Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification Alton R. Johnson Jr. 1 , Junpeng Yan

1. Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification Alton R. Johnson Jr.1, Junpeng Yan2, Qing Li2, and Jianxin You2 Barry University, Miami Shores, FL 331611, University of Pennsylvania, Philadelphia, PA 191042 Abstract Materials and Methods Conclusion The papillomavirus E2 protein is a viral regulatory protein that is essential for viral transcription, DNA replication, and genome maintenance. In previous studies, the cellular bromodomain protein Brd4 was identified as a major bovine papillomavirus (BPV) E2 interacting protein. Brd4 tethers E2 and BPV viral genomes to mitotic chromosomes in dividing cells. However, in the case of human papillomavirus (HPV), additional cellular factors may be involved in episomal maintenance. In order to better understand the mechanisms involved in HPV episomal maintenance, the identification of additional HPV16-interacting proteins is therefore necessary. To achieve this goal, we employed several strategies for isolating interacting proteins, including the production of 6X HIS- and GST-tagged proteins for affinity purification. The use of GST-tagged E2 proteins proved successful at pulling down the interacting protein Brd4 as well as three previously unidentified proteins. Our hope is that the identification of these newly isolated interacting proteins will provide clues about how HPV infection arises and how it may be mitigated. (Supported by NIH-NIGMS MBRS RISE grant R25 GM059244, Barry University) The usage of GST-tagged E2TA resin was proven valid in binding the viral episome. The binding was successful when using the viral episome extracted from W12 cell lysate. The W12 episome bound to the E2TA was washed using a phosphate buffer solution (PBS) and eluted using 2M KCl the episome. The eluate attached to the resin was analyzed via SDS-PAGE. The proteins of interest were stained using the silver stain method. The next phase is to identify these proteins using mass spectrometry. The identification of these novel proteins can provide an understanding of the mechanisms involved in the tethering of the viral episome to chromosomes. There is also the potential for developing a drug that can inhibit this protein binding to reduce the spread of the viral episome during mitotic division. Future Direction Introduction • Identify proteins of interest using mass spectrometry. In the United States, currently 20 million Americans are infected with Human Papillomavirus (HPV); an estimated 6.2 million people are infected with HPV each year (You et al.,2004). HPV infection can lead to the formation of papillomas, warts, and certain cancers. There are approximately 140 different strains of HPV; 40 of these strains are responsible for infecting the genital tract. The four major papillomaviruses (PVs) that infect the genital tract are classified as either being low risk such as HPV6 and HPV11 or high risk such as HPV16 and HPV18. The high risk types are the subject of interest because DNA from these high risk types can be found in 98% of the cervical cancer cases. This abundance of high-risk HPV DNA presence is a strong indicator that HPV is the causative agent for the cancer. The severity of cervical cancer is rapidly growing being the second most common cancer among women worldwide; in addition, there are nearly 500,000 new cases annually (Parkin et al., 2005). The papillomavirus E2 protein is a critical viral protein that is necessary for transcription, DNA replication and genomic maintenance. In previous studies, the cellular bromodomain protein Brd4 has been identified as a major bovine papillomavirus (BPV) E2-interacting protein. The Brd4 tethers the E2 and BPV viral genome to mitotic chromosomes. However, in HPV there may be additional proteins that allow the viral genome to bind to the mitotic chromosome. Therefore, we conducted this experiment to investigate further what additional cellular factors may assist in the tethering of E2 and the HPV viral genome to the mitotic chromosome or other cellular proteins. In this study using DNA affinity purification, we hoped to isolate the viral genome using the lacO/LacI system and E2/W12 binding system. Once the viral genome isolated the protein of interest that is bound to the viral genome could then be eluted and identified using mass spectrometry. References Results Dostatni N., Thierry F., and Yaniv M. 1988. A dimer of BPV-1 E2 containing a protease resistant core interacts with its DNA target. EMBO J. 7:3807-3816. Howley P.M., and LowyD.R. 2001. Papillomaviruses and their replication. In Fields Virology, D.M. Knipe and P.M. Howley, eds. (Philadelphia, PA: Lippincott Williams & Wilkins), pp. 2197. Parkin, D.M., F Bray, J. Ferlay, and Pisani P. 2005. Global cancer statistics, 2002. CA Cancer J. Clin 55:74-108 Weinstock H, Berman S, Cates W.  Sexually transmitted disease among American youth: Incidence and prevalence estimates, 2000.  Perspectives on Sexual and Reproductive Health 2004; 36: 6-10. You, J., Croyle J.L., A. Nishimura A., Ozato K., and Howley P.M. 2004 Interaction of the bovine papillomarvirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes. Cell 117:349-360. Zur Hansen, H. 1996. Papillomavirus infections-a major cause of human cancers. Biochem. Biophys. Acta1288:55-78. A. B. HPV episome from W12 cell lysate successfully binds to GSH-E2TA Resin HPV16-lacO from lysate did not bind HIS-LacIResin Wash 10mM Imidazole Elute 150mM Imidazole WASH (PBS) ELUTE (2M KCl) Wash PBS Fig.2: Agarose Gel Confirmation of Episomal Binding Aim GSH/GST-E2TA PM PM GSH/GST To identify additional cellular factors that may be involved in the episomal maintenance. Acknowledgements kDa University of Pennsylvania Susan Ross, PhD Edward Marshall III Susan Sheng Barry University FlonaRedway, PhD Peter Lin, PhD Teresa Petrino,PhD ChristophHengartner, PhD 170- 130- Proteins of Interest Protein of Interest 95- GST-E2TA 72- Protein of Interest ? 55- Contact Information ? ? 43- Brd4 Department of Biology 11300 NE 2nd Ave. Miami Shores, FL 33161 Phone: (305) 899-3035 Alton.Johnson@mymail.barry.edu Protein of Interest E2 34- Protein of Interest 26- Fig. 1 Episomal Binding Complex 17- Fig. 3: Silver Stain of Proteins of Interest