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The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates. / Chemistry

Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate. / Chemistry

Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen. / Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.

Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...

Diverses études ont souligné le rôle prépondérant du dialogue entre les cellules NK et les cellules dendritiques au cours des réponses immunes. Cependant, les récepteurs impliqués dans ce processus restent incertains. Au cours de ce travail, nous nous sommes attachés à identifier les récepteurs mis en jeu lors de la reconnaissance virale à l’aide de modèles humains et murins. Pour cela, nous avons mimé l’infection virale en utilisant deux ARN bicaténaires synthétiques – poly(AU) et poly(IC) – et montré qu’ils sont tous deux capables d’activer TLR3 mais que seul poly(IC) engage les hélicases RIG-I et MDA5. Les deux ARN induisent l’activation des cellules NK au sein des PBMC humaines, mais seul poly(IC) induit la production d’IFN-gamma. Les DC myéloïdes (mDC) sont requises pour cette activation sans nécessité d’un contact cellulaire entre les cellules NK et les mDC. En outre, les IFN de type I et l’IL-12 secrétés par les DC sont respectivement nécessaires à l’initiation du potentiel lytique et à la production d’IFN-gamma. Poly(IC), au contraire de poly(AU), a une action synergique avec l’IL-12 produite par les mDC pour induire la production d’IFN-gamma en agissant directement sur les cellules NK. Enfin, l’activation conjointe de TLR3 et des hélicases RLR sur les mDC et RIG-I sur les cellules NK, nécessaire à la production d’IFN-gama en réponse à l’ARN bicaténaire, a été confirmée à l’aide de souris déficientes pour TLR3 et Cardif et d’un ligand spécifique de RIG-I. En conclusion, nous rapportons pour la première fois la nécessité pour un composé microbien d’engager deux familles de récepteurs sur deux populations cellulaires distinctes pour induire une réponse innée éfficace. / Crosstalk between NK cells and DC is critical for the response to the microbial mimic poly(IC) but the dsRNA receptors involved in each cell types remained to be defined. We show herein that two dsRNA, poly(AU) and poly(IC), similarly engaged TLR3 while only poly(IC) triggered the RIG-I and MDA-5 helicases. Both dsRNA triggered NK cell activation within PBMC but only poly(IC) induced IFN-gamma. mDC were required for NK cell activation by the two dsRNA, suggesting that they triggered at least TLR3 on mDC. DsRNA induction of cytolytic potential and IFN-gamma production in NK cells did not require contact with mDC but was dependent on the secretion of type I IFN and IL-12, respectively. Poly(IC) but not poly(AU) synergized with mDC-derived IL-12 for high IFN-gamma production by acting directly on NK cells. Finally, the requirement of TLR3 and the RLR on mDC and the involvement of the RIG-I but not TLR3 on NK cells for the production of IFN-gamma induced by dsRNA was confirmed using TLR3 and Cardif deficient mice and RIG-I specific activator. This cooperation was further confirmed using inactivated FLU virus infected-target cells both in human and mouse system demonstrating that NK cells were able to sense viral material by a direct transfer from infected cells likely through lytic immunological synapse without prior infection of NK cells. Thus, we report for the first time the requirement of cotriggering

Certains motifs moléculaires associés à la mort cellulaire semblent identifier les cancers prompts à répondre à une thérapie cytotoxique. Ceci en élaborant une réponse anti-tumorale basées sur une réponse T protectrice. Mon travail de thèse montre que le traitement par chimiothérapie immunogène active des voies moléculaires mimant une infection virale. Ceci conduit au niveau des cellules tumorales à une signalisation autocrine via l’IFNαβ / IFNAR1/2, initiée par la reconnaissance d’ARN double brin (dsRNA) endogène par les Récepteurs endosomaux de Reconnaissance des Motifs (PRRs). De façon plus détaillée, nous montrons que les axes TLR3/TRIF (senseurs endosomaux de dsRNA) et IFNAR1/2 (Récepteurs de l’IFN de Type I) doivent signaliser au niveau de la cellule tumorale pour que la chimiothérapie puisse aboutir à l’induction de l’axe CXCL10/CXCR3 et éliciter une réponse efficace in vivo. L’analyse du profil ARN de cellules tumorales Tlr3+/+ (mais pas Tlr3-/-) exposées aux anthracyclines a révélé une forte empreinte virale/IFN, indispensable à l’efficacité/activité anti-tumorale. Le fait d’affecter les axes TLR3 ou IFNAR1/2 au niveau tumorale soit à l’aide d’anticorps neutralisants, soit à l’aide de modèles KO abroge le relarguage de CXCL10 induit par la chimiothérapie, et ainsi la capacité à contrôler la pousse tumorale à moins que de l’IFNαβ ou du CXCL10 exogène soit co-administré aux anthracyclines. De plus la chimiorésistance des tumeurs traitées par des molécules n’induisant pas de signature virale peux être réversée par de l’IFN de Type I exogène. Enfin, la détection d’une signature IFN au niveau de biospies de cancers du sein humains permet de prédire la bonne réponse au traitement adjuvant par anthracyclines. D’un point de vue de l’évolution, alors que les tumeurs (comme les virus) ont élaboré des mécanismes pour échapper aux réponses IFN, la signature virale induite par la chimiothérapie devrait contribuer à contrecarrer cette immunoédition. / Distinct cell death-associated molecular patterns might define cancers proned to respond to a cytotoxic therapy by mounting a protective T cell-based anticancer immunity. My PhD Thesis work shows that immunogenic chemotherapy phenocopies viral infection leading to autocrine IFNαβ/IFNAR1/2 signalling in tumor cells initiated by recognition of self dsRNA by endosomal pattern recognition receptors (PRRs). In detail, TLR3/TRIF (endosomal dsRNA sensors) and IFNAR1/2 (Type I IFN receptors) must signal within the tumor cells so that chemotherapy can induce downstream CXCL10/CXCR3 axis and elicit therapeutic responsiveness in vivo. RNA profiling of Tlr3+/+ (but not Tlr3-/-) tumor cells exposed to anthracyclines revealed a strong IFN/viral fingerprint, indispensable for the tumoricidal activity. Neutralization by antibodies or genetic defects affecting tumor –associated TLR3 or IFNAR1/2 compromised chemotherapy-induced CXCL10 release and tumor control unless exogenous IFNαβ or CXCL10 are concomitantly supplied to anthracyclines. Moreover, chemoresistance of tumors treated by drugs failing to induce a viral signature can be reversed by exogenous Type I IFN. Finally, the IFN fingerprint of human breast cancers allowed to predict tumors proned to benefit from adjuvant anthracyclines. From an evolutionary viewpoint, while tumors (like viruses) have evolved mechanisms to evade an IFN response, chemotherapy-induced viral mimicry might contribute to bypass such as immunoediting.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013