FIG 2.

The transactivation domain of HPV31 E2 is required for the control of SRSF1, -2, and -3 and the production of L1 protein. (A) SRSF levels are increased in differentiated HPV31-infected CIN612-9E cells. Semiquantitative Western blot analysis was performed to determine the levels of SR proteins in undifferentiated (U) and differentiated (D) CIN612-9E cells. Protein extracts (5, 10, or 20 μg) were loaded as indicated above the blots. SRSF1, -3, and -7 were detected with specific monoclonal antibodies, as indicated on the right-hand sides of the blots. SRSF1, -2, -4, -5, and -6 were detected with Mab104, which detects phospho-epitopes of all of the classical SR proteins except SRSF9. Involucrin was detected as a control for differentiated epithelial cells. Involucrin is detected in the undifferentiated cells due to ca. 20% of these having undergone differentiation. GAPDH was detected as a protein loading control. A single gel was blotted and probed with antibodies against SRSF1 and involucrin. The same samples were electrophoresed on identical gels for probing with GAPDH/SRSF7 or with Mab104/SRSF3. This experiment was repeated three times, with very similar results from each Western blot. (B) Keratinocytes containing a transactivation-negative E2 point mutant HPV31 genome have reduced levels of SRSF1, -2, and -3. Western blot analysis of SRSF1, -2, and -3 levels in normal human foreskin keratinocytes (NFKs) stably transfected with wild-type E2 (E2wt) or point mutant E2:I73L HPV31 genomes (E2:IL-73). SRSF7 was detected as a control for an SR protein whose levels are not changed upon epithelial differentiation or significantly transactivated by HPV16E2. The levels of L1 protein in the two lines are also shown. The experiment was carried out three times using two different clones of each E2-expressing keratinocyte line. Very similar results were obtained in each experiment. (C) Western blot analysis of levels of E2 protein and differentiation status of NFKs stably transfected with wild-type E2 (E2wt) or point mutant E2:I73L HPV31 genomes (E2:IL-73). As a marker of differentiation, the levels of involucrin are shown in undifferentiated (U) and differentiated (D) CIN6129E keratinocytes for comparison. The experiment was carried out twice using two different clones of each E2-expressing keratinocyte line. Very similar results were obtained in each experiment. (D) Quantification of levels of the SR proteins shown in panel B. The graph shows the means and standard deviations from three separate experiments. Values were calculated relative to the GAPDH levels. Very similar data were obtained with two different clones of each keratinocyte line. (E) Western blot quantification of levels of L1 protein in differentiated NFKs stably maintaining wild-type HPV31 and in NFKs stably maintaining point mutant E2:I73L HPV31 genomes. The graph shows the means and standard deviations from three separate experiments. Values were calculated relative to the GAPDH levels. P values were calculated using a Student t test.