Assembly of Hepatitis Delta Virus Ribonucleoprotein Complexes: Uncovering an RNA Nucleosome
Griffin, Brittany Lynn
Casey, John L
The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which internal loops and bulges are interspersed between short helical segments. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virally encoded protein, hepatitis delta antigen (HDAg), perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. This dissertation examines the structural organization of HDV RNPs, the RNA features that determine HDAg specificity for HDV RNA, and the stoichiometry of RNPs assembled with genome RNAs. HDAg binds only to quasi-double-stranded RNAs that are at least ~311 nt. Using a minimal length RNA, I have characterized the assembly of the basic complex formed between HDV RNA and HDAg. Analysis of free and HDAg-bound RNAs by selective 2'OH acylation analyzed by primer extension indicates that the characteristic secondary structure of HDV RNA is preserved within RNPs. However, many predicted unpaired positions in the RNA became more dynamic in the context of the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. These results support a model in which unpaired regions in HDV RNA contribute flexibility that allows HDV RNA bending and condensing by HDAg. Towards extending this study to longer HDV RNAs, my work has advanced the in vitro analysis of HDV RNPs in two important ways. I have characterized, for the first time, highly specific, high-affinity binding of full-length HDAg to HDV RNA. Additionally, I have demonstrated sequential binding of HDAg multimers to the genome RNA that saturates at high concentrations of the protein. Analysis of RNPs formed with full-length HDAg reveals that ~4 to 5 HDAg multimers sequentially bind and encapsidate the HDV genome RNA.
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