Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Hypertension is a major health problem worldwide. In 1999-2000, 29% or 3.6 million Australians aged 25 yrs and over had high blood pressure (> 140 / 90 mmHg) or were on medication for the condition. It is estimated that about one billion of the world’s population has hypertension and that this will increase to 1.56 billion by 2025. Although antihypertensive drugs have been relatively successful in attenuating elevated blood pressure (BP) and in reducing adverse outcomes, control of BP depends on continuation of therapy. Drugs may have undesirable side effects which diminish compliance and BP may be resistant to treatment. Gene transfer approaches may potentially provide a tool to control BP. RNA interference (RNAi) is a new tool for the study of gene function, producing specific down regulation of protein expression. I tested the hypothesis that angiotensin II type 1 receptor (AT1R) inhibition using RNAi technology would result in sustained reduction of blood pressure in the spontaneously hypertensive rat (SHR). To enable in vivo gene delivery into animal models of hypertension, I have developed small interfering RNA (siRNA) inhibition of AT1R mRNA delivered in a DNA plasmid (pPlasRi-AT1R). Transfection of the recombinant plasmid into a mammanlian cell line resulted in strong expression of the transgenes and a significant reduction in the level of AT1R expression. pPlasRi-AT1R plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at 1.5mg/kg. Telemetric blood pressure transducers were implanted into eight month old male SHR for long-term recording of blood pressure. Twenty-four hour intra-arterial blood pressure was measured weekly. After a 2 week control period animals were injected via the tail vein with AT1R DNA plasmid (n=6), control plasmid containing green fluorescent protein (GFP, n=6) or saline (NaCl, n=6)) and followed for 8 weeks. Additional animals were treated with the DNA plasmid or saline and euthanized at 0, 1, 2, 4, 6 and 8 weeks for determination of tissue AT1R expression using RT-PCR. Aims: (i) To develop an accurate radio-telemetry BP recording method in the SHR, (ii) To design rational siRNA sequences and select of methods for effective silencing in vitro, (iii) To measure the expression of DNA delivered RNAi-AT1R plasmid in vitro and in vivo, and (iv) To determine the in vivo effect of systemic delivery of DNA AT1R plasmid on BP. Methods: Continuous 24 h arterial BP was recorded by radio-telemetry using Maclab hardware and a transducer fixed in the abdominal aorta connected to a transmitter in the abdominal cavity. Data was analyzed using software specifically written for the project. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect AT1R transcripts in various tissues following in vivo AT1R gene delivery. BP was monitored weekly for 8 weeks following 1.5 mg DNA delivered RNAi -AT1R plasmid delivery into 8-month-old SHR by tail vein injection. SHR injected with DNA enhanced green fluorescent protein (eGFP) plasmid or saline served as controls. Results: Weekly 24 h BP was successfully recorded for up to 10 weeks. Following transfection with DNA delivered RNAi -AT1R plasmid in vitro, expression of AT1R in transfected cells was determined by western blot, immunofluorescence and flow cytometry. Furthermore, RT-PCR was employed to confirm the AT1R mRNA levels. Following systemic delivery of RNAi-AT1R plasmid into middle-aged SHR, in animals injected with RNAi plasmid control blood pressure (150 +/- 1mmHg) was reduced 1week after injection (145 +/- 0.5 mm Hg, p<0.05) with maximal reduction 4 weeks after injection (127 +/- 1 mmHg, p<0.01). Blood pressure returned to control level by 8 weeks. There was no change in blood pressure in GFP plasmid or saline injected animals. Tissue expression of AT1R in heart, lung, kidney and liver was reduced following AT1R plasmid injection and was associated with reduction in pressure (r=0.99, p<0.05 for each tissue). There were no significant adverse clinical or biochemical effects. AT1R silencing resulted in significant blood pressure reduction in 8 month old male SHR for approximately 2 months. There was a significant decrease in endogenous AT1R gene expression in tissues as determined by RT-PCR. The results suggest that the systemic delivery of siRNA against AT1R mRNA by DNA-based plasmid vector may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant DNA vector for a long-lasting siRNA effect are warranted. RNAi technology with inhibition of AT1R offers a potential new paradigm for the management of high blood pressure. Conclusions: Transfection of cells with DNA delivered RNAi -AT1R plasmid resulted in detection of AT1R transcript in transfected cells confirming a silencing effect in vitro. Significant BP reduction was induced in a group of middle-aged SHR following systemic delivery of DNA plasmid incorporating the siRNA against the AT1R gene. This correlated with significant decrease of endogenous AT1R in various tissues which supported the role of the gene therapy approach in producing a reduction in BP. In summary, the thesis lays the foundation for DNA delivered RNAi mediated AT1R gene delivery as a therapeutic strategy for hypertension. Future work should consider the possible benefits of DNA vector driven AT1R shRNA plasmid containing a regulated tissue-selective promoter and explore approaches which might extend the time during which the hypotensive effect is present

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/29). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references (p. 98-107).

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of Dicer, a key component of RNA silencing mechanisms. Our work demonstrates that adenovirus encodes two miRNAs within the loci of the virus-associated RNA I (VA RNA I). We find that one of these miRNAs, miR-VA “g”, enters into a functional, Argonaute-2 (Ago-2)-containing silencing complex during infection. Currently, the cellular or viral target genes for these miRNAs remain unidentified. Inhibition of the function of the miRNAs during infection did not affect viral growth in a highly cytopathic cell culture model. However, studies from other viruses implicate viral miRNAs in the establishment of latent or chronic infections. Additionally, we find that adenovirus infection leads to the reduced expression of Dicer. This downregulation does not appear to be dependent on the presence of VA RNA or its associated miRNAs. Rather, Dicer levels appear to inversely correlate with the level of viral replication, indicating that another viral gene product is responsible for this activity. Misregulation of Dicer expression does not appear to influence viral growth in a cell culture model of infection, and also does not lead to gross changes in the pool of cellular miRNAs. Taken together, our results demonstrate that RNA silencing pathways are active participants in the process of infection with human adenovirus. The production of viral miRNAs and the regulation of cellular Dicer levels during infection implicate RNA silencing mechanisms in both viral fitness as well as potential host defense strategies.

Aim of this work was to find out how mitochondrial processing peptidases are working in the mitochondrion of Trypanosoma brucei. I have shown by RNA interference that mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) are essential for procyclic stages. Moreover, processing of human frataxin in T. brucei has a similar pattern as in human cells.

SmSP1 is a chimerical serine protease consisted of three domains (cub, LDLa and trypsin-like) and found in Schistosoma mansoni. Its characterization was performed by molecular techniques such as PCR screen, qRT-PCR and RNA interference (RNAi) to gain information about expression profile, level expression and susceptibility to RNAi. Further, protein expression was carried out to gain an antigen for immunization and recombinant for biochemical studies. Results of PCR screen and qRT-PCR suggested possible function of SmSP1 in egg and adult stages but SmSP1 gene was not found susceptible to RNAi in NTS. Recombinant from E. coli was successfully used for immunization. Active recombinant was likely expressed in Pichia pastoris but expression conditions are unstable and expression optimization is necessary.

Doctor of Philosophy / Department of Plant Pathology / Anna E. Whitfield / The Bunyaviridae is a family of animal and plant viruses that pose a threat to human, animal, and plant health worldwide. In nature, the dissemination of these viruses is dependent on arthropod vectors (genera Orthobunyavirus, Nairovirus, Phlebovirus, and Tospovirus) or rodent vectors (genus Hantavirus). The genus Tospovirus is the only one within this virus family that is composed of plant-infecting viruses transmitted by thrips. Tomato spotted wilt virus (TSWV), the type species of the Tospovirus genus, is one of the ten most devastating plant viruses known. It is most efficiently transmitted by the western flower thrips, Frankliniella occidentalis Pergande, in a persistant propagative manner. The insect molecules associated with virus infection and transmission by the thrips vector remain unidentified to date. The aim of this work was to identify F. occidentalis larval thrips proteins that are differentially expressed during TSWV infection of the insect vector and those that directly interact with TSWV. To achieve these goals, I used two-dimensional (2-D) gel electrophoresis and mass spectrometry coupled with Mascot searches. I identified 26 protein spots that displayed differential abundances in response to TSWV infection, which contained 37 proteins. Sixty two percent of these proteins were down-regulated by the viral infection demonstrating a complex response. Moreover, 8 and 11 protein spots that directly interacted with purified TSWV virions and a TSWV glycoprotein (GN), respectively, were identified in overlay assays of larval thrips proteins resolved by 2-D gel electrophoresis. A total of five proteins were identified from these spots. These interacting proteins might play roles in attachment and entry, endocytosis/exocytosis, and escape from different tissues for transmission to occur. Injection of double-stranded RNA (dsRNA) into adult female thrips triggered an RNAi response that resulted in 23% reduction of the target gene transcript level. This significant reduction resulted in increased mortality and decreased fertility compared to insects injected with control dsRNA or water and non-injected insects as well. The work presented here provides new insights on the molecular basis of this virus-vector interaction and describes new tools to conduct functional genomic assays to study gene function and design control strategies of F. occidentalis.

L'interférence à l'ARN (RNAi) est un mécanisme cellulaire connu pour inhiber l'expression de gènes avec une grande spécificité de séquence. Chez la levure Schizosaccharomyces pombe, ce processus induit des modifications de structure de la chromatine et implique une interaction entre un ARN naissant et un petit ARN associé au complexe du RNAi, RITS (RNA-induced Initiation of Transcriptional gene Silencing). RITS cible les régions répétées et non codantes et joue un rôle essentiel dans l'intégrité de l'hétérochromatine de ces sites génomiques. Une étude a mis en évidence la présence de la sous-unité Argonaute 1 du complexe RITS, ainsi qu'une marque de l'hétérochromatine, la méthylation de la lysine 9 de l'histone H3 (H3K9me), au niveau de la chromatine de deux gènes méiotiques, mei4 et ssm4. Ceci suggérait une nouvelle fonction du RNAi dans la différenciation sexuelle. Au cours de ma thèse, j'ai montré que la protéine de liaison à l'ARN Mmi1 (Meiotic mRNA interception protein 1), permet à RITS de s'associer spécifiquement à la chromatine et à l'ARN messager de ces gènes méiotiques. La protéine Mmi1 orchestre une répression post-transcriptionnelle de gènes méiotiques spécifiques, une activité de « silencing » essentielle au contrôle de la différenciation sexuelle. Nous avons mené une analyse de l'ensemble du transcriptome dans une souche déficiente pour Mmi1, ce qui nous a conduits à l'identification de nouveaux ARNm méiotiques ciblés directement par Mmi1 et le RNAi. Curieusement, la chromatine des gènes méiotiques correspondants ne présente pas systématiquement la marque épigénétique répressive H3K9me, ce qui suggère que le RNAi pourrait réprimer certains gènes codants seulement au niveau post-transcriptionnel. En parallèle, en combinant des techniques de génétique, de biologie moléculaire et de physiologie cellulaire, nous mettons en évidence un probable rôle direct du RNAi dans l'inhibition de la différenciation sexuelle. Nous proposons que le RNAi pourrait coopérer avec Mmi1 pour bloquer de manière efficace une partie du programme transcriptionnel méiotique durant le cycle végétatif. Cette régulation serait essentielle pour l'activation appropriée de ce programme au cours de la progression de la différenciation sexuelle. / RNA interference (RNAi) is a cellular process known for inhibiting gene expression in a sequence-specific manner. In the fission yeast Schizosaccharomyces pombe, this process induces modifications in chromatin structure and is assumed to involve an interaction between nascent transcripts and a small RNA contained in the RNAi complex, RITS (RNA-induced Initiation of Transcriptional gene Silencing). RITS targets repeated and non-coding regions, and is essential for heterochromatin integrity at these genomic sites. In one study, RITS complex subunit Argonaute 1, and a heterochromatin mark, methylation of histone H3 on lysine 9 (H3K9me), were detected on chromatin of two meiotic genes, mei4 and ssm4. This finding suggested a possible new function for RNAi in sexual differentiation. During my PhD studies, I found that a RNA-binding protein, Mmi1 (Meiotic mRNA interception protein 1), enables RITS to specifically associate with the chromatin and messenger RNAs of these meiotic genes. Mmi1 protein triggers a post-transcriptional repression of specific meiotic genes, a silencing activity essential for the control of sexual differentiation. We conducted a genome wide transcriptomic analysis from a mmi1Δ strain, and uncovered additional meiotic mRNAs that are directly targeted by both Mmi1 and RNAi. Intriguingly, chromatin of the corresponding meiotic genes does not necessarily display the repressive epigenetic mark H3K9me, suggesting that RNAi might silence some protein-coding genes only at a post-transcriptional level. In parallel, combining genetic, molecular biology and physiological techniques, we highlighted a potentially direct role for RNAi in the inhibition of sexual differentiation. We propose that RNAi cooperates with Mmi1 to efficiently block expression of the early meiotic transcriptional programme during vegetative growth. This regulation might be essential for the proper timing of activation of this programme during sexual differentiation progression.