Structural characterization of HBV capsid – envelope interaction through cryo-EM
Restricted (Penn State Only)
- Author:
- Garg, Sonal
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 09, 2024
- Committee Members:
- Joseph Wang, Thesis Advisor/Co-Advisor
Jianming Hu, Thesis Advisor/Co-Advisor
Lisa Shantz, Program Head/Chair
Matthew T Swulius, Committee Member - Keywords:
- Hepatitis B virus (HBV)
HBV capsid
HBV envelope protein
Cryo-EM - Abstract:
- HBV infection has affected millions of people worldwide, leading to liver damage, cirrhosis, and hepatocellular carcinoma. Even with HBV vaccines, current treatments for patients already infected with HBV rarely lead to a complete cure. This is partially due to the complexity of the virus infection cycle. HBV is a small double-stranded DNA virus that hijacks lipids from its host cells during viral envelopment. Previous mutagenesis studies have identified several regions within the HBV capsid and surface proteins that play essential roles in capsid envelopment. However, we still need the structural information to fully understand the detailed interaction between the HBV capsid and surface proteins. This study aims to structurally characterize the direct interaction between HBV capsid and envelope proteins. In our preliminary study, we utilized cryo-EM to investigate the structure of the purified HBV virions isolated from chronic HBV-infected patients. The cryo-EM reconstruction analysis revealed the distinct features of the HBV capsid and envelope at resolutions of 3.9Å and 13Å, respectively. The integration of these structures has unveiled a comprehensive HBV virion architecture. We identified a potential density adjacent to the interdimer interface outside the hydrophobic pocket. The sequence information directly derived from the cryo-EM density suggests that this density corresponds to a 22aa region from the pre-S domain of the large surface protein. To further investigate this, we synthesized peptides corresponding to the pre-S and S domains of the HBV envelope surface protein, along with the pCI-HBc plasmid encoding the core protein (HBc), enabling the production of empty capsids upon transfection into mammalian HepG2 cells. Next, we iv investigated the interaction between the empty capsid and S peptide of HBV using cryoEM. The structures of the capsid-peptide complex and the control empty capsid were resolved to 3.1Å and 3.8Å in overall resolution, respectively, Despite the high resolutions, we observed no discernible differences in the icosahedral symmetry between the two structures. This suggests that the S-peptide may not induce significant conformational changes in the capsid upon interaction, or the interaction might be transient. Consequently, further exploration of the S envelope protein with the capsid is required. Since cryo-EM requires significant time for data processing, we employed the native agarose gel electrophoresis technique. Through this method, we observed a mobility shift of capsids when mixed with the pre-S1 peptide, indicating the formation of a complex between the capsid and the pre-S1 peptide. Notably, this observation aligns with our cryo-EM structure of an empty HBV virion, further supporting the interaction between the capsid and the pre-S1 peptide. To delve deeper into the molecular interactions, we synthesized a biotin-tagged peptide with specificity for streptavidin. This peptide is a crucial tool for conducting streptavidin-biotin assays, facilitating in-depth exploration of the binding dynamics between the capsid and peptide. Moreover, our ongoing efforts are directed toward obtaining a high-resolution structure of the purified HBV empty capsid complexed with the pre-S1 peptide. These experiments aim to elucidate the details of the capsid-peptide interaction at the atomic level. Overall, our findings shed light on the fundamental molecular processes driving the HBV envelopment and will offer valuable insights into potential targets for therapeutic intervention.