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Antiviral activity of 3D8 single chain Fv against RNA virus infection

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dc.contributor.author전, 혜령-
dc.date.accessioned2012-10-26T04:12:30Z-
dc.date.available2012-10-26T04:12:30Z-
dc.date.issued2012-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/7544-
dc.description.abstractCatalytic antibodies against viruses have been constantly developed in preventive or therapeutic fields to target viral antigens effectively. 3D8 is an anti-DNA catalytic antibody that binds to DNA and RNA then degrades them without sequence specificity as well known as Abzymes. Previously it was reported that 3D8 single chain variable fragment (scFv) has cell-penetrating activity by electrostatic interaction with cellular membrane and it localizes in cytosol resulting in cell death by cellular rRNA degradation. These result mean that 3D8 scFv would also have potential activity as an intracellular antiviral antibody. Since 3D8 scFv has RNase activity, 3D8 scFv could hydrolyze almost viral RNA transcripts or genomes replicated in cytoplasmic space without sequence specificity. Above all, RNA genome viruses preferentially replicate their genes and transcripts in cytoplasmic space. The 3D8 scFv localized in cell cytoplasm could target these viral RNA genomes as a therapeutic reagent. So far, antiviral drugs are limited in specific viral antigens or specific virus species. The advantage of many viruses is their fast mutation so that can make variant mutants from one genus of viruses. Herein I show viral RNA degradation by 3D8 scFv without sequence specificity resulting in suppressed replication of various RNA genome viruses; classical swine fever virus, vesicular stomatitis virus, and influenza A virus. The method for detection of viral RNA degradation is based on click chemistry. Consequently, it is demonstrated that the antiviral activity of RNA-hydrolyzing 3D8 scFv against RNA viruses by using a 3D8 scFv expressing cell lines and by the exogenous treatment of 3D8 scFv protein to cells. 3D8 scFv strongly suppressed virus replication from the viral RNA replication level without involving IFN-β induction. The antiviral mechanism of action is inhibition of propagating viral RNA transcripts by binding or degrading on viral RNA due to RNase activity of 3D8 scFv itself.-
dc.description.tableofcontentsABSTRACT i

TABLE OF CONTENTS iii

LIST OF TABLES vi

LIST OF FIGURES vii

Ⅰ. INTRODUCTION 1

A. Literature review 1

B. Abzyme 5

C. 3D8 single chain variable fragment 7

D. Antiviral activity of RNases 10

E. Research progression of 3D8 scFv and other RNases 16

F. Purposes of this research 17

Ⅱ. MATERIALS AND METHODS 23

A. Cells 23

B. Viruses 23

C. Propagation of viruses 24

D. Infection of viruses 25

E. Plaque assay (VSV titration) 25

F. Hemagglutination assay (H9N2 titration) 26

G. Peroxidase-linked assay (PLA, CSFV titration) 26

H. Antibodies and reagents 27

I. Bacterial expression and purification of proteins 27

J. Establishment of stable cell lines 28

K. Enzyme-linked immunosorbent assay (ELISA) 28

L. FRET (fluorescence resonance energy transfer)-based RNA cleavage assay 29

M. Reverse transcription-PCR 29

N. Confocal Microscopy 32

O. Flow cytometry 32

P. Click-iT assay 33

Q. Analysis of RNA degradation 33

R. Cytoslic fractionation 34

Ⅲ. RESULTS 36

A. Establishment of stable cell lines 36

B. Relatively low expression level of 3D8 scFv in stable cell lines 39

C. Relative growth level of 3D8scFv-expressing stable cell lines 42

D. RNA hydrolyzing activity of 3D8 scFv 45

E. Intracellular catalytic activity of 3D8 scFv-expressing stable clones 47

F. Intracellular presence of 3D8 scFv confers resistance to CSFV infection 49

G. CSFV replication is down-regulated at the viral RNA level by 3D8 scFv 52

H. Constitutive expression of 3D8 scFv confers resistance to VSV infections 54

I. Intracellular presence of 3D8 confers suppression of VSV replication 56

J. Expression of 3D8 scFv in A549 cells exerts antiviral activity against

Avian Influenza Virus 58

K. Influenza A virus replication is down-regulated at the viral RNA levels

by 3D8 scFv 61

L. Antiviral activity of 3D8 scFv is not associated with induction of type I IFN 64

M. Purified 3D8 scFv protein penetrates cells and localizes in cytosol 66

N. Cell penetrating 3D8 scFv do not confer cytotoxicity to A549 cells 68

O. 3D8 scFv endocytosed to cells confers resistance to virus replication 70

P. Virus replication is down-regulated at the viral RNA level by 3D8 scFv

protein taken up to PK15 cells 72

Q. The degraded cellular RNAs release from the fixed/permeabilized cells 74

R. Comparison of RNase A and 3D8 scFv in real time RNA degradation

by Ribogreen 77

S. The down-regulated intracellular RNAs by 3D8 scFv added to

fixed/permeabilized cells 79

T. Direct evidence of viral RNA degradation by 3D8 scFv 82

Ⅳ. DISCUSSION 84

Ⅴ. CONCLUSION 90

REFERENCES 91

국문요약 101



LIST OF TABLES



Table. 1. Published RNases that have antiviral activity. 15

Table. 2. Primers for Reverse-Transcription PCR 31



LIST OF FIGURES



Fig. 1. Interferon (IFN) induction or double-stranded (ds) RNA signaling 12

Fig. 2. Overview of the IFN pathway and viral-counteracting strategies 13

Fig. 3. Simplified schematic of the influenza-virus life cycle 18

Fig. 4. Model for preferential translation of newly appearing mRNAs during

VSV infection. 21

Fig. 5. Establishment of stable cell lines 39

Fig. 6. Confocal images of 3D8 scFv-expressing stable clones 42

Fig. 7. Comparative cellular growth levels of 3D8 scFv-expressing clones 44

Fig. 8. In-vitro RNA-hydrolyzing activity of 3D8 scFv 46

Fig. 9. Catalytic activity of C26 cells and its RNA hydrolyzing activity 48

Fig. 10. Suppressive effect on CSFV replication in 3D8 scFv-expressing

stable cell line 50

Fig. 11. Immunoperoxidase assay for detection of CSFV E2 protein 51

Fig. 12. Down-regulation of viral RNAs in 3D8 scFv-expressing

stable cell line (C26) 53

Fig. 13. Suppression of VSV replication and inhibition of apoptosis in C26 clone 55

Fig. 14. Constitutive expression of 3D8 scFv confers resistance to VSV infections 57

Fig. 15. Suppression of influenza A virus replication in AC7 clone 60

Fig. 16. Influenza A virus replication is down-regulated at the viral RNA levels

by 3D8 scFv 63

Fig. 17. Antiviral activity of 3D8 scFv is not associated with induction of

type I IFN 65

Fig. 18. Cytosolic localization of 3D8 scFv 67

Fig. 19. Non-cytotoxicity of 3D8 scFv taken up to A549 cells 70

Fig. 20. 3D8 scFv protein taken up to PK15 cells gives resistance against

CSFV infections 72

Fig. 21. Down-regulated CSFV viral RNA levels by 3D8 scFv protein

taken up to PK15 cells 74

Fig. 22. Direct detection of degraded cellular RNAs 77

Fig. 23. Detection of decreased cellular RNA level in relation with released,

degraded RNA fragments from fixed and permeabilized cells 79

Fig. 24. Measurement oreal time RNA degradation by Ribogreen 82

Fig. 25. Direct detection of reduced viral RNA by 3D8 scFv 84
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dc.language.isoen-
dc.titleAntiviral activity of 3D8 single chain Fv against RNA virus infection-
dc.title.alternative핵산 가수분해 촉매 항체의 RNA 바이러스에 대한 염기서열 비특이적 항바이러스 활성 메커니즘-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000012687-
dc.description.degreeDoctor-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor전, 혜령-
dc.date.awarded2012-
dc.type.localTheses-
dc.citation.date2012-
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