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 coverage.

Using RNase R to degrade linear RNA resulted in significantly more reads of the HDV genome compared to untreated RNA. Additionally, genome coverage analysis revealed that regions with complex secondary structures, such as the ribozyme region, had lower quality reads. Areas with lower secondary structure, like the open reading frame, exhibited better coverage. GC content did not significantly affect sequencing quality, though we had expected that it would.

The depletion of linear RNA proved effective in enhancing HDV sequencing by increasing the read count. This improvement is critical as natural infections typically have much lower HDV concentrations. The dip in quality reads around the ribozyme region will likely be more pronounced in natural infections and this extra processing with RNase R could help detect HDV infections that previously would have not been detected. Future research will focus on further optimizing sequencing techniques to improve resolution in these challenging regions.

Personal Reflection

One of the most critical lessons I learned was the importance of understanding each phase of the experimental procedure. Every step and chemical used in the process has a specific purpose, refined through extensive scientific analysis. Doing this project specifically aiming at the refinement of sequencing HDV has given me a deeper appreciation of the work that goes into basic scientific processes.

Working on this project under Dr. Weller’s mentorship was an invaluable experience that broadened my understanding of the processes involved in viral genome sequencing. Dr. Weller’s expertise and guidance were a great resource throughout this experience, and she helped me discover a greater appreciation for the meticulous process of scientific research.

I also gained experience in the collaborative nature of scientific research. Discussing protocols, troubleshooting issues, and interpreting our results with Dr. Weller and others in the lab fostered a great environment where everyone was willing to learn from mistakes and help each other to increase our understanding and achieve our research goals. This project was a great experience that emphasized the importance of precision and collaboration in scientific research. When I go on to graduate school and pursue my PhD in virology, I will certainly be utilizing the experience I have gained from this research program.

Footnotes

1. Lai, Michael M. C. “The Molecular Biology of Hepatitis Delta Virus.” Annual Review of Biochemistry 64, no. 1 (1995): 259–86. https://doi.org/10.1146/annurev.bi.64.070195.001355.

2. Perez-Vargas, Jimena, Fouzia Amirache, Bertrand Boson, Chloé Mialon, Natalia Freitas, Camille Sureau, Floriane Fusil, and François-Loïc Cosset. “Enveloped Viruses Distinct from HBV Induce Dissemination of Hepatitis D Virus in Vivo.” Nature Communications 10, no. 1 (May 8, 2019): 2098. https://doi.org/10.1038/s41467-019-10117-z.

3. Weller, Melodie L., Matthew R. Gardener, Zoe C. Bogus, Michael A. Smith, Elisa Astorri, Drew G. Michael, Donald A. Michael, et al. “Hepatitis Delta Virus Detected in Salivary Glands of Sjögren’s Syndrome Patients and Recapitulates a Sjögren’s Syndrome-Like Phenotype in Vivo.” Pathogens & Immunity 1, no. 1 (May 2016): 12–40. https://doi.org/10.20411/pai.v1i1.72.

4. Chang, Wei-Shan, John H.-O. Pettersson, Callum Le Lay, Mang Shi, Nathan Lo, Michelle Wille, John-Sebastian Eden, and Edward C. Holmes. “Novel Hepatitis D-like Agents in Vertebrates and Invertebrates.” Virus Evolution 5, no. 2 (July 2019): vez021. https://doi.org/10.1093/ve/vez021.

5. Kuhn, Jens H., Leticia Botella, Marcos de la Peña, Eeva J. Vainio, Mart Krupovic, Benjamin D. Lee, Beatriz Navarro, Sead Sabanadzovic, Peter Simmonds, and Massimo Turina. “Ambiviricota, a Novel Ribovirian Phylum for Viruses with Viroid-like Properties.” Journal of Virology 0, no. 0 (June 10, 2024): e00831-24. https://doi.org/10.1128/jvi.00831-24.

6. Zheludev, Ivan N., Robert C. Edgar, Maria Jose Lopez-Galiano, Marcos de la Peña, Artem Babaian, Ami S. Bhatt, and Andrew Z. Fire. “Viroid-like Colonists of Human Microbiomes.” BioRxiv, January 21, 2024, 2024.01.20.576352. https://doi.org/10.1101/2024.01.20.576352.

7. Alberts, Bruce. Molecular Biology of the Cell. 6th ed. 1 vols. New York, NY : Garland Science, Taylor and Francis Group, 2015.