Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification

1. Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification Alton R. Johnson Jr. Barry University, Miami Shores, FL Summer Research Institution: University of Pennsylvania, Philadelphia, PA

2. History of Human Papillomavirus (HPV) • At least 140 different strains • 40 of these strains affect the genital tract • Low risk types such as HPV6 and HPV11 • High risk types such as HPV16 and HPV18 • Strain examined in this study: HPV16

3. History of HPV • DNA from high risk strands (HPV16 and HPV18) is found in 98% of cervical cancer cases • Suggesting HPV increases the production of these malignancies • Cervical cancer is the 2nd most common cancer in women worldwide

4. E2 Protein • Plays critical role in viral transcription, DNA replication, and genome maintenance • Binds as a dimer to viral DNA • Strong binding affinity with viral DNA and Brd4 protein • Tethers to the episome to host chromosomes during mitosis

5. Brd4 Protein • Contains a bromodomain and extraterminal domain (BET) • A major E2- interacting protein • Remains bound to condensed chromatin during mitosis • Associates with histones H3 and H4 • Mediates Transcriptional Activation

6. Research Purpose To identify additional cellular factors that may be involved in the episomal maintenance. We must first identify the other binding proteins that are bound to the HPV16 other than the Brd4 protein.

7. Brd4/E2 interaction allows stable genomic maintenance Proteins of Interest HPV16 Episome ? Nucleus ? Parent Cell ? HPV16 Episome HPV16 Episome Nucleus Nucleus Daughter Cell Daughter Cell Mammalian Cell

8. LacO/LacI Binding Mechanism NO HPV16 LacO HPV16 Mammalian Cell (NHOK) - Mammalian Cell (NHOK) + Cell Lysis Cell Lysis to Extract Viral DNA Background Proteins H i s - L Hi s -L LacO HPV16 6xHis LacI 6xHis LacI Background Proteins Wash Wash Proteins of Interest Elute Elute Identify Background Proteins Identify Proteins of Interest

9. E2/HPV16 Binding Mechanism HPV16 Episome Nucleus Nucleus Mammalian Cell (HFK) Mammalian Cell (W12) - + Cell Lysis Cell Lysis to Extract Viral DNA Detect HPV16 by PCR E2 - R ESIN E2 - R ESIN Background Proteins Background Proteins Background Proteins HPV16 Proteins of Interest Run DNA Lysate through resin containing E2 Protein Run DNA Lysate through resin containing E2 Protein Wash Resin Wash Resin Elute Proteins Elute Proteins Identify Proteins of Interest Identify Background Proteins

10. HPV episome from W12 cell lysate successfully binds to GSH-E2TA Resin WASH (PBS) ELUTE (2M KCl) July 31, 2009 250 µl of W12 cell lysate and 250 µl was added to the 10 µg of GSH-E2TA bead resin M- 100bp Ladder C- pUC19-HPV16-W12

11. Silver Stain of Proteins of Interest GSH/GST-E2TA PM PM GSH/GST (kDa) 170 - 130 - Proteins of Interests 95 - GST-E2TA 72 - Proteins of Interests 55 - 43 - Proteins of Interests 34 - Proteins of Interests 26 - 17 - August 5, 2009

12. Conclusion The proteins of interest have now been stained and molecular weights identified. The next phase is to identify these proteins using massspectrometry.

13. Significance The identification of these novel proteins can provide better data to understand the mechanisms involved in the tethering of the viral episome to chromosomes. If identified there is a possibility of developing a drug that can inhibit this protein binding to reduce the spread of the viral episome during mitotic division.

14. P.I. and Mentor

15. Laboratory Staff

16. Research in the Lab

17. Presenting Research

18. Culture/Sightseeing/Fun

19. The View

20. Carolina Pombo-Martinez and Me

21. Acknowledgements University of Pennsylvania Jianxin You, PhD Junpeng Yan, PhD Qing Li, PhD Susan Ross, PhD Edward Marshall III Susan Sheng Barry University Flona Redway, PhD Peter Lin, PhD Teresa Petrino,PhD Christoph Hengartner, PhD Supported by NIH-NIGMS MBRS RISE grant R25 GM059244, Barry University