Title Page

Abstract

Contents

Chapter 1. Introduction 14

Chapter 2. Materials and Methods 24

2.1. Cell cultures 24

2.2. Virus 28

2.3. Chemicals and media components 35

2.4. Western blot analysis 38

2.5. Production of mRFP-EGFP-LC3B stably expressing cell lines 43

2.6. Immunofluorescence 46

2.7. Production of ATG5 KO cell lines 48

2.8. In vitro cell viability assay 53

2.9. Plaque assay 53

2.10. Statistical analyses 56

Chapter 3. Results 57

3.1. Identification of autophagic flux in HeLa cells in response to nutrient deprivation 57

3.2. The cleavage of the SQSTM1/p62 protein is induced independently of the autophagic flux pathway in HeLa cells infected with CVB3 at a high MOI 59

3.3. CVB3 infection does not trigger a complete autophagic flux, including the formation of autophagosomes 62

3.4. CVB3 infection does not affect Akt/mTOR/p70S6K signaling pathway phosphorylation 68

3.5. Autophagosome formation is not essential for CVB3 infection 71

3.6. Autophagic flux is critical for the inhibition of CVB3 replication 75

3.7. Restoring of CVB3 inhibition through modulation of autophagic flux by compound C treatment in ATG5 KO HeLa cells 81

Chapter 4. Discussion 83

Reference 92

국문 초록 118

Table 2.1. Chemicals and media components used in the experiments 37

Table 2.2. Primary antibodies used in western blot analysis 41

Table 2.3. Secondary antibodies used in western blot analysis 42

Figure 1.1. Classification of the virus family Picornaviridae. Picornaviridae is one of the most genetically diverse families of positive-sense and single-stranded RNA viruses, classified into 29... 19

Figure 1.2. Genome organization of coxsackievirus B3 (CVB3) [3]. The CVB3 genome contains a single open reading frame (ORF) with highly structured untranslated regions (UTR) at the 5'-... 20

Figure 1.3. Structure of the coxsackievirus B3 (CVB3) capsid. (A) A schematic model of the icosahedral CVB3 capsid structure composed of 60 asymmetric units, each composed of one... 21

Figure 1.4. Schematic model of the three main types of autophagy described in mammalian cells. (A) Macroautophagy degrades soluble proteins, organelles, and protein aggregates upon their... 22

Figure 1.5. The process of macroautophagy. A portion of cytoplasm, including organelles, is enclosed by a phagophore or isolation membrane to form an autophagosome. The outer membrane... 23

Figure 2.1. Morphology of HeLa cells. HeLa cells were sub-cultured in the Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and a 100 ㎍/ml... 26

Figure 2.2. Morphology of Vero cells. Vero cells were sub-cultured in Minimum Essential Medium α, no nucleosides medium supplemented with 5% fetal bovine serum and a 100 ㎍/ml... 27

Figure 2.3. Virus taxa infecting vertebrates. Viruses are broadly classified according to the type of nucleic acid (DNA viruses or RNA viruses), which are then further classified according to their... 30

Figure 2.4. Phylogenetic tree based on the complete genomes of coxsackievirus B3 (CVB3) strains from around the world. The tree was constructed using the maximum-likelihood method... 32

Figure 2.5. Cytopathic effect (CPE) observed in HeLa cells after coxsackievirus B3 (CVB3) infection. HeLa cells were infected with CVB3 at a multiplicity of infection (MOI) of 10⁻¹ for 24... 33

Figure 2.6. Cytopathic effect (CPE) observed in Vero cells after coxsackievirus B3 (CVB3) infection. Vero cells were infected with CVB3 at a multiplicity of infection (MOI) of 10⁻¹ for 24... 34

Figure 2.7. Schematic representation of the tandem mRFP-EGFP-LC3B reporter to monitor autophagic flux. (A) Representation of the reporter construct mRFP-EGFP-LC3B and the... 44

Figure 2.8. Plasmid map of ptfLC3. ptfLC3B plasmid encoding the monomeric red fluorescent protein (mRFP) and the enhanced green fluorescent protein (EGFP) fused in tandem with light... 45

Figure 2.9. The molecular structures of Lysotracker red. Lysotracker red DND-99 is a fluorescent marker in the molecular structure of a conjugated multi aromatic ring containing a... 47

Figure 2.10. The molecular biology of autophagy. Activation of AMPK in response to low cellular energy status inhibits mTOR and activates ULK1 via phosphorylation. Coordinated activation of... 50

Figure 2.11. Schematic representation of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system. The Cas9... 52

Figure 2.12. Schematic representation of virus quantification using the plaque assay. This assay is based on a microbiological method conducted in a plate. Specifically, a confluent monolayer of... 55

Figure 3.1. Monitoring of autophagic flux in starvation-induced HeLa cells by western blot. The expression levels of SQSTM1/p62 (p62) and LC3B (LC3BI and LC3BII) proteins were detected... 58

Figure 3.2. Monitoring of autophagic flux in HeLa cells infected with coxsackievirus B3 (CVB3) at a high multiplicity of infection (MOI) by western blot. The expression levels of SQSTM1/p62... 60

Figure 3.3. Evaluation of autophagic flux following chloroquine (CQ) treatment. (A) The expression levels of p62 and LC3B (LC3BI and LC3BII) proteins were detected by western blot... 61

Figure 3.4. Assessment of autophagic flux by different short-term stimuli conditions in HeLa cells stably expressing exogenous mRFP-EGFP-LC3B. HeLa cells stably expressing mRFP-EGFP-... 64

Figure 3.5. Assessment of autophagic flux by different long-term stimuli conditions in HeLa cells stably expressing exogenous mRFP-EGFP-LC3B. HeLa cells stably expressing mRFP-EGFP-... 66

Figure 3.6. Comparison of autophagic flux in autophagy-stimulated HeLa cells by fluorescence microscopy. HeLa cells treated with serum-deprivation, EBSS, 20 μM chloroquine (CQ), or... 67

Figure 3.7. Tracking of the Akt/mTOR/p70S6K signaling pathway in starvation-induced HeLa cells by western blot. The expression levels of Akt/mTOR/p70S6K pathway related proteins (p-... 69

Figure 3.8. Tracking of the Akt/mTOR/p70S6K signaling pathway in HeLa cells infected with coxsackievirus B3 (CVB3) at a high multiplicity of infection (MOI) by western blot. The... 70

Figure 3.9. Generation of ATG5 knockout (KO) HeLa cell lines. (A) DNA sequencing analysis revealed the presence of mutation in ATG5 KO HeLa cell lines. The black arrow indicates the... 73

Figure 3.10. Monitoring of autophagic flux in coxsackievirus B3 (CVB3) infected ATG5 knockout (KO) HeLa cells by western blot. (A) The expression levels of SQSTM1/p62 (p62),... 74

Figure 3.11. Assessment of cell viability of HeLa cells after treatment with autophagy modulating drugs. Cell viability was quantified using the water soluble tetrazolium salt assay in HeLa cells... 77

Figure 3.12. Evaluation of the antiviral activity of autophagy modulating drug on coxsackievirus B3 (CVB3) infected HeLa cells. HeLa cells infected with CVB3 at a multiplicity of infection of... 78

Figure 3.13. Effect of compound C (CC) on autophagic flux in coxsackievirus B3 (CVB3) infected HeLa cells stably expressing exogenous GFP-RFP-LC3B. HeLa cells stably expressing... 80

Figure 3.14. Restoring coxsackievirus B3 (CVB3) inhibitory effect by compound C (CC) in autophagy deficient ATG5 knockout (KO) cells. Wild type (WT) HeLa cells and ATG5 KO HeLa... 82

Figure 4.1. Schematic representation of the stages of autophagy and the regulation of autophagy by coxsackievirus B3 and compound C. Autophagy begins when a membrane known as the... 91