Wild type HPV18 E6 forms large soluble oligomers and antibodies against them reveal a homogeneous distribution of the oncoprotein in HeLa cells

MARÍA GARCÍA ALAI; K. DANTUR; L. ALONSO; C. SMAL; G. DE PRAT GAY

Churchil College, Cambridge, England

Congreso; Tumour Virus Meeting 2005; 2005

The E6 protein of the high-risk human papillomavirus type 18 is involved in the tumorigenesis of human cervical cells by targeting the p53 tumour suppressor and PDZ domain-containing proteins for degradation. It is a putative Zn binding protein of 158 amino acids that contains four copies of a Cys-X-X-Cys motif and a total of 10 cysteine residues. The recombinant production of wild type HPV E6 is known to be difficult due to the precipitation dependent on the oxido-reduction conditions. Recombinant E6 has been stably produced as fusion proteins for solubility enhancement or by mutating cysteine residues. In this work we show that, under certain conditions, wild type HPV-18 E6 refolds completely to soluble high molecular weight oligomers (E6 Spherical oligomers, E6SO).  Size exclusion chromatography indicates that these oligomers are larger than 300 KDa and they appear as spherical particles of 40 nm average diameter under the atomic force microscope.  E6SOs are stable to heat denaturation and once assembled, they undergo intermolecular oxidation that further stabilize the macromolecular structure. E6SO are highly immunogenic and we obtained rabbit polyclonal antibodies that readily detect the oncoprotein in HeLa cells by indirect immunofluorescence microscopy, showing that endogenous E6 is evenly distributed in the cytoplasm and the nucleus