No description available.

No description available.

Virus infection represents an intracellular invasion of host cells for the sole purpose of multiplication. Successful virus replication requires entry into tropic cells, usurping of the cellular machinery, and the production of progeny virions to initiate further rounds of infection. Establishment of the antiviral state in response to virus begins at the site of infection and requires the initiation of a preexisting signaling network designed to inhibit virus replication and aid in the establishment of this coordinated immune response. Integral components of this network include the IRF-3 and IRF-7 transcription factors that play essential roles in the cellular response to infection through virus induced phosphorylation by an unknown virus activated kinase. The objective of this research was to elucidate the viral antigen(s) required to induce the phosphorylation and subsequent activation of IRF-3 and IRF-7, to identify the molecular component(s) required in this activation process, and to decipher the mechanism(s) by which cellular recognition of virus infection initiates this antiviral response. We demonstrate that activation of this signaling network, following RNA virus infection, is dependent on viral entry, viral transcription, and viral translation and propose that the molecular requirements governing activation of IRF-3 and IRF-7 are the result of viral byproducts formed during the process of cytoplasmic RNA self replication; including both the formation of double stranded RNA and ribonucleoprotein complexes. Following cellular recognition of these motifs, signal transduction networks converge onto an IKK-related kinase structure composed of adaptor proteins bound to two kinase subunits, TBK-1 and IKKepsilon, which we propose to be essential components of the virus activated kinase. We demonstrate that TBK-1 or IKKepsilon activation results in the establishment of an antiviral state that renders cells non permissive to viral replic

With the great promise that metagenomics holds in exploring virome composition and discovering novel virus species, there is a pressing demand for comprehensive and up-to-date reference databases to enhance the downstream bioinformatics analysis. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation of RNA virus genomes downloaded from the three major virus sequence databases including NCBI, ViralZone, and ViPR. To reduce viral sequence redundancy caused by multiple identical or nearly identical sequences, sequences were first clustered and all sequences except one in a cluster that have more than 98% identity to one another were removed. Other identity cutoffs were also examined, and Hepatitis C virus genomes were studied in detail as an example. Using the 98% identity cutoff, sequences obtained from ViPR were combined with the unique RNA virus references from NCBI and ViralZone to generate the final RVRD. The resulting RVRD contained 23,085 sequences, nearly 5 times the size of NCBI RNA virus reference, and had a broad coverage of RNA virus families, with significant expansion on circular ssRNA virus and pathogenic virus families. Compared to NCBI RNA virus reference in performance evaluation, using RVRD as reference database identified more RNA virus species in RNAseq data derived from wastewater samples. Moreover, using RVRD as reference database also led to the discovery of porcine rotavirus as the etiology of unexplained diarrhea observed in pigs. RVRD is publicly available for enhancing RNA virus metagenomics. / Master of Science / Next-generation sequencing technology has demonstrated capability for the detection of viruses in various samples, but one challenge in bioinformatics analysis is the lack of well-curated reference databases, especially for RNA viruses. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation from the three commonly used resources: NCBI, ViralZone, and ViPR. While RVRD was managed to be comprehensive with broad coverage of RNA virus families, clustering was performed to reduce redundant sequences. The performance of RVRD was compared with NCBI RNA virus reference database using the pipeline FastViromeExplorer developed by our lab recently, the results showed that more RNA viruses were identified in several metagenomic datasets using RVRD, indicating improved performance in practice.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19192号 / 農博第2131号 / 新制||農||1034(附属図書館) / 学位論文||H27||N4938(農学部図書室) / 32184 / 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 / Doctor of Agricultural Science / Kyoto University / DFAM

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.

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.

Two species of bees in the genus Apis, real honey bees, has long been of interest for man. These two are the European honey bee, Apis mellifera, and the Asian honey bee, Apis cerana. In Vietnam, beekeeping is of great importance, both with A.cerana and A.mellifera. The aim of this project was to investigate if the introduction of the European honey bee in Asia has affected the Asian honey bee, and whether different pathogens from A.mellifera have been transferred to A.cerana. Totally 40 samples, 20 from every species, were analysed for 8 different viruses. RNA was extracted and analysed with qRT-PCR. The results showed that 5 different viruses were present in the samples, DWV, CBPV, BQCV, SPV and SBV. SPV and SBV were only found occasionally while DWV, CBPV and BQCV were present in the majority of the samples. Differences in virus titres between the two bee species were significant for CBPV and BQCV, however the result for DWV titres was not considered significant. DWV therefore seem to be a ubiquitous virus in Vietnamese beekeeping irrespective of species. Further, the results cannot describe the influence or origin of the viruses but only confirm their presence. Additional investigations are needed in order to answer this question.

The presented work is the application of recent methodologies on modeling and simulation of single stranded RNA viruses. We first present the methods of modeling RNA molecules using the coarse-grained modeling package, YUP. Coarse-grained models simplify complex structures such as viruses and let us study general behavior of the complex biological systems that otherwise cannot be studied with all-atom details. Second, we modeled the first all-atom T=3, icosahedral, single stranded RNA virus, Pariacoto virus (PaV). The x-ray structure of PaV shows only 35% of the total RNA genome and 88% of the capsid. We modeled both missing portions of RNA and protein. The final model of the PaV demonstrated that the positively charged protein N- terminus was located deep inside the RNA. We propose that the positively charged N- terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into an icosahedral virus. Third, we simulated T=1 empty capsids using a coarse-grained model of three capsid proteins as a wedge-shaped triangular capsid unit. We varied the edge angle and the potentials of the capsid units to perform empty capsid assembly simulations. The final model and the potential are further improved for the whole virus assembly simulations. Finally, we performed stability and assembly simulations of the whole virus using coarse-grained models. We tested various strengths of RNA-protein tail and capsid protein-capsid protein attractions in our stability simulations and narrowed our search for optimal potentials for assembly. The assembly simulations were carried out with two different protocols: co-transcriptional and post-transcriptional. The co-transcriptional assembly protocol mimics the assembly occurring during the replication of the new RNA. Proteins bind the partly transcribed RNA in this protocol. The post-transcriptional assembly protocol assumes that the RNA is completely transcribed in the absence of proteins. Proteins later bind to the fully transcribed RNA. We found that both protocols can assemble viruses, when the RNA structure is compact enough to yield a successful virus particle. The post-transcriptional protocol depends more on the compactness of the RNA structure compared to the co-transcriptional assembly protocol. Viruses can exploit both assembly protocols based on the location of RNA replication and the compactness of the final structure of the RNA.

Positive strand RNA viruses, inculding tombusviruses, are known to utilize cellular membranes to assemble their replicase complexes (VRCs). Two tombusviruses , Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), replicate on different organellar membranes, peroxisomes or endoplasmic reticulum (ER) for TBSV and mitochodria outer membranes in case of CIRV. I showed that both TBSV and CIRV replicase proteins could assemble VRCs and replicate viral RNA on purified microsomes (ER) and mitochondria. Different efficiencies of assembly was shown determined by multiple domains on TBSV or CIRV replication proteins. To study why VRC assembly could occur on an alternative organellar membranes, I focused on the phospholipids, key lipid components in ER or mitochondria membranes. Phospholipids directly interact with viral replicases, however, their specific roles during (+)RNA virus replication are far less understood. I used TBSV as a model (+) RNA virus, and established a cell-free TBSV replication system using artificial membranes prepared from different phospholipids. I showed that phosphatidylethanolamine (PE) is required for full cycle replication of the viral RNA.Moreover, PE is enriched at the sites of TBSV replication in plant and yeast cells, and was up-regulated during TBSV replication. Furthermore, up-regulation of total cellular PE content in yeast due to deletion of CHO2 leads to dramatically stimulated TBSV replication. Overall, I identified PE as the key lipid component of membranes required for TBSV replication, and my data highlighted that PE, an abundant phospholipid in all eukaryotic cells, not only serves as a structural component of membrane bilayers, its interaction with the viral replication proteins also stimulates (+)RNA virus replication. Further experiments indicated both early secretory pathway and endocytic pathway are involved in PE re-distribution to site of replication. In addition to lipids and subcellular membranes, certain host proteins are also involved in (+) RNA virus replication and VRC assembly. I identified Hop-like stress- inducible protein 1 (Sti1p), which interacts with heat shock protein 70, is required for the inhibition of CIRV replication. My findings indicate that Hop/Sti1 co-chaperone could act as a virus restriction factor in case of mitochondrial CIRV, but not against peroxisomal tombusvirus.