der einzelnen Proteine und das Verständnis der molekularen Grundlagen der coronaviralen Replikation noch in ihren Anfängen stecken, ist bereits jetzt klar, dass die an der Replikation beteiligten Mechanismen deutlich komplexer sind als bei den meisten anderen RNA-Viren. Man hofft, dass aus der Untersuchung der einzelnen an der Replikation beteiligten Proteine Erkenntnisse zu den Besonderheiten des Lebenszyklus dieser ungewöhnlich großen RNA-Viren abgeleitet werden können und dass sich daraus auch Ansatzpunkte für die Entwicklung von Inhibitoren einzelner Proteine/Enzyme ergeben, die für eine zukünftige antivirale Therapie genutzt werden könnten. In der vorliegenden Arbeit wurden zwei enzymatische Aktivitäten von Coronaviren, eine Helikase und eine Endonuklease, die Teil der coronaviralen Nichtstrukturproteine nsp13 bzw. nsp15 sind, in vitro untersucht. Zur Etablierung allgemeingültiger Prinzipien coronaviraler Enzymaktivitäten wurden die homologen Proteine von HCoV-229E und SARS-CoV, also von Vertretern unterschiedlicher serologischer und genetischer Coronavirus-Gruppen, parallel untersucht und ihre Eigenschaften miteinander verglichen. Die nsp13-Helikase des SARSCoronavirus wurde als bakterielles Fusionsprotein exprimiert, und die nsp13-Helikase des humanen Coronavirus 229E wurde in Insektenzellen mittels baculoviraler Vektoren exprimiert. Beide Proteine zeigten Polynukleotid-stimulierbare NTPase- und 5'-3'-Helikase-Aktivitäten. Darüber hinaus besaßen sie vergleichbare Hydrolyseaktivitäten gegenüber den 8 getesteten Ribound Desoxyribonukleosidtriphosphaten. Die Anwesenheit von poly(U) führte zu einer 3-fachen Erhöhung der katalytischen Effizienz (kcat/Km) und einer etwa 100-fachen Steigerung der Hydrolysegeschwindigkeit (kcat). Es wurde am Beispiel von HCoV-229E-nsp13 gezeigt, dass Nukleinsäuresubstrate mit hoher Affinität (K50 ≈ 10-8 M), jedoch ohne erkennbare Präferenz für einzel- oder doppelsträngige DNA- oder RNA-Substrate gebunden werden. Solch eine feste Bindung ist typisch für Enzyme, die prozessiv mit Nukleinsäuren interagieren. Sie korreliert darüber hinaus mit der beobachteten effizienten Entwindung (Trennung) von RNA- und DNADuplexen mit langen, doppelsträngigen Bereichen von 500 Basenpaaren und mehr. Dies legt eine Funktion als replikative Helikase nahe, wie sie beispielweise bei der effektiven Entwindung doppelsträngiger replikativer Intermediate benötigt werden könnte. In dieser Arbeit wurde darüber hinaus eine neue enzymatische Aktivität coronaviraler Helikasen entdeckt. Die gefundene RNA-5'-Triphosphatase-Aktivität nutzt das aktive Zentrum der NTPase-Aktivität und katalysiert wahrscheinlich die erste Reaktion innerhalb der Synthese der Cap-Struktur am 5’- Ende viraler RNAs. Die sehr ähnlichen biochemischen Eigenschaften der HCoV-229E- und SARS-CoV-Helikasen lassen vermuten, dass die Enzymologie der viralen RNA-Synthese (trotz relativ geringer Sequenzidentität der beteiligten Enzyme) unter den Vertretern unterschiedlicher Gruppen von Coronaviren konserviert ist. Der zweite Teil der Arbeit beschäftigte sich mit der biochemischen Charakterisierung des Nichtstrukturproteins nsp15, für das eine Endonuklease-Aktivität vorhergesagt worden war. Auch in diesem Fall wurden die entsprechenden Proteine von HCoV-229E und SARS-CoV charakterisiert. Beide (bakteriell exprimierten) Enzyme zeigten identische enzymatische Eigenschaften. In-vitro-Experimente bestätigten, dass diese Proteine eine Mn2+-abhängige RNA- (jedoch nicht DNA-) Endonukleaseaktivität besitzen. Sie spalten doppelsträngige RNA deutlich effektiver und spezifischer als einzelsträngige RNA. Die Enzyme spalten an Uridylat-Resten und erzeugen Produkte mit 2', 3'-Zyklophosphat-Enden. Bei doppelsträngigen RNA-Substraten wurde eine Spezifität für 5'-GU(U)-3' gefunden. Die Tatsache, dass diese Sequenz in den nidoviralen transkriptionsregulierenden Sequenzen (TRS) der Minusstränge konserviert ist und auch die Endonuklease bei allen Nidoviren konserviert ist, unterstützt die Hypothese, dass die Endonukleaseaktivität eine spezifische Funktion innerhalb der coronaviralen (nidoviralen) diskontinuierlichen Transkription besitzt. / Human coronaviruses are important pathogens that are mainly associated with respiratory (e.g. SARS) and enteric diseases. With genome sizes of about 30 kilobases, coronaviruses are the largest RNA viruses currently known. The replication of the genome RNA and the synthesis of multiple subgenomic (sg) RNAs, which encode structural and accessory (probably virulenceassociated) proteins, is mediated by the viral replicase. The coronaviral replicase is a multienzyme complex, which is produced from viral precursor polyproteins (pp1a and pp1ab) that are autoproteolytically processed into 16 nonstructural proteins (nsp). It also involves several cellular proteins. Although the functional characterization of most of these proteins and, more generally, the understanding of the molecular mechanisms involved in coronavirus replication are still at an early stage, it is already clear that these mechanisms are much more complex than those used by most other RNA viruses. The investigation of the proteins involved in virus replication is anticipated to result in a better understanding of the specific features of the replication cycle of these unusually large RNA viruses, potentially providing novel approaches to the development of enzyme (protein) inhibitors that, in the long run, may be developed into drugs suitable for antiviral therapy. In this work, two coronavirus enzymatic activities, a helicase and an endonuclease, residing in the coronavirus nonstructural proteins nsp13 and nsp15, respectively, were investigated in vitro. In order to establish potentially existing common principles of coronavirus enzymatic activities, the homologous proteins of HCoV-229E and SARS-CoV, which belong to different serological and genetic coronavirus groups, were studied in parallel and their properties were compared with each other. The SARS-CoV helicase was expressed in bacteria as a fusion protein and the helicase of HCoV-229E was expressed in insect cells using baculovirus vectors. Both proteins were shown to have polynucleotide-stimulated NTPase and 5’-to-3’ helicase activities. Furthermore, they had comparable hydrolysis activities with all eight (natural) ribo- and deoxyribonucleoside triphosphates. The presence of poly(U) led to a 3-fold increase of the catalytic efficiency (kcat/Km) and an about 100-fold acceleration of the hydrolysis rate (kcat). Using HCoV-229E nsp13 as an example, it was shown that the coronavirus helicase has a high binding affinity for nucleic acids (K50 ≈ 10-8 M). No preference for single-stranded (ss) versus double-stranded (ds) substrates could be established for this protein. Such a tight binding is typical for enzymes acting highly processively on nucleic acids (e.g., polymerases). Furthermore, coronavirus helicases proved to be able to unwind long RNA and DNA duplexes (of 500 bp and more) highly effectively. Together, these data support the idea that coronavirus nsp13s are “replicative helicases” that are involved in the unwinding of long double-stranded replicative intermediates. In this study, yet another enzymatic activity, namely an RNA-5’-triphosphatase activity, was established for coronaviral helicases. The activity, which employs the NTPase active site, probably mediates the first step in the formation of the 5’-cap structures present on coronaviral RNAs. The HCoV-229E and SARS-CoV helicases were found to have very similar biochemical features, suggesting that, despite the relatively low sequence identity among these enzymes, the enzymology involved in viral RNA synthesis is well conserved among members of different coronavirus groups. The second part of the study was devoted to the biochemical characterization of coronavirus nsp15, a protein with predicted endonuclease activity. Also in this case, the homologous proteins from HCoV-229E and SARS-CoV were studied in parallel. Bacterially expressed forms of both enzymes showed essentially identical enzymatic properties. In vitro experiments confirmed that nsp15 possesses a Mn2+-dependent RNA (but not DNA) endonuclease activity. The proteins cleaved double-stranded RNAs much more effectively and specifically than ssRNA substrates. Cleavage was shown to occur at uridylates, generating products with 2’,3’-cyclophosphates. In the case of dsRNA substrates, nsp15 was confirmed to be specific for 5’-GU(U)-3’ sequences. The fact that (i) the GUU sequence is conserved among the negative-strand complements of coronavirus transcription-regulating sequences and (ii) the endonuclease domain is conserved among all nidoviruses supports the hypothesis that the endonuclease activity has a specific function in coronavirus (nidovirus) discontinuous transcription.

Influenza A is a single-stranded, multi-segmented, negative sense RNA virus of the family Orthomyxoviridae and is the causative agent of seasonal Influenza. Influenza viruses cause significant impacts on a global scale regarding public health and economics. Annual influenza virus infections in the United States account for over 200,000 hospitalizations, up to 49,000 deaths, and an $87.1 billion economic burden. Influenza A virus has caused several pandemics since the turn of the 20th century. The effects of Influenza on public health and economics, compounded with low efficacy of the annual vaccine and emerging antiviral resistance, brings to light the need for an effort to stem these impacts, prevent pandemics, and protect public health by developing novel treatments. This project proposes an alternative approach to combatting Influenza by targeting host factors hijacked during infection that, if inhibited, significantly impair viral RNA expression, but result in low host toxicity. The host factors we examined include RNA export factors (XpoT and Xpo5) and RNA helicases (UAP56 and URH49). We selected paralogs URH49 (DDX39A) and UAP56 (DDX39B) because previous studies suggest differing roles during infection, but we theorize that their high degree of sequence similarity, similar function, and association with many of the same cellular factors may allow them to substitute for one another if one is inhibited. CRISPR was considered as the primary method to evaluate the effect of knockout of these factors on viral RNA expression and host cell toxicity. CRISPR is an RNA-guided mechanism for gene editing and can be used to make null mutations in targeted host genes. However, CRISPR proved to be a significant challenge and, while we could not conclusively confirm whether the CRISPR plasmids were effective at targeting our genes of interest, our initial results were not promising and we did not pursue this approach further. As an alternative, host RNA export factors were evaluated using siRNA to knockdown the factor prior to influenza infection. RNA was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The potential of inhibiting UAP56 or URH49 as a novel therapeutic target was determined using a visual assessment of cell death. We found that siRNA-mediated knockdown of XpoT and Xpo5 did not have any impact on viral RNA synthesis early during infection. siRNA against UAP56 and DDX39 (targets both UAP56 and URH49) resulted in significant impairment in viral RNA synthesis, confirming previously established work suggesting that UAP56 and URH49 have important roles during infection. Importantly, these helicases play an interferon (IFN) independent role to enhance viral replication, as indicated by analysis in IFN deficient VERO cells. A viability assay relying on trypan blue exclusion did not yield trustworthy results, so a visual assessment of cell death was done. The visual assessment confirms previously-established observations that Nxf1 siRNA treatments result in a high degree of cell death, indicating the toxic nature of Nxf1 inhibition. Cells treated with UAP56 or DDX39 siRNAs demonstrated little to no additional toxicity compared to the non-target control, suggesting they can be inhibited to serve as antiviral targets.

Steroid hormones and receptors play a role in regulating biological events underlying brain development and sexual differentiation. Current evidence indicates that circulating sex steroid hormones are not entirely responsible for development of neural sex differences in song birds such as the zebra finch. p68, as a coactivator specific for estrogen receptor alpha (ERα) and an essential factor in early tissue development and maturation might play a role in sexual differentiation. Zebra finches have a sexually dimorphic song control nuclei in the brain, males have larger song nuclei than females, and are ideal model for investigating the mechanisms controlling sexual differentiation of the brain and behavior. Western blot analysis showed a significant sex difference at post hatch day 10 (P10). Immunohistochemistry showed localization of p68 immunoreactive cells in the ZF brain including nuclei that compose the avian song system. p68 is probably developmentally regulated and may be modulated by endogenous estrogen and estrogen receptors suggesting a role for p68 in sexual differentiation. INDEX WORDS: p68, coactivator, RNA helicase, steroid receptor, song control nuclei, zebra finch (ZF)

<p>Topoisomerase III&alpha; (Top3&alpha;) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans. Previous work has shown that Top3&alpha; and Bloom's helicase (Blm) are able to convergently migrate the dHJ to create solely non-crossover products, thus preserving genomic integrity. However, many questions remain about the details of this process. Using a combination of biochemical and genetic tools, including dHJ substrate assays, gel electrophoresis, EMSA, pulldowns, fly crosses, and electron microscopy, this work expands our knowledge of the dissolution reaction. Tail mutants of Top3&alpha; were created and tested in a series of <italic>in vitro</italic> assays. Through these experiments, I discovered that the C-terminus of Top3&alpha; is important for binding Blm, interacting with DNA, conveying RPA stimulation, and <italic>in vivo</italic> functionality. I also observed that dissolution is an extremely processive reaction, with no accumulation of intermediates prior to product formation. When a non-specific topoisomerase was used (Top1, a type IB), accumulation of an intermediate was evident; however, contrary to predicted models, direct observation revealed that this intermediate is not a hemicatenane structure and still requires branch migration. Modifications were also made to the dHJ substrate creation method so that multiple types of HJ substrates could be produced efficiently.</p> / Dissertation

<p>RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.</p> / Dissertation

Univ., Diss., 2006--Kassel

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Genetic competence is a developmental process in bacteria that allows natural transformation. Competent Gram positive bacteria such as Bacillus subtilis carry a cytosolic helicase which is required for efficient transformation. In this work ComFA is confirmed as a DEAD-box helicase. I also describe a new accessory motif in ComFA that contributes to transformation independently of the helicase activity in ComFA. The newly discovered metal-binding motif consists of four cysteines which are required for transformation and zinc binding. While the zinc finger is required for full function, it is not required for DNA binding. As DEAD-box family helicases are generally non-processive, it appears that at least part of the rapid DNA uptake process is mediated by a non-processive helicase. Active uptake using the ComFA helicase motor may be required to maintain the integrity of the incoming DNA to allow subsequent recombination.

Cardiomyopathies are the most common form of genetic disorders featuring primary abnormalities in the structure and function of the heart. Over the past few decades, tremendous progress has been made in elucidating the genetic basis of cardiac disorders. However, the development of specific and effective therapies remains largely limited due to the lack of suitable therapeutic targets. Nucleoli are polyfunctional subnuclear domains that are heavily involved in ribosomal RNA production. Recent studies have identified nucleolar structure perturbations and functional defects associated with different types of cardiomyopathies. Additionally, several mutations have been identified in several ribosomal genes that are linked to cardiomyopathy in human patients. We previously identified a nucleolar DEAD-box RNA helicase, DDX27, as a critical regulator of myogenesis. This study aimed to investigate the role of ddx27 deficiency in cardiac muscle and expand the understanding of DDX27 mediated pathways that are involved in myopathies. In this study, we used zebrafish models to investigate ddx27 deficiency in cardiac muscle. Phenotype characterization, cardiac function testing, transmission electron microscopy and histological analysis of ddx27 mutants revealed corresponding dilated cardiomyopathy and skeletal muscle hypotrophy. Furthermore, knockdown of DDX27 ortholog, Rs1, in cardiac muscle was fatal for Drosophila larvae. However, other tissues (i.e., neural or gastrointestinal) were unaffected suggesting that abnormalities caused by Ddx27 deficiency are specific to cardiac and skeletal muscle. Immunofluorescence, northern blotting and polysomal profiling of ddx27 zebrafish myofibers revealed that DDX27 is necessary for preserving nucleolar architecture and ribosome biogenesis. Here we have shown that DDX27 is essential for normal function of cardiac and skeletal myogenic processes due to its critical role in ribosomal regulation. Additionally, we provide novel evidence for DdX27 deficiency contributing to dilated cardiomyopathy. Overall, the findings of this study provide further evidence for the role of RNA helicases, specifically DDX27, in cardiac and skeletal muscle pathogenesis as well as provide novel insight into the molecular pathways of therapeutic benefit for afflicted patients of these diseases. / 2022-06-04T00:00:00Z