Adenosine deaminase acting on RNA (Adar1) is a dsRNA binding protein responsible for catalyzing the C-6 deamination of adenosine (A) to produce inosine (I) in RNA editing. Adar1 has been shown to play either proviral or antiviral roles in many different viruses. Japanese encephalitis virus (JEV) belongs to the family Flaviviridae and is known to cause human encephalitis. Functions of Adar1 in JEV life cycle remain unknown. In this study, upregulation of Adar1 expression in both A549 and HEK-293T cells infected with JEV was found. Knocking down Adar1 increased viral translation and production significantly, indicating that Adar1 plays an antiviral role in JEV infection. Double-stranded RNA is a known inducer of interferon. Infection of JEV induced much less interferon in the Adar1 knocked out cells than in the wild type cells suggesting that Adar1 functions as an essential activator of JEV induced interferon responses. Interestingly, ADAR1 colocalized with nucleolin in the nucleolus of uninfected cells while characteristically redistributed within the nucleus upon JEV infection. ADAR1 also colocalized with viral core and NS5 proteins in the nucleus. Nucleolus has been shown to play roles in ribosome biogenesis and the cellular response to stress suggesting a pathway of ADAR1 in antiviral signaling. Results from this study provides a first insight into the antiviral function of Adar1 in JEV infected mammalian cells.

I. INTRODUCTION……………………1
1. Japanese encephalitis……………………………………1
2. Japanese encephalitis virus………………………………………1
3. Viral life cycle…………………………………………………2
4. Adenosine deaminase acting on RNA (ADAR)….……………3
5. Nucleolus……………………………………………………5
6. Aim of this study…………………………………………………….....……………..7

II. MATERIAL AND METHODS……………………………9
1. Cell lines and virus strains……………………………………9
2. Preparation of viral stocks………………………………………………..…….……..9
3. Virus infection...………………………………………………………...…..….....…10
4. Transfection…………………………………………………………...….…...…......10
5. Western blotting……………………....…………………….………..…..……….…11
5.1 Protein extraction………………………………….…………….…..…….........11
5.2 SDS PAGE …………………………………….………………….…......……..11
5.3 Transfer...………….……………………...…………………………....……….12
5.4 Detection……………….……………………………………………...………..12
6. RNA interference…………………………………………………………...……….13
7. Plaque Assay……………………………………………………………..…….........13
8. Immunofluorescence Assay……………………………………………..…………..13
9. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)….......14
9.1 RNA extraction…………….………………………………………..…...……..14
9.2 Reverse transcription……..……..…………….…………………………...........15
9.3 Quantitative polymerase chain reaction……..……….....………….…...............15

III. RESULTS…….………………………..…………….…………………..………..……17
1. JEV infection upregulates Adar1 expression in A549 and HEK-293T cells ……......17
2. Adar1 plays an antiviral role against JEV infection…….……….………….…....….18
3. Adar1 is responsible for increased interferon response in JEV infected cells..…….....19
4. JEV infection causes upregulation and redistribution of Adar1.…………..……..….19
5. ADAR1 co-localizes with nucleolin.……………………………………….……..…20
6. NS5 co-localizes with Adar1 in the nucleolus….………………………………….…20

IV. DISCUSSION.………….…………………………………………….……………..….23

V. REFERENCES…………………...……...………………………….…….…..…..….…29

VI. TABLES AND FIGURES…………………..………………….……………………...35

VII. Appendix.……………….……………..………………….………….………………..55
1. Restriction map of pmGFP-ADAR1-P110…………………………………...........55
2. Restriction map of pmGFP-ADAR1-P150………………………………….....…..57

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