We investigated whether differentiation-dependent expression of papillomavirus (PV) L1 genes is influenced by the cell cycle state in keratinocytes (KCs) grown in vitro or in vivo. In primary keratinocytes, flow cytometry revealed a clear shift from predominantly G0/G1 to G2/M cells from day 1 to day 7, with a three-fold increase in G2/M-like cells in day 7 keratinocytes that showed approximately 50% of the cells expressed a terminal differentiation marker involucrin. The correlation between the levels of the L1 proteins expressed from authentic (Nat) L1 genes of HPV6b and BPV1 and the frequencies of the G2/M-like KCs was significantly positive, while in contrast, a significantly negative correlation in the levels of L1 proteins expressed from codon-modified (Mod) L1 genes of HPV6b and BPV1 with the frequencies of the G2/M-like KCs was observed. Experiments using cell cycle arrest reagents (all-trans retinoic acid (RA) and colchicine) confirmed that L1 proteins expressed from PV Nat L1 genes were facilitated in G2/M-like KCs upon differentiation. Using immunofluorescence microscopy, it appears that L1 proteins from PV Nat L1 genes were co-expressed with cyclin B1, while the L1 proteins expressed from PV Mod L1 genes were preferentially associated with cyclin D2 in KCs in vitro and in mouse skin. Our results demonstrate that (1) expression of the L1 proteins from Nat L1 genes of HPV6b and BPV1 that have strong codon usage bias with A or T at codon third position dependent on KC differentiation is favoured by the G2/M-like environment and (2) codon modifications can alter the cell differentiation-dependent and cell cycle-associated patterns of expression of the PV L1 proteins in KCs.
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