School of Dentistry
3 Optimizing Sequencing Processes for the Hepatitis Delta Virus Genome
Weston Hall and Melodie Weller
Faculty Mentor: Melodie Weller (Dentistry, University of Utah)
Introduction and Background
During the SPUR summer research program, I worked on a project aimed at optimizing the sequencing techniques for the Hepatitis Delta Virus (HDV) genome. HDV is an interesting virus for several reasons. HDV is a satellite virus, meaning that it is unable to produce its own structural proteins which allow viruses to exit cells and infect other cells. Instead, HDV requires a helper virus, the most well-studied of which is the Hepatitis B Virus. HDV co-opts the surface glycoproteins of HBV and the plasma membrane of the host cell that make up an infectious HDV particle [1].
HDV infection and HDV-like infectious agents may be more prevalent than currently known. Although HDV is most commonly associated with HBV as its helper virus, recent studies have shown that there may be a wide diversity of helper viruses for HDV transmission. In vivo experiments show a diversity in HDV-packaging viruses [2] and patients without a history of HBV infection show evidence of HDV infection [3]. Additionally, HDV-like particles have been found in organisms all across the tree of life, from vertebrates to invertebrates [4], from fungi [5] to bacteria [6].
Another interesting aspect, and the focus of my research this summer, is the structure of the HDV genome. HDV has a high amount of self-complementarity, meaning that its single stranded genome forms intramolecular base-pairing with itself, resulting in extensive secondary structures. This feature of HDV causes it to condense down into a tightly packed rod-shaped particle, characteristic of HDV and HDV-like entities [4,5]. A specific region of very complicated secondary structure on HDV and HDV-like sequences is the ribozyme, a catalytic domain of RNA, which is an integral part of HDV’s replication and circularization mechanism [1].
HDV’s genome also has a high GC content, meaning that it has a high proportion of guanine and cytosine base pairs. GC base pairs have more bonds and stabilizing interactions than adenine and thymine or uridine base pairs. The high GC content makes it more difficult to pull apart the highly structured genome and it requires special considerations when performing the molecular techniques required for HDV research [7].
Hypothesis and Results
Our hypothesis for this project was that areas of high secondary structure would have lower quality reads through Illumina sequencing. We predicted that these secondary structures would impede replication enzyme activity.
To study this, we cultured cells and transfected them with a plasmid to produce HDV RNA genomes. After several passages, the RNA was isolated and processed with various enzymes that target the degradation of certain RNA molecules to enrich for circular RNAs like the HDV genome. Illumina sequencing was used to assess genome coverag