In this communication we present experimental evidence demonstrating expression in HEK-293T cells of a codon-adapted version of the HPV16 E5α protein, as well as of a series of N- or C-terminal deletions. Expression of mutant protein hR58E5 could not be demonstrated in western blots. Whether the failure to identify expression of this mutant is due to inherent protein instability is unknown. Immunofluorescence experiments with markers specific for different cellular compartments show colocalisation of HPV16 hE5α with calnexin, indicating that the protein is mainly localised at the endoplasmic reticulum. This finding is in agreement with previous results obtained also using codon-adapted versions of the E5 gene [14, 17]. Interestingly, these results contrast with published reports showing the protein mainly associated with the Golgi apparatus [2, 3]. It must be noted however that the initial reports refer to genes encoding the original, non-human-adapted viral codons. A possible explanation for this difference may lie in the large amounts of E5 protein synthesised from the codon-adapted genes, which will probably accumulate in the ER with the cell being unable to further transport the protein to the Golgi compartment. Similar remarkable shifts in cellular localisation and/or function have also been reported for the L1 , E6  and E7  proteins from different PVs after humanisation of the codon usage. Experimental differences between the effects of the expression of wild-type and humanised PV genes highlights again the physiological importance of the biased codon usage in PVs [24, 25]. The colocalisation demonstrated for E5α with the early endosomal marker EEA-1 is most interesting since a physical association between HPV16 E5α and the 16 K subunit of the proton ATPase has already been demonstrated. This association has been made responsible for the modulation of the internal pH value of the endosomes [4, 6]. In addition, the presence of HPV16 E5α in early endosomes is in agreement with the functional observation of an E5-dependent pH alteration in endosomes, as previously described [17, 26].
The observation that the presence of the first hydrophobic region is necessary for localisation of the protein to the ER, early endosomes or lysosomes is noteworthy, mainly because of the lack of a canonical signal peptide. Deletion of the first 30 amino acids (recombinant R30hE5) results in changes in both localisation pattern and biological effects of the protein. This mutant showed no loss colocalisation of the Flag epitope with 58 K, calnexin, EEA-1 or Lamp-2. Moreover, in the anchorageindependent growth experiments this mutant strongly inhibited colony growth and the size of the few growing colonies was clearly reduced in comparison with that of the full length gene. Further, the immunofluorescence pictures showed a punctuated distribution for this mutant, suggesting that the protein was associated with some kind of vesicular structure, although we were not able to identifiy it.
The results shown in this communication demonstrate that the expression of theHPV16 E5 protein increases the number and the size of HaCaT cell colonies growing in soft-agar. The results were the same for both the wild-type version of the E5 gene and for a codon-optimised version, despite strong differences in expression intensity between these two genes. This suggests that the amount of expressed E5 protein is not decisive for colony number but determines the number of cells and therefore the colony size (see Fig. 6). The data here detailed represent the first quantitative description of the ability of HPV16 E5 to promote growth in soft agar. A previous qualitative description in the same sense had been provided by Suprynowicz and coworkers, in primary keratinocytes . The effects of HPV16 E5 on uncontrolled cell growth and malignisation seem therefore to be multiple. Thus, although an increased expression of the protein seems to shorten the in vitro life span of primary keratinocytes , the expression of HPV16 E5 allows human keratinocytes to grow in soft agar ( and the present paper). Finally, as a correlate at the organismic level, the expression of HPV16 E5 in transgenic mice leads to the development of endophytic papillomas, precursors to carcinomas, and contributes to the promotion and progression stages of carcinogenesis .
Regarding the dissection of the differential implication of the three transmembrane domains of HPV16 E5 in the biological effects of the protein, our finding that Nterminal deletion mutant R30hE5 was unable to promote growth in soft-agar is interesting since this mutant did not show colocalisation with any of the markers used in our immunofluorescence experiments. The molecular mechanisms responsible for this effect are unknown. Computer analysis of the protein identifies the first hydrophobic region of HPV16 E5 as a putative trans-membrane segment [1, 28]. It may be speculated that interactions of with other membrane proteins determine the biological activity of the viral protein. This is further supported by recently published work showing that E5 interacts in the ER with calnexin through the first hydrophobic segment . This interaction results in retention of the HLA class I molecules in the Golgi apparatus, with concomitant down-regulation of its plasma membrane expression [11, 13, 14].
Thus, we can conclude that the first 30 amino acids of the HPV16 E5α protein play a crucial role in the biological properties of the protein. This region determines cellular localisation of the protein, binding to the chaperone calnexin, and anchorageindependent growth in a human keratinocyte cell line.