The Cantell strain of Sendai virus (SeV-C) represents a typical laboratory attenuated virus which is less virulent and able to induce large amount of interferon in infected cells. This strain has widely been used in the laboratory for immunological studies due to its extraordinary ability to stimulate type I interferon production. Nevertheless, the underlying mechanism by which SeV-C is sensed by immune receptors as an invading microorganism is still largely unknown. Meanwhile, during the course of infection, Sendai virus is known to generate substantial amount of non-infectious viral particles consisting of defective interfering RNAs (DI RNAs) from replication errors. It was known that one major form of DI RNAs generated by some negative stranded RNA viruses may adopt a stem-loop panhandle structure with a relatively long double stranded region. Therefore, we hypothesized that some SeV-C DI RNAs may bear structures similar to intermediate-length dsRNA recognized by cytosolic immune receptor RIG-I, thus triggering interferon production. In this study, three DI RNAs were successfully isolated from SeV-C infected cells. Particularly, one of them designated T4 was found to contain a double stranded region of 93 base pair, and it was capable of stimulating interferon β production when transfected to reporter cells. The immunostimulatory activity of T4 alone was as potent as that of SeV-C, suggesting that T4 would be the major component in attenuated virus SeV-C that activates type I interferon production. Furthermore, cellular dsRNA binding protein PACT was shown to play a role in T4 recognition by RIG-I. T4 binds to PACT and potently stimulates PACT-induced activation of RIG-I. The identification and characterization of T4 reveals the major immunostimulatory component in SeV-C. Our work defines a RIG-I agonist naturally produced during the course of viral infection and provides a new mechanism to explain virus attenuation. We also demonstrate the role of PACT in immune sensing of a viral RNA. In addition, our findings also provide new strategies for the design and development of vaccines, vaccine adjuvants and other immunostimulatory agents. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph. D.)--The University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

A Thesis Submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy in Medicine Johannesburg 1986 / An investigation was done to examine the role of IFN in viral persistence at the cellular level. For this purpose two types of persistent infections were chosen. The first type was cell lines which contained hepatitis B virus (HBV) DNA (PLC/PRF/5 and Hep 3B cells) uninfected control hepatoma cells, (Mahlavu, HA22T and Hep G2 cells) or simian virus 40 (SV40) DNA (C2, C6, Cll cells) and control uninfected (CV-1 cells). In the second type of infection Vero cells persistently infected with SSPE or Sendai virus were used. / IT2018

Thesis (Ph. D.)--University of Florida, 2002. / Typescript. Vita. Includes bibliographical references (leaves 98-110).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20609号 / 医博第4258号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 伊佐 正, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 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 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23812号 / 医博第4858号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 山下 潤, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Die für die Vermehrung der Paramyxoviren notwendige Freisetzung des Virusgenoms in die Wirtszelle findet nach einer Verschmelzung der Virushülle mit der Zellmembran statt. Die Membranfusion wird durch eine Konformationsänderung des membranständigen Fusionsproteins (F-Protein) der Paramyxoviren vermittelt. Der Auslöser der Strukturumwandlung des F-Proteins ist bislang unbekannt. Man nimmt an, daß eine Wechselwirkung mit dem zweiten membranständigen Protein der Hämagglutinin-Neuraminidase (HN-Protein) die Strukturumwandlung des F-Proteins induziert. Das F-Protein kann jedoch auch in Abwesenheit des HN-Proteins eine Membranfusion vermitteln. Für das Verständnis des Mechanismus der F-proteinvermittelten Fusion ist die Kenntnis der dreidimensionalen Struktur des F-Proteins notwendig. In der vorliegenden Arbeit wurden die F-Proteine der Paramyxoviren, Sendaivirus und Simianvirus 5, in fusionskompetenter Form isoliert und in kleine Lipidvesikel rekonstituiert, um deren Struktur mittels Kryoelektronenmikroskopie und Einzelpartikelanalyse aufzuklären. Die 3D-Struktur des Sendaivirus-F-Proteins konnte mit einer Auflösung von 16 Angström aufgeklärt werden. Es ist die erste 3D-Struktur des F-Proteins eines Paramyxovirus in der fusionskompetenten Form. Um geeignete Bedingungen herauszufinden, die das Auslösen der Konformationsänderung der F-Proteine bzw. das "Einfangen" von Strukturintermediaten während der Fusion ermöglichen, wurde das Fusionsverhalten von Sendaivirus und Simianvirus 5 bei unterschiedlichen Temperatur- und pH-Werten sowie in Anwesenheit von Lysolipiden mittels Fluoreszenzdequenchingassays untersucht. Ein signifikanter Anstieg der Fusionsaktivität der untersuchten Viren konnte durch eine Erhöhung der Temperatur erreicht werden. Mittels ESR-Spektroskopie unter Einsatz von spinmarkierten Lysolipiden konnte gezeigt werden, daß Lysolipide die proteinvermittelte Fusion von Hüllviren in einem späten lipidabhängigen Schritt hemmen. Diese Untersuchungen bilden damit eine Grundlage zur Aufklärung der 3D-Struktur des F-Proteins im fusionsaktiven Zustand. Desweiteren wurde die Rolle der transmembranalen und zytoplasmatischen Domäne des F-Proteins bei der Membranfusion und der Wechselwirkung mit dem HN-Protein mittels Fluoreszenzmikroskopie untersucht. Die Befunde der 3D-Strukturaufklärung und der fluoreszenzmikroskopischen Studien wurden unter anderem in Hinblick auf die Bedeutung der Wechselwirkung zwischen den F- und HN-Proteinen für die Fusion diskutiert. / Paramyxoviruses infect their host cells by fusion of the viral envelope with the cell membrane. The membrane fusion is mediated by a confomational change of a viral envelope glycoprotein called the fusion (F) protein. The trigger of the F protein conformational change is still unknown. It is suggested, that an interaction of the F protein with the second envelope glycoprotein hemagglutinin-neuraminase (HN) induces its conformational change. However the F protein can mediate membrane fusion in absense of HN. The knowledge of the three dimensional structure of the F protein is reqiured to understand the F mediated membrane fusion. In the present work the fusion competent form of the fusion proteins of the paramyxoviruses Sendai virus and Simian virus 5 were isolated and incorporated each of them into small lipid vesicles. The 3D-structure of the entire ectodomain of the Sendai virus F protein has been determined in fusion potential conformation by cryo electron microscopy of single molecules and 3D-reconstruction at a resolution of 16 Angström. To detect usefull conditions for triggering the conformational change of F, the fusion of Sendai virus and Simian virus 5 have been studied at different temperature and pH, respectively, using a fluorescence dequenching assay. A significant increase of virus fusion activity has been found due to temperature enhancement. Using ESR-spectroscopy and spin-labeled lysolipids it has been shown that lysolipids inhibit the protein mediated fusion of enveloped viruses at a late lipid-dependent intermediate. Thus lysolipids are capable to freeze a conformational intermediate of the F protein during fusion. Furthermore the role of the transmembrane and the cytoplasmic domain of the Sendai virus F protein for membrane fusion was investigated using fluorescence microscopy. The results of the fluorescence microscopy study and the detection of the 3D-structure have been discussed in view of the relevance of F-HN-interaction for membrane fusion.

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