IDENTIFICATION OF CONSERVED HYDROPHOBIC C-TERMINAL RESIDUES OF THE HUMAN-PAPILLOMAVIRUS TYPE-1 E1-BOOLEAN-AND-E4 PROTEIN NECESSARY FOR E4 OLIGOMERIZATION IN-VIVO

Previous studies have shown that human papillomavirus (HPV) E4 protein s undergo oligomerisation, although the precise sequences involved hav e not been identified. Using the yeast two-hybrid system we have ident ified HPV 1 E4 sequences that are critical to multimerisation. Fusion proteins were created by linking wild-type and mutant E4 proteins to a LexA DNA-binding domain or a B42 transactivation domain. HPV 1 E4:E4 interactions were examined by expression of these fusion proteins in S accharomyces cerevisiae. This assay showed that (1) amino acid residue s 95 to 115 at the carboxy-terminus were critical for oligomerisation and (2) hydrophobic residues (isoleucine 107, phenylalanine 114) in th is domain are major determinants in the formation of oligomers. Intere stingly, the carboxy-terminal domain shares homology with other E4 pro teins of cutaneous HPV types and, furthermore, positions 107 and 114 a re conserved residues. Substitution of the conserved aspartate amino a cids (residues 110 and 112) did not abrogate 54 oligomerisation. Chemi cal cross-linking of wart and recombinant (baculovirus-expressed) HPV 1 E4 protein indicated that in solution this viral protein forms compl exes consistent in size with either trimers or tetramers. These comple xes were resistant to urea denaturation and are not dependent on the f ormation of disulphide linkages. A mutant protein containing a deletio n of residues 110 to 115 was unable to form oligomers following cross- linking supporting a role for this region in mediating E4:E4 interacti ons. We conclude that oligomerisation of the HPV 1 E4 protein is likel y to be mediated by carboxy-terminal residues and that conserved hydro phobic residues of this domain play a major role in E4 oligomerisation . (C) 1998 Academic Press.