Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...

Mycoviruses are viruses that infect and replicate in fungal cells, but unlike most known viruses of plants and animals, they exceptionally produce deleterious effects on their host. Nonetheless, the last discoveries showed that some mycoviruses can decrease the virulence of their phytopathogenic fungal hosts, making them very attractive for their possible use as biological control agents. Most mycoviruses have dsRNA genomes and are widespread in all major taxa of fungi. Beauveria bassiana is one of the most studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as biocontroller against invertebrates in agriculture. In the present work, a collection of 137 isolates of B. bassiana obtained at different locations and from different habitats in the Czech Republic was analysed. These isolates were analysed for the presence of dsRNA elements indicative of viral infections. The results revealed a high prevalence of viral infections in Czech B. bassiana isolates, with 22.6% of the isolates containing dsRNA elements with viral characteristics. Obtained dsRNA electropherotypes showed that virus diversity in infected isolates was high and that mixed virus infections occurred among them. Based on the characteristics of the electrophoretic band patterns, it could be hypothesized that B. bassiana isolates collected in the Czech Republic could harbour members of the viral families Totiviridae, Partitiviridae, Chrysoviridae and Hypoviridae.

African horsesickness virus (AHSV) is a dsRNA virus that belongs to the Orbivirus genus within the Reoviridae family. Each of the ten viral dsRNA segments encodes one virus-specific protein. During its life cycle AHSV replicates both in an insect vector and in a mammalian host, but while it has no detrimental effect on insect cells the virus is highly pathogenic to mammalian cells. It is postulated that this relates to different viral release mechanisms. Currently the main candidate for mediating viral release in both insects and mammals is the viral nonstructural protein NS3. In bluetongue virus (BTV), the prototype virus of the Orbivirus genus, it has been shown that NS3 interacts with both the viral outer capsid protein VP2 and a cellular exocytosis protein. For AHSV, we investigated whether the same mechanism was involved in viral release. This study aimed to identify and map possible protein-protein interaction between AHSV NS3 and VP2, and AHSV NS3 and unknown insect cellular proteins. For investigating the NS3-VP2 interactions a eukaryotic expression system (yeast twohybrid), a column binding assay utilising bacterially expressed NS3 and recombinant baculovirus expressed VP2 as well as a membrane flotation assay utilising recombinant baculovirus expressed VP2 and NS3-GFP, were used. A number of problems were encountered and no conclusive results were obtained. For investigating viral-cellular protein interactions the yeast two-hybrid system was also used, utilising NS3 as bait to screen proteins expressed from a Drosophila cDNA library. Results showed an interaction between the N-terminal region of AHSV NS3 and ubiquitin, an essential protein for the trafficking and degradation of membrane proteins from the endoplasmic reticulum. It also acts as a sorting signal in both the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. It has been shown that ubiquitin could play a role in the pinching off of budding vesicles. An AHSV infected cell could therefore potentially use ubiquitin in its vesicular budding pathway, therefore giving the opportunity for viruses to use this to release them from the cell. The Hsp70 was another protein identified that interacts with AHSV NS3. This protein plays a role in folding reactions, protein translocation across membranes of organelles and protein assembly. It has been reported in other studies done that both ubiquitin and Hsp70 play roles in regulating the bioavailability of viral proteins, which could explain the different levels of NS3, high in insect cells and low in mammalian cells, which indirectly control the viral exit pathway used, budding versus lytic release. These results lay the foundation for explaining the potential role of NS3 in the AHSV life cycle in insect cells. / Dissertation (MS)--University of Pretoria, 2007. / Genetics / unrestricted

Cell wall is the major barrier in the delivery of biomolecules such as nucleic acids into the plant cell. Biological (bacteria or viruses) and biolistic (particle-based) methods are used to deliver nucleic acids into the plant cell. However, these methods have significant limitations when it comes to species range, scalability, and field assays. In this work, we report the use of layered double hydroxide (LDH) topically applied to deliver RNA molecules into the plant cell. LDH were assembled by methanol-based co-precipitation of magnesium and aluminum nitrate solution with sodium hydroxide and finally dispersed in deionized water. The assembled LDH were physically characterized by AFM, zeta-sizer and their binding to RNA was confirmed by gel electrophoresis. LDH complexed with double stranded RNA (dsRNA) was topically applied to Nicotiana benthamiana leaves. As a model system, virus specific dsRNA-LDH complexes were used to activate cellular RNAi machinery against Tomato Yellow leaf Curl Virus (TYLCV) in N. benthamiana plants. Our results demonstrated that topical application of the TYLCV specific dsRNA-LDH complexes reduce viral genome accumulation and viral symptoms development. Similarly, dsRNA-LDH protected plants produce typical leaves, flowers, and seeds, confirming efficient virus resistance compared unprotected TYLCV infected plants. Topical application and noninvasive delivery of nucleic acid has several advantages, as these methods are specie independent, easy to scale up, applied with low-pressure spray, requires no tissue culture and no sophisticated equipment. The LDH based noninvasive delivery of nucleic acids has the capability to overcome the cell wall barrier limitations and will open new opportunities to exploit the full potential of cellular machinery to produce resilient plants and insure sustainable food production.

No description available.

Submitted by VANESSA SANTOS MOURA null (vanessa_smoura@hotmail.com) on 2017-08-29T04:50:50Z No. of bitstreams: 1 Dissertação_Vanessa_Santos_Moura.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-29T18:18:44Z (GMT) No. of bitstreams: 1 moura_vs_me_jabo.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) / Made available in DSpace on 2017-08-29T18:18:44Z (GMT). No. of bitstreams: 1 moura_vs_me_jabo.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) Previous issue date: 2017-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O bolor verde e a podridão azeda destacam-se entre as doenças de pós-colheita em frutos cítricos, causados por Penicillium digitatum e Geotrichum citri-aurantii, diminuindo a qualidade e a quantidade dos frutos e, consequentemente, resultando em significativas perdas econômicas. Uma alternativa para controle destes fungos é através da toxinas killer produzidas por algumas espécies de levedura, capazes de matar fungos filamentosos. Saccharomyces cerevisiae produz toxinas killer proteicas que são letais para células sensíveis de levedura. Estas toxinas foram agrupadas em quatro tipos, K1, K2, K28 e Klus, codificado por elementos extra cromossomais associados a partículas virais na forma de dsRNA. Este trabalho tem como objetivo caracterizar a toxina killer de S. cerevisiae ACB-K1 e testar sua atividade antagônica em patógenos pós-colheita de citros. O isolado ACB-K1 apresentou atividade killer, sobre levedura sensível (S. cerevisae NCYC 1006) além do fitopatógeno P. digitatum, não apresentando porém inibição contra o patógeno G. citri-aurantii. A toxina apresentou máxima atividade em pH 4,1 a 22 °C, tanto para a levedura sensível quanto para o fitopatógeno P. digitatum. A toxina apresentou estabilidade em diferentes pH de 4,1 a 6,0, após a incubação de 24h a 22 °C sobre o fungo. O isolado ACB-K1 apresentou dsRNA, sendo detectadas duas formas (LA e M-dsRNA), sugerindo que a base genética para a produção da toxina é extra cromossomal, dado confirmado pela cura do fenótipo killer a 40 °C. As frações obtidas por cromatografia de exclusão molecular em gel de Sephadex G75 demonstraram características de biocontrole contra o fitopatógeno P. digitatum. / Green mold and sour rot are among post-harvest diseases in citrus fruits, caused by Penicillium digitatum and Geotrichum citri-aurantii, reducing a quality and quantity of fruits and, consequently, resulting in significant economic losses. An alternative for the control of fungi is using killer toxins produced by some species of yeasts, capable of killing filamentous fungi. Saccharomyces cerevisiae produces protein killer toxins that are lethal to yeast sensitive cells. These toxins were grouped into four types, K1, K2, K28 and Klus, encoded by extrachromosomal elements associated with viral particles in the form of dsRNA. This work aims to characterize a killer toxin of S. cerevisiae ACB-K1 and to test its antagonistic activity in post-harvest citrus pathogens. The isolate ACB-K1 showed activity killer on sensitive yeast (S. cerevisae NCYC 1006) besides the phytopathogenic P. digitatum, but did not present inhibition against the pathogen G. citri-aurantii. The killer toxin showed maximum activity at pH 4.1 at 22 ° C for both a sensitive yeast and the phytopathogenic P. digitatum. The toxin presented stability at pH range from 4.1 to 6.0, after a 24h incubation at 22 ° C on the fungus. The ACB-K1 isolate showed dsRNA and two forms were detected (LA and M-dsRNA), suggesting that a genetic basis for a toxin production is extrachromosomal, confirmed by curing the killer phenotype at 40 ° C. The fractions obtained by exclusion chromatography Sephadex G75 gel, demonstrated biocontrol characteristics against the phytopathogen P. digitatum.

Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Thanks to environmental changes, globalization, long distance trade and plant transport, invasive organisms have become a major threat for world biodiversity and ecosystem services. Over the last 20 years, common European ash trees (Fraxinus excelsior L., Fraxinus angustifolia Vahl. etc.) have been subjected to heavy dieback and mortality because of the introduction and spread of the ascomycetous fungal pathogen Hymenoscyphus pseudoalbidus Queloz (syn. Chalara fraxinea Kowalski). Once the disease is established, its management is hardly possible. Therefore, one of the main objectives of European researchers is to find effective and respectful control methods, such as biological control. The discovery of viruses which reduce the virulence of the chestnut blight fungus Cryphonecria parasitica (Murr.) Barr., has intensively stimulated the research of fungal viruses as potential biological control agents (BCA). The occurrence of putative dsRNA particles in the decaying fungus H. pseudoalbidus was investigated as an important indicator of the mycoviruses' presence. In total, 106 samples of this pathogen were obtained from eight different European countries. According to the results, dsRNA segments were confirmed in 32.1 % of examined samples (two similarly sized at 2--2.5 kb and a third one of approximately 5 kb). Statistical results have revealed no significant relation between the presence of dsRNA and growth rate, colour or any other characteristic of the mycelium.