Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification

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

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

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

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

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

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.

Brd4/E2 interaction allows stable genomic maintenance

Parent Cell

HPV16 Episome

Nucleus

Mammalian CellDaughter Cell

HPV16 Episome

Nucleus

Daughter Cell

Proteins of Interest

??

?

HPV16 Episome

Nucleus

LacO

Mammalian Cell (NHOK)

+ -

NO HPV16

6xHis LacI HPV16H i s - L

6xHis

Hi s -L

Wash Wash

Elute

Proteins

of Interest

Elute

LacI

Background Proteins

Background Proteins

Cell Lysis to Extract Viral DNA

Identify Proteins of Interest Identify Background Proteins

HPV16

Cell Lysis

LacO

LacO/LacI Binding Mechanism

Mammalian Cell (NHOK)

Mammalian Cell (W12)

HPV16 Episome

Nucleus

Cell Lysis to Extract Viral DNA

Detect HPV16 by PCR

E2

-

R

ESIN

HPV16

Wash Resin

Run DNA Lysate through resin

containing E2 Protein

Elute Proteins

Identify Proteins of Interest

Cell Lysis

E2

-

R

ESIN

Wash Resin

Run DNA Lysate through resin

containing E2 Protein

Elute Proteins

+

Background

Proteins

Background

ProteinsProteins of Interest

Identify Background Proteins

Nucleus

Mammalian Cell (HFK)

-

E2/HPV16 Binding Mechanism

Background

Proteins

HPV episome from W12 cell lysate successfully binds to GSH-E2TA Resin

July 31, 2009M- 100bp LadderC- pUC19-HPV16-W12

250 µl of W12 cell lysate and 250 µl was added to the 10 µg of GSH-E2TA bead resin

WASH (PBS)

ELUTE (2M KCl)

PM

170 -

130 -

95 -

72 -

55 -

43 -

34 -

26 -

17 -

(kDa)PM GSH/GST

GSH/GST-E2TA

August 5, 2009

GST-E2TA

Silver Stain of Proteins of Interest

Proteins of Interests

Proteins of Interests

Proteins of Interests

Proteins of Interests

Conclusion

The proteins of interest have now been stained and molecular weights identified. The next

phase is to identify these proteins using mass spectrometry.

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.

P.I. and Mentor

Laboratory Staff

Research in the Lab

Presenting Research

Culture/Sightseeing/Fun

The View

Carolina Pombo-Martinez and Me

Acknowledgements University of PennsylvaniaJianxin You, PhDJunpeng Yan, PhDQing Li, PhDSusan Ross, PhDEdward Marshall IIISusan Sheng

Barry UniversityFlona Redway, PhDPeter Lin, PhDTeresa Petrino,PhDChristoph Hengartner, PhD

Supported by NIH-NIGMS MBRS RISE grant R25 GM059244, Barry University