Analysis of hepatitis delta virus RNA structure: Effects on RNA-protein interactions and viral replication

Hepatitis delta virus (HDV) is a unique human pathogen whose RNA structures are critical to viral function. The genome is a circular single-stranded RNA with high levels of intramolecular complementarity (~ 74%) leading to formation of the unbranched rod structure characteristic of HDV RNA. The complementary antigenome, which has similar structural features, encodes the open reading frame from which mRNAs of the two forms of HDV protein are transcribed: the short form of the delta antigen (HDAg-S) is absolutely required for initiation and maintenance of viral replication, and the long form (HDAg-L) is necessary and sufficient for virus particle production in cells expressing hepatitis B surface antigens (HBsAg). HDV is considered a subviral satellite of hepatitis B virus (HBV) due to its dependence on HBsAg. In fact, HDV also relies on host functions to complete its replication cycle. Exploitation of cellular functions may be directly related to the structure of the ribonucleoprotein complex (RNP) formed through interaction of HDV RNA and viral proteins. This dissertation analyzes the effects of HDV RNA structure on RNA-protein interactions and viral replication. The first half examines the direct biochemical interaction between native bacterially expressed HDAg and in vitro transcribed HDV RNA. These data identify features of the RNA structure that are critical to binding by HDAg. Through electrophoretic mobility shift assay, it was discovered that HDV RNAs containing at least 311 total nucleotides (nt) were stably bound while those with fewer than 298 nt were not bound. This feature was shown to be biologically relevant in transfected cells. Furthermore, micrococcal nuclease digestion indicates the size of the RNP protected is the same as that formed by the smallest RNA bound by HDAg. The second half of this dissertation focuses on effects of RNA structure on viral function specifically, replication. Mutational analysis, specifically designed to preserve the predicted RNA structure of the interior of the unbranched rod structure, indicates sensitivity to nucleotide deletion. Deletions of various sizes (totaling 6 -- 53 nt) in 6 different unrelated segments showed reduced viral replication. These results, and others, suggest most of the HDV RNA structure is pivotal to RNP formation and viral function.