An Investigation Into the Effects of pH on Human Papillomavirus Capsid Structure and Applications
Open Access
- Author:
- Langley, Caroline
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- October 31, 2023
- Committee Members:
- Moriah Louise Szpara, Thesis Advisor/Co-Advisor
Joyce Jose, Committee Member
Santhosh Girirajan, Program Head/Chair
Susan Hafenstein, Thesis Advisor/Co-Advisor
Ganesh S Anand, Committee Member - Keywords:
- virus
papillomavirus
HPV16
virology
cryo electron microscopy
cryoEM
structural biology - Abstract:
- Human papillomavirus (HPV) is the most common sexually transmitted infection and a causative agent of cancer. Vaccine studies have led to the development of an effective vaccine against nine HPV serotypes, which include the two most prevalent and harmful serotypes, HPV16 and HPV18. However, there are hundreds of other serotypes, which the vaccine does not protect against, and there are no antivirals that will clear an HPV infection. There are many unanswered questions in the field about HPV structure, entry, trafficking, and particle assembly and release, and hopefully research in these areas will lead to development of a vaccine that protects against a wider range of serotypes. In this thesis, an introduction on the current knowledge in the field of HPV structure, entry, and capsid disassembly is presented in Chapter 1. An investigation into the effects of pH on HPV capsid structure is presented in Chapter 2. The primary methods used in this work are cryoEM single particle analysis and dynamic light scattering. The history, theory, and workflow behind cryoEM are explained, along with the considerations that must be addressed to solve high resolution structures. The flexibility of the HPV capsid limited resolution initially, and the solutions that allowed for high resolution structures of HPV are presented here. One specific area of the HPV replication cycle examined in this thesis is the effects of transportation of HPV from the endosome to the late endosome, or lysosome. During this transition, the pH decreases from neutral pH to a pH of less than 5. Disassembly or degradation of the HPV capsid occurs in the late endosome or lysosome, and it is possible that these events are linked. The goal of this project was to determine if conformational changes occur in the HPV capsid when exposed to low pH. Here I present three cryoEM structures at various pH and the model built from the structure at pH 5.45. The diameter of the capsid did not change due to pH under these conditions. The capsid was degraded or disassembled at a pH of 2.0 based on negative stain TEM images. At a pH of 5.45, the HPV capsid was not disassembled, indicating that a pH change to 5.45 alone is not sufficient to cause capsid disassembly. This indicates that proteases in the late endosome/lysosome may be involved in capsid disassembly or that disassembly occurs at a lower pH. The lack of degradation due to low pH has implications for HPV purification strategies, indicating that HPV could be concentrated and purified with an affinity column and eluted with a low pH buffer. Dynamic light scattering (DLS) results showed that the capsid diameter increased at low pH when the capsid was resuspended in potassium phosphate buffer (PPB). Our current hypothesis is that conformational changes to L2 occur as pH decreases, which results in observed capsid diameter increasing with DLS. Changes to L2 would not be observed with cryoEM single particle analysis or negative stain TEM imaging. Future experiments will be done to further investigate this hypothesis. This thesis explores the trafficking mechanisms of HPV and the effects of low pH on capsid structure, which will provide information for future HPV biochemical studies.