The Mechanism of Action of a Human Papilloma Virus Oncoprotein

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The Mechanism of Action of a Human Papilloma Virus Oncoprotein
Source: Imai, Y., Y. Matsushima, T. Sugimura, M. Terada. 1991. Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product. Journal of Virology 65(9): 4966–4972.
Corresponding chapter(s) in the textbook: Chapter 19 (and 17)Review the following terms before working on the problem: human papilloma virus, [35S]methionine labeling, [32P]phosphate labeling, phorbol ester, retinoblastoma protein, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, autoradiography, pull-down assay, Western blotting
Read the paper and answer the questions below that refers to the data described in Figures 4 and 5 of the paper.
Be prepared to discuss the other experiments described, in class.

Experiment

Human papilloma virus strain 16 (HPV-16) is one of the causative agents of cervical cancer in women. The purpose of the research presented in the figure was to study the mechanism of action of the E7 oncoprotein of HPV-16.
Graph A shows the results of a preliminary experiment. Cells of a human leukemia cell line were cultured without (samples 1 and 3) or in the presence of phorbol ester (samples 2 and 4) and concurrently labeled with [35S]methionine (samples 1 and 2) or [32P]phosphate (samples 3 and 4). (Note: Phorbol ester stimulates protein kinase C.) Cell extracts were immunoprecipitated with an antibody specific for the retinoblastoma protein (anti-RB). SDS-polyacrylamide gel electrophoresis was performed, followed by autoradiography.
Graph B shows the results of the experiment designed to analyze the function of the E7 protein. Human leukemia cells were cultured in the absence (samples 1 to 3) or presence of phorbol ester (samples 4 to 6). Cell extracts were prepared, and aliquots were incubated with agarose beads to which E7 protein molecules had been covalently attached. After incubation, the beads were sedimented by centrifugation, and Western blot analysis was performed using the anti-RB antibody on the supernatants (S; samples 2 and 5), pellets (P; samples 3 and 6), or total cell extracts (T; samples 1 and 4).
Figure

A) Analysis of RB-immunoprecipitates from human leukemia cell extracts
B) Analysis of RB and E7 protein interactions by pull-down assay (T: total cell extract; S: supernatant; P: pellet)
Questions
1. What was the goal of [35S]methionine and [32P]phosphate labeling?
2. What do bands a and b represent?
3. What does the state of RB protein (graph A) tell you about the cell cycle conditions of the leukemia cells?
4. How did phorbol ester treatment affect the synthesis and phosphorylation of the RB protein?
5. How would the effect of phorbol ester treatment affect the leukemia cell culture?
6. What phenomenon was studied in the experiment that produced graph B?
7. Which form of the RB protein is affected by the E7 oncoprotein?
8. What effect would expression of the E7 protein in the leukemia cells have?
9. Summarize the mechanism of action of the E7 oncoprotein.

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