아데노바이러스 구조 안정성에서 펜톤 베이스 모티프 변화의 영향 = The Impact of Penton Base Motif Variations on the Adenovirus Structure Stability

다국어 초록 (Multilingual Abstract)

Adenovirus is distinguished as a non-enveloped virus with an icosahedral capsid comprising 252 capsomeres; 240 hexons and 12 penton bases. Specifically, each penton base is associated with a fiber that extends outwardly from the virion. Adenovirus entry into host cells occurs through a sequence of two protein-receptor interactions. The primary interaction is related to the binding of the knob domain on the fiber to the coxsackievirus and adenovirus receptor (CAR) on the host cell surface. The secondary interaction occurs between the Arginine-Glycine-Aspartic acid (RGD) motif located in the penton base and cellular integrins to facilitate viral internalization.
This study investigates the structure and infectivity of adenoviruses, examining the impact of these motifs on the structure and infectivity of adenoviruses. This was achieved through a comparative analysis of the penton base structures of wild-type human adenovirus D type 37 (HAdV-D37) which includes the RGD loop and Tyrosine-Glycine-Aspartic acid (YGD) and its engineered variants, including rHAdV-D37-NoRGD (No RGD, YGD), rHAdV-D37-AAD (RGD, AAD (YGD replaced with AAD)), and rHAdV-D37-NoRA (No RGD, AAD). First, to elucidate the structural basis for the observed outcome, in silico analysis was conducted using AlphaFold3, with a specific focus on examining the interaction dynamics and stability of the penton base.
This study provides critical molecular insights into how minor alterations in capsid surface motifs can impact viral assembly and cellular infectivity. Furthermore, this work underscores the essential importance of rational capsid protein engineering in advancing the development of safe and effective adenovirus-based vaccines and gene delivery platforms.

목차 (Table of Contents)

• Contents 1
• List of Figure 3
• Abstract 5
• I. Introduction 7
• 1.1. Introduction of human adenovirus 7
• 1.2. Adenovirus structure 9
• 1.3. Adenovirus internalization 11
• 1.4. Deep learning-based protein structure prediction 12
• 1.5. Aim of this study 13
• Ⅱ. Materials and Methods 16
• 2.1. Cell culture 16
• 2.2. Construction of HAdV-pIX-mNeon fusion virus 16
• 2.3. Recombinant virus transfection and rescue 17
• 2.4. Virus genome extraction and PCR analysis 17
• 2.5. Running AlphaFold3 and PAE Analysis 18
• 2.6. Model Confidence Evaluation: pLDDT, pTM and ipTM 19
• Ⅲ. Results 21
• 3.1. Generation of penton base motif variants and construction of recombinant adenoviral genomes 21
• 3.2. Construction of mNeon-tagged recombinant adenoviruses and virus rescue 22
• 3.3. AlphaFold3-based structural analysis of penton base 24
• 3.4. Structural comparison of penton base-fiber complexes predicted by AlphaFold3 25
• Ⅳ. Discussion 49
• Reference 53
• Acknowledgement 63