Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643–8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro.

Here, we first wished to establish for mouse primary keratinocytes (KCs) the Ca(2+) concentrations that were associated with KC differentiation in vitro. Using the range of Ca(2+) concentrations (0-6 mM) to differentiate primary KCs in culture to varying extents for 2 days, we then examined how KC differentiation impacted on expression of papillomavirus (PV) native (Nat) and codon modified (Mod) L1 genes. L1 mRNAs transcribed from either Nat or Mod L1 genes were present in similar amounts in KCs exposed to six Ca(2+) concentrations. However, expression of the L1 proteins from two Mod L1 genes were down-regulated, with no L1 signal detected in KCs exposed to 6 mM Ca(2+). In contrast, L1 proteins expressed from the two Nat L1 genes were not detectable in KCs without Ca(2+), but dramatically up-regulated as the KC cultures exposed to Ca(2+) from 0.5 to 2 mM, then down-regulated in KCs exposed to Ca(2+) from 4 to 6 mM. The different regulatory roles of the Ca(2+) in L1 protein expression from Nat and Mod L1 genes in cultured KCs were confirmed by TGF-beta1 experiments. We observed that aminoacyl-tRNAs (aa-tRNAs) from the 2 mM Ca(2+)-treated KCs only significantly enhanced the Nat L1 mRNAs translation in vitro, suggesting that aa-tRNAs play a differentially regulatory role in translations of the PV Nat and Mod L1 mRNAs. Importantly, the Ca(2+) experimental model provides evidence that mouse primary KCs could be transiently infected by BPV1 virus to express L1 mRNA and protein, which is very useful for future HPV virus infection study.

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.