Using Single Molecule FRET to Study Hepatitis C Virus NS3h andNS3-NS4A Helicase Unwinding Pubblico

Abstract

Hepatitis C virus infection results in development of chronic liver diseases. A potential target for drugs to prevent the development of the virus is the NS3 protein that has helicase and primase activities. In the last few years several studies have described mechanistic aspects of the helicase, but the role of the interaction with NS4A cofactor in the NS3 function is not clear. In this paper, we describe the use of fluorescence and single molecule fluorescence resonant energy transfer (FRET) to understand the mechanisms of binding and unwinding of DNA-DNA substrates and RNA-DNA substrates by the NS3 helicase domain and the NS3-NS4A complex. Our results indicate that multiple units bind to single strand substrates with very high affinity and that unwinding of RNA-DNA occurs at faster rates than unwinding of DNA-DNA. Our results provide the basis for more complete and systematic studies that will help determine the mechanisms of these proteins.

Table of Contents

1. Abstract ................................................................................................................1

2. Introduction ..........................................................................................................1

a. Hepatitis C virus ......................................................................................1

b. Helicases ..................................................................................................2

c. NS3-NS4A ...............................................................................................4

d. Fluorescence Resonance Energy Transfer (FRET)..................................5

e. Single Molecule FRET (smFRET) ..........................................................6

3. Objective ..............................................................................................................7

4. Material and Methods ..........................................................................................7

a. Experimental Setup ..................................................................................7

b. DNA Substrates .......................................................................................9

c. Bulk Solution Experiments ......................................................................10

d. PEG Slide .................................................................................................10

e. Data Analyses .........................................................................................11

5. Results ..................................................................................................................11

a. smFRET Binding Experiments ................................................................11

b. Unwinding Experiments ..........................................................................14

c. Ensemble Experiments.............................................................................14

6. Discussion ............................................................................................................17

7. Acknowledgements ..............................................................................................20

8. References ............................................................................................................21

9. Appendix A: Protocols .........................................................................................24

10. Appendix B: Figures ............................................................................................40

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biophysics, General
Parola chiave	NS3h FRET NS3/NS4A smFRET NS3 helicase NS3-NS4A
Committee Chair / Thesis Advisor	Rasnik, Ivan, Emory University
Committee Members	Crouse, Gray, Emory University Stokes, Darrell R, Emory University

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