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

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.

No description available.

Etwa 3% der Weltbevölkerung sind von dem Hepatitis-C-Virus-Infektion betroffen. Phänotyp der HCV-induzierten Lebererkrankung variiert stark von einem Patienten zum anderen. Die Wahrnehmung der viralen doppelsträngigen RNA (dsRNA) und einzelsträngigen RNA (ssRNA) durch den Toll-like-Rezeptor 3 (TLR3) bzw. TLR7 scheinen an der Früherkennung der Pathogene und an der Wirtsantwort auf viraler Infektion beteiligt zu sein. Darüber hinaus ist die membran-assoziierte Form des Endotoxin-Rezeptor-Bestandteils CD14 (mCD14) mit TLR3 in Intrazellulärräumen kolokalisiert und erweitert die dsRNA-Erkennung und TLR3-Signalleitung. Die vorliegende Arbeit analysiert epidemiologische und klinische Daten von Patienten kaukasischer Abstammung mit einer chronischen Hepatitis C in Bezug auf bestimmte Einzellnukleotidpolymorphismen (SNPs) mit relevanten minor allele frequencies (MAFs) in Genen, die für obengenannte Rezeptoren kodieren. Es wurde keine Assoziation von dem TLR3-Promotor-Polymorphism rs5743305 (T/A) mit TLR3-Genexpression gefunden, weder in peripheren mononukleären Zellen des Blutes (PBMCs) noch in der Leber; keine weitere Korrelation mit epidemiologischen und klinischen Parametern der chronischen Erkrankung waren zu beobachten. Andererseits, T-homozygote Patienten am rs3775291-(C/T)-Polymorphismus (der in Exon 4 lokalisierter nicht-synonymer SNP) zeigen Tendenz zu einer höheren TLR3-Genexpression in der Leber. Außerdem, unter HCV-subtyp-1a-infizierten Patienten sind keine T-Homozygoten zu finden. Im Unterschied zur Lage bei alkoholischer Lebererkrankung wurde in chronischen Hepatitis-C-Patienten keine Assoziation zwischen den Fibrosegrad und CD14-Gen-C-159T-Polymorphismus gefunden. Bei T-homozygoten Patienten wurden jedoch häufiger portale lymphoide Aggregaten gefunden als bei C-Allele-Trägern. Außerdem das Vorhandensein von portalen lymphoiden Aggregaten korrelierte eng mit der Leberentzündung und mit Gallengangsläsionen. Am Ende wurde der funktionelle nicht-synonyme SNP in Exon 3 des X-gekoppelten TLR7 Gens, rs179008/Gln11Leu, untersucht. Die Analyse war auf homo- und hemizygoten Personen, die mittels Allelspezifischentranskriptquantifizierung (ASTQ) in heterozygoten weiblichen Personen eingeordnet wurden, eingeschränkt. Es zeigte sich dabei ein individueller verzerrter Mosaizismus in PBMCs. Das variante T-Allel war nur mit der Anwesenheit der portalen lymphoiden Aggregaten assoziiert. Hepatische Viruslast und Expression der Gene, die bekannterweise bei einer chronischer HCV-Infektion induziert sind, unterschieden sich zwischen Wildtyp- und Variantallelträger nicht. Jedoch eine signifikant niedrigere Expression der interleukin-29 (IL-29)/lambda1 interferon (IFN-λ1) und beider Untereinheiten seines Rezeptors (IL-10 Rβ and IL-28Rα) war bei T-homo- und hemizygoten Patienten zu beobachten. Diese Tatsache könnte eher eine Auswirkung auf die Ansprechbarkeit auf zukünftige IFN- λ-basierte Therapie haben, als auf eine Vorhersage des Ausgangs der gängigen IFN-α-basierten Therapie.

No description available.

Afin de se répliquer et de se propager efficacement, les virus ont développé de multiples stratégies leur permettant d’échapper au système de défense innée : le système IFN de type I. Ce travail de thèse a alors consisté à étudier les interactions entre protéines virales et protéines de ce système de défense afin de mieux comprendre les mécanismes de subversion virale et d’identifier d’éventuelles cibles cellulaires thérapeutiques. La reconstruction d’un réseau d’interactions entre ces protéines nous a permis d’identifier des stratégies différentielles de subversion pour 4 familles virales et de montrer un ciblage massif et significatif des protéines du système IFN de type I par les virus. Les protéines en interaction directe avec ces protéines sont également fortement touchées par les virus et sont de potentiels modulateurs du système IFN de type I. Parmi ces modulateurs, le processus biologique sur-représenté est le transport nucléocytoplasmique et la protéine KPNA1 impliquée dans ce processus a retenu notre attention. L’étude fonctionnelle de l’interaction entre la protéine KPNA1 et la protéine NS3 du VHC a montré que la protéine NS3 associée à son cofacteur NS4A inhibe partiellement la réponse IFN de type I en empêchant l’import nucléaire de STAT1. Ce phénotype pourrait résulter de la dégradation de KPNA1 par NS3/4A. Par ailleurs, l’identification de nouveaux inter-acteurs de la protéine NS1 du virus influenza par criblage double-hybride levure a révélé la protéine induite par les IFN de type I, ADAR1, comme partenaire de la protéine NS1 de multiples souches virales et nous avons montré qu'ADAR1 est un facteur pro-viral dont la fonction editing est activée par NS1 / To replicate and propagate efficiently, viruses have developed multiple strategies allowing them to escape the innatedefense system: the type I IFN system, This work of thesis then consisted in studying the interactions between viralproteins and proteins of this defence system in order to understand better the mechanisms of viral subversion andidentifY possible therapeutic cellular tatgets. The reconstruction of a network of interacting proteins involved in the typeI IFN system allowed us to identifY differentiai subversion strategies for 4 viral families and to show a massive andsignificant targeting of proteins of the type I IFN system by viruses. Proteins directly interacting with the type Iinterferon system network are also strongly targeted by viruses and are potential modulators of the type I IFN system.Among these modulators, the most tatgeted function conesponds to the transport of NLS-bearing substrates to thenucleus and the KPNAI protein involved in this process held our attention. The functional study of the interactionbetween KPNA1 and NS3 protein of the HCV showed that NS3 protein associated with its cofactor NS4A inhibitsprutially the type I IFN response by preventing the nuclear translocation of ST A Tl. This phenotype could result fromthe degradation of KPNAI by NS3/4A. Besides, the identification of new cellular prutners ofNS 1 prote in of influenzavirus by yeast two-hybrid screens revealed ADARI, an interferon-stimulated prote in, as partner of NS 1 of ali testedvirus strains and we showed that ADARI is an essential host factor for viral replication and its editing function isactivated by NS 1 protein

Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) which has had a severe impact on the urban landscape in Canada. This research program focused on developing molecular genetic strategies to control this pathogenic fungus. The first strategy involved the development of RNA interference (RNAi) for the down-regulation of genes involved in pathogenicity. An efficient RNAi cassette was developed to suppress the expression of the endopolygalacturonase (epg1) locus which encodes a cell-wall degrading enzyme. This epg1-RNAi cassette significantly reduced the amount of polygalacturonase activity in the fungus and resulted in almost complete degradation of epg1 mRNA. The need for a native promoter to selectively down-regulate specific gene loci was addressed by developing a carbon-catabolite regulated promoter (alcA) to drive the expression of the epg1-RNAi cassette. The expression of an alcA-driven epg1-RNAi cassette resulted in the down-regulation of epg expression under glucose starvation but normal levels of expression in high glucose. The expression could therefore be controlled by culture conditions. The second strategy explored the potential of using dsRNA viruses to vector disruptive RNAi cassettes. An isolate of O. novo-ulmi strain 93-1224 collected in the city of Winnipeg, was infected by two dsRNA mitoviruses which upon sequence characterization were named OnuMV1c and OnuMV7. To assess the transmissibility of this dsRNA virus the infected isolate 93-1224 was paired with three naive isolates of the related fungi O. ulmi and O. himal-ulmi. Through the use of nuclear and mitochondrial markers it was determined that the virus OnuMV1c may not rely on mitochondrial fusion for transmission but may have a cytoplasmic transmission route. This investigation of gene expression and manipulation has provided tools to help understand gene regulation in O. novo-ulmi. It has also added to our knowledge of mitoviruses, their transmission and potential use as a biological control. By enhancing our understanding of transmissible hypovirulence this work contributes to efforts to develop a new approach to target DED as well as a potential model for the control of other fungal diseases. / Graduate / 0307 / 0306 / 0369 / jscarneiro@hotmail.com