Mutational analysis of bovine papillomavirus type 1 E5 peptide domains involved in induction of cellular DNA synthesis

Abstract

Early gene E5 of bovine papillomavirus type 1 encodes a 44-amino-acid protein whose expression can transform immortalized mouse cell lines. We have previously reported that a chemically synthesized ES peptide functions to induce cellular DNA synthesis upon microinjection into growth-arrested mouse cells. We further defined the two E5 domains essential for the full DNA synthesis induction activity by the analysis of E5 deletion and amino acid substitution mutant peptides. The first domain is the C-terminal 13-amino-acid core which is sufficient to activate DNA synthesis at high peptide concentration and contains two essential, highly conserved cysteine residues. The second domain is the 7-amino-acid hydrophobic sequence contiguous to the core domain which is sufficient to confer a 1,000-fold higher molar specific activity to the E5 peptide. A random hydrophobic sequence, but not charged amino acids, fulfills the function of the second domain.

Mutational analysis of human papillomavirus type 11 E5a oncoprotein

“…It was reported that the structure of the E5 protein of BPV-1 is required for it to exert its transformation function (15,16,23). In our study, the introduction of amino acids of different charges at the COOH terminus may cause a transformation defect.…”

“…The HPV-6 and HPV-11 E5a proteins share structural similarities with the BPV-1 E5 protein: they both contain a Cys-X-Cys sequence near the carboxyl terminus and a Gln residue within the hydrophobic domain. The Cys-X-Cys sequences of the BPV-1 E5 protein play an essential role in transformation as well as in mediation of dimer formation (23). Mutation of either Cys in BPV-1 E5 leads to a partial loss of dimerization and complete abolishment of transformation activity (15).…”

Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

“…Dimerization of E5 is considered to be an essential step for productive interaction with the PDGFR. Early studies had demonstrated the functional importance of the two conserved cysteines at position 37 and 39, and it was commonly assumed that E5 homodimerizes via two intermolecular disulfide bridges (17)(18)(19)(20)(21). A recent study on the truncated transmembrane segment, however, reported that this part of E5 per se is able to dimerize in vitro (9).…”

“…The location of the cysteines appeared to be less important and compatible with a nonhelical conformation, as it was possible to move them from their original positions 37 and 39 to positions 34 and 42 or 36 and 40 and retain function, although when they were moved to positions 35 and 41 or 33 and 43, activation was lost (19). Furthermore, single-cysteine mutants were still able to transform cells and enhance DNA synthesis, albeit with decreased intensity (18,20). In conclusion, it was postulated that the cysteines are needed for dimerization of E5 before its interaction with PDGFR.…”

“…Several mutagenesis studies have shown that the two cysteines in the C-terminal luminal Cys 37 -Ser 38 -Cys 39 (CSC) motif of E5 are important for induction of total DNA synthesis, host-cell transformation, and PDGFR binding (17)(18)(19)(20)(21). Double mutation of the two cysteines completely abolished the transforming activity of E5 and interfered with receptor interaction.…”