NMR STUDIES OF THE GLYCOPROTEIN CYTOPLASMIC TAILS OF HANTAVIRUS AND CRIMEAN CONGO HEMORRHAGIC FEVER VIRUS

Estrada, David Fernando

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NMR STUDIES OF THE GLYCOPROTEIN CYTOPLASMIC TAILS OF HANTAVIRUS AND CRIMEAN CONGO HEMORRHAGIC FEVER VIRUS

Estrada, David Fernando

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Abstract

The Bunyaviridae family of viruses is a diverse grouping of approximately 350 members that is present throughout the world. Viruses range from the innocuous to the severely pathogenic and impact not only public health, but crop production and the live-stock industry as well. Collectively, Bunyaviruses represent a serious health and economic risk to not only the United States, but also to Europe, Asia, and Africa. Humans infected by the most pathogenic Bunyaviruses, such as Hantaviruses and Nairoviruses, can have a collection of symptoms that include hemorrhagic fevers, pulmonary edema, severe ecchymosis of the extremities, and in the most serious cases, respiratory failure. Outbreaks of the Hantavirus in the U.S. in 1993 and the Crimean Congo Hemorrhagic Fever virus in Turkey in 2005 have had mortality rates as high as 40%. Bunyaviruses are enveloped anti-sense RNA viruses that contain three genomic segments (Small, Medium, and Large). The M segment produces a glycoprotein precursor that is post-translationally cleaved into a Gn and Gc glycoproteins. Gn and Gc form a heterodimer with the ectodomain representing the surface spike proteins, and the a cytoplasmic domain that extends into the interior of the assembled virus. The Gn cytoplasmic tail in particular is present in a variety of lengths in different Bunyaviruses. Recent studies strongly suggest the Gn tail participates in assembly of the mature virion. Prior to this dissertation, the atomic structure for a Bunyaviridae Gn tail was unknown. This work describes the solution structures of two such cytoplasmic domains in the Andes (Hantavirus) and the Crimean Congo Hemorrhagic Fever virus (Nairovirus), as well as characterize the structure of a third domain from the non-pathogenic Prospect Hill Virus (Hantavirus). As it turns out, a conserved repeating CCHC motif in these domains folds into a unique arrangement of back-to-back ββα type zinc fingers. Whereas classical ββα zinc fingers typically form an extended "beads on a string" arrangement, the Bunyavirus-type zinc fingers form a compact domain in which each zinc finger folds interdependently. Furthermore, while the core zinc finger fold is conserved in different Bunyaviruses, the distribution in the conserved surface electrostatics can vary between genera. Lastly, this work contains preliminary electrophoretic mobility shift assays that suggest that these cytoplasmic zinc finger domains of the Bunyaivurs Gn cytoplasmic tails may contribute to a protein-RNA interaction, thus providing further evidence for a role in binding the ribonucleocapsid complex during assembly. The work presented in this dissertation has expanded our knowledge of the Bunyavirus life cycle by bringing attention to the structure and the function of these conserved Gn cytoplasmic domains. Understanding the behavior of these domains sheds light on critical assembly events that until know have remained largely unknown.