44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

Abstract

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the ES gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the ES protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the ES protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.The E5 gene of bovine papillomavirus type 1 (BPV1) encodes a short hydrophobic protein required for efficient in vitro transformation of established mouse cell lines. Frameshift mutations or the insertion of a translation termination codon in open reading frame (ORF) ES markedly decreases the ability of the viral DNA to induce foci in mouse C127 cells (5,9,16,20,24). Detailed mutational and biochemical analysis of this gene indicates that the portion of ORF E5 downstream of the first methionine codon in the reading frame is translated to produce a 7-kilodalton polypeptide required for efficient focus formation (5, 9, 29). In addition, expression of this portion of the E5 gene from a strong heterologous promoter is sufficient to induce focus formation (27,28,32). The ES protein is predicted to be only 44 amino acids long (Fig. 1). Its amino-terminal two-thirds consists almost exclusively of strongly hydrophobic amino acid residues, and the carboxyl-terminal third contains several charged and polar amino acid residues. There is no apparent similarity between the papillomavirus ES proteins and other sequenced proteins including known viral or cellular transforming proteins.An antiserum generated against the carboxyl-terminal third of the BPV1 E5 protein has been used to immunoprecipitate the 7-kilodalton E5 protein from BPV1-transformed mouse and hamster cells (5,29,33). In accordance with its predicted hydrophobic composition, the E5 protein is recovered predominantly in the membrane fraction of mechanically disrupted cells (29). Moreover, isolation of the E5 protein under nonreducing conditions results in the detection of E5 dimers and higher-order oligomers that can be converted to monomers by treatment with dithiothreitol (DTT) (5). We have proposed that the E5 protein oligomerizes via disulfide bonds at conserved cystei...

Mechanisms of cell transformation by papillomavirus E5 proteins

“…It is localized largely to the membranes of the endoplasmic reticulum and Golgi apparatus of transformed cells and exists as a dimer of two identical subunits linked by disul®de bonds involving cysteine residues in the carboxyl-terminal third of the protein (Burkhardt et al, 1989;Horwitz et al, 1988;Schlegel et al, 1986). The BPV E5 protein does not have intrinsic enzymatic activity but rather induces transformation by modulating the activity of cellular membrane proteins that regulate cell growth.…”

“…On the basis of extensive mutational analysis, we proposed that BPV E5/PDGF b receptor complex formation requires an electrostatic bond between a juxtamembrane lysine in the PDGF b receptor and aspartic acid 33 of the BPV E5 protein and a hydrogen-bond between a transmembrane threonine in the receptor and glutamine 17 of the BPV E5 protein (Figure 2) (Horwitz et al, 1988Klein et al, 1998Klein et al, , 1999Kulke et al, 1992). In this model, the type II E5 protein is in the opposite orientation to the type I PDGF b receptor.…”

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

“…Furthermore, dimerization of the E5 protein is required for stable complex formation with the PDGF b receptor and receptor activation (Horwitz et al, 1988;Meyer et al, 1994;Mattoon et al, 2001). Finally, because the E5 protein and the PDGF receptor are thought to traverse the membrane in opposite orientations, the transmembrane domains of these two proteins evidently align in an antiparallel orientation.…”

“…In transformed cells, the E5 protein exists as a disulfidelinked type II transmembrane homodimer localized largely to the membranes of the endoplasmic reticulum and Golgi apparatus (Schlegel et al, 1986;Burkhardt et al, 1987Burkhardt et al, , 1989Surti et al, 1998). Transformation requires Gln17, Asp33, and two conserved cysteine residues in the carboxy-terminus that mediate disulfide bond formation, but activity is not impaired by a variety of hydrophobic substitution mutations in the central hydrophobic domain of the E5 protein (Horwitz et al, 1988;Meyer et al, 1994). The E5 protein induces transformation by activating the PDGF b receptor, a type I transmembrane receptor tyrosine kinase (Petti et al, 1991;Nilson and DiMaio, 1993;Lai et al, 2005).…”

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours

“…Therefore, BPV-1 E5E6 replication-incompetent VEEV-VRPs were constructed by subcloning a fusion gene product comprising BPV-1 E5 and E6 into the replicon plasmid, pVR200 (Alphavax). Before subcloning, point mutations were inserted at the C terminus of E5 (Horwitz et al, 1988) and the four CxxC motifs of E6 (Thomas et al, 2006), regions critical for proper gene function, in order to avoid transforming effects in the host and to enhance safety. To test the immunogenicity of the BPV-1 E5E6 VRPs in mice, female C57BL/6 mice (Taconic Farms) were vaccinated with 0.5610 8 IU BPV-1 E5E6 VRPs or empty VRPs (n55 per group).…”