Figure 6. Integrity of RXL tripeptides is required for HPV18 E1^E4 induced G2/M arrest in primary foreskin keratinocytes.

(A) Cell cycle profiles of primary human foreskin keratinocytes (HFK) infected with recombinant adenoviruses expressing β-galactosidase (βGal), HPV18 E1^E4 (WT) or the mutants T23I or RXLD for 36 h. The percentages of cells in G1 and in G2/M were used to calculate the G2+M:G1 ratios. Results are show for keratinocytes derived from two different donors and are representative of at least four independent experiments. (B) Western blot analysis of cell lysates showing the level of expression of the different proteins using β-actin levels as a loading control. (C) Cellular distribution of cyclin B1 (red) in HFK mock-infected (i), infected with the virus expressing (βGal) (ii), or with viruses expressing E4WT, E4T23I or E4RXLD (βGal and E4 proteins shown in green). Cyclin B1 is sequestered to both fully collapsed E4 filamentous networks (iii) as well as those that have undergone a partial collapse (iv). Mutation of threonine 23 (v) did not abrogate the association. Cyclin B1 was not sequestered to the collapsed filamentous networks of RXLD (vi and vii), although occasional cells showed a partial co-distribution (viii). Nuclear stain (DAPI) is shown in blue. (D) Cyclin A (red) distribtion in HFK, either mock-infected or infected with viruses expressing βGal, E4WT or E4RXLD proteins (all shown in green). Nuclear stain (DAPI) is shown in blue. Bar, 10 μm. The percentages of dual-positive cells is given as the means ± standard deviation, in the top-right of the panel s. Data was derived from three counts, each of a minimum of 100 cells.