Viruses are the only organisms known to store their genetic information solely in the form of RNA, and have thus evolved unique machinery to replicate an RNA genome and initiate viral gene expression in the infected cell. The large polymerase protein (L) of negative-strand (NS) RNA viruses is a particularly intriguing model for viral replication, where all of the enzymatic activities required for mRNA transcription, RNA modification, and genomic RNA replication are contained within a single polypeptide. Whereas the host cell requires a suite of enzymes to accomplish these tasks, L alone is the catalytic engine driving NS RNA viral replication. Here we demonstrate purification of functional L protein from Machupo virus (MACV) and reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified recombinant components. Through single-molecule electron microscopy analysis of MACV L, we provide the first structural information of viral L proteins. Comparative analysis with nonsegmented NS RNA viral L proteins reveals how the various enzymatic domains are arranged into a conserved architecture shared by both polymerases. Our in vitro RNA synthesis data defines the basis of arenavirus sequence-specific polymerase recruitment and how inter-termini interactions regulate template recognition. Moreover, we discover a new role for the arenaviral matrix protein in regulating viral RNA synthesis by locking a polymerase-template complex. The inhibitory matrix-L-RNA assembly functionally links transcription regulation and polymerase packaging, and reveals a mechanism for NS RNA viruses to ensure polymerase incorporation during virion maturation. Reconstitution of RNA synthesis in vitro establishes a new framework to understand the arenaviral polymerase complex, and our structural and biochemical experiments provide a basis for mechanistic analysis of the NS RNA viral replication machinery.

Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan® systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases. The use of QPCR as a tool for diagnostic virological and viral research laboratories has greatly increased in recent years. It often replaces conventional PCR and amplicon detection systems which are more complex and laborious, with a higher risk of amplicon carry-over contamination. The new QPCR methods presented here utilize broadly targeted primers and probes for rational and sensitive detection and quantification of variable RNA viruses. They take advantage of the dual properties, both RNA and DNA dependent DNA polymerase activities, of the rTth thermostable polymerase, and thermolabile UNG with dUTP to protect against inadvertent contamination of samples with amplimers. In paper one, a novel QPCR approach to detect and quantify human enteroviral (EV) RNA in patients with neurological disorders such as aseptic meningitis is presented. In the second paper, the development of a novel serological technique, quantitative PCR enhanced immunoassay (QPIA), for serodiagnosis of EV infection, is described. In paper three the subject is the development of a touch-down QPCR (TD-QPCR) for detection and preliminary genogrouping of norovirus (NV), a group of Caliciviruses. In paper four a rational, broadly targeted, system for detection of diverse influenza viruses, yet being able to discriminate between influenza A, B and C, is designed and evaluated. In the last paper, another rational broadly targeted system, for detection of corona viruses in humans and animals, is described. The technologies described in this collection of papers have common features. They are a platform for further development of diagnostic tools for screening and detection of viruses in known viral diseases, maybe also for discovering new viruses.

As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR). In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer. In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential. Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that assembles its membrane-bound replication complexes at mitochondria in plant cells. This process is accompanied by extensive inward invagination of the mitochondrial outer membrane, leading to the formation of cytosol-filled spherules, wherein viral RNA synthesis occurs. The mechanism by which CIRV is able to induce spherule formation is unknown, however growing evidence suggests that the host-cell ESCRT (Endosomal Sorting Complex Required for Transport) machinery – a multi-protein complex normally involved in late endosome maturation – may be involved. ESCRT consists of ~30 soluble proteins that form sub-complexes assembled at the late endosomal surface, and function in multivesicular body (MVB) biogenesis. While ESCRT is relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Hence, as an initial step towards understanding the potential role of ESCRT in CIRV replication, we examined the protein-protein interaction network, subcellular localization, and gene expression profiles of the Arabidopsis thaliana ESCRT components. Overall, the results from these studies suggest that ESCRT organization and function is relatively well conserved in plants compared to other eukaryotes. We also observed that ESCRT is important for CIRV replication, as expression of dominant-negative versions of several key ESCRT components reduced CIRV replication efficiency in plant cells. Moreover, the Arabidopsis ESCRT-I component, Vps23A is recruited from late endosomes to mitochondria in plant cells expressing the CIRV replicase protein, p36, and recruitment of Vps23A was shown to be mediated by sequences located at the N terminus of p36. It was also shown that recruitment of Vp23A to mitochondria by p36 does not require the Ubiquitin E2 Variant domain of Vps23A, which is in contrast to recruitment of ESCRT by retroviruses during viral budding in mammalian cells. Taken together, these results support the hypothesis that CIRV recruits ESCRT by a novel mechanism in order to carry out its replication, a finding that may lend important insight to aspects of normal ESCRT function in plants.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18333号 / 農博第2058号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4840(農学部図書室) / 31191 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当

No description available.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks. / United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) / North Dakota State Agricultural Products Utilization Committee (ND APUC) / North Dakota State Board of Agricultural Research (ND SABRE)

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19907号 / 生博第354号 / 新制||生||47(附属図書館) / 32984 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 米原 伸, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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