Crimean-Congo hemorrhagic fever virus: Identification of an NS(M) protein and oligomerization of the viral glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus within the family Bunyaviridae, and can cause severe disease in humans with a fatality rate between 5-30%. The surface glycoproteins, GN and GC, are responsible for CCHFV attachment to and entry into host cells. Additionally, they likely play critical roles in assembly and budding of virions. However, the mechanisms by which they mediate these events and their structural organization on the virion surface remain undefined. These glycoproteins are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. In order to explore the roles of GN and GC in diverse aspects of CCHFV replication, a thorough understanding of the processes that lead to their generation and subsequent interactions with other proteins is needed. In this dissertation, we have investigated the proteolytic event that defines the C terminus of the GN glycoprotein and have found that G N likely undergoes cotranslational intramembranous cleavage within its second transmembrane domain. This cleavage liberates a cell-associated nonstructural protein termed NSM, which traffics to the Golgi compartment, the site of CCHFV assembly. Sequence analysis of the transmembrane domains flanking NSM has indicated that both are candidates for signal peptidase (SPase) cleavage. Mutagenesis of the SPase cleavage site blocked N-terminal cleavage of NSM, confirming this hypothesis. Using transiently expressed M polyproteins lacking NSM, we found that gross deletion of NSM results in improper trafficking and aberrant processing of the viral glycoproteins, suggesting a possible chaperone-like role for NSM. We have also begun to probe the composition of the viral spikes by examining the oligomerization of the GC glycoprotein. We have found that GC molecules from several geographically diverse CCHFV strains existed in both monomeric and oligomeric forms, with the oligomeric form being preferentially recognized by a conformation-dependent antibody. These oligomers formed in the presence and absence of GN, and we have confirmed that the GC ectodomain is sufficient for oligomerization. Possible roles of NSM in viral assembly and GC containing oligomers in viral entry are discussed in the final chapter, along with future approaches to dissect mechanisms of entry, replication, and assembly for CCHFV