MUTATIONAL ANALYSIS OF THE HUMAN PAPILLOMAVIRUS TYPE-16 E1-BOOLEAN-AND-E4 PROTEIN SHOWS THAT THE C-TERMINUS IS DISPENSABLE FOR KERATIN CYTOSKELETON ASSOCIATION BUT IS INVOLVED IN INDUCING DISRUPTION OF THE KERATIN FILAMENTS

The function of the human papillomavirus (HPV) E4 proteins is unknown, In cultured epithelial cells the proteins associate with the keratin intermediate filaments (IFs) and, for some E4 types, e.g., HPV type 16 (HPV-16), induce collapse of the keratin networks. An N-terminal leuc ine-rich motif (LLXLL) is a conserved feature of many E4 proteins, In a previous study we showed that deletion of this region from the HPV-1 and -16 E4 proteins abrogated the localization of the mutant proteins to the keratin cytoskeleton in a simian virus 40-transformed human ke ratinocyte cell line (S. Roberts, I. Ashmole, L. J. Gibson, S. M. Rook es, G. J. Barton, and P. H. Gallimore, J. Virol, 68:6432-6445, 1994). The E4 proteins of HPV-1 and -16 have little sequence homology except at the N terminus, Therefore, to establish the role of sequences other than those at the N terminus, we have performed a mutational analysis of the HPY-16 E4 protein, The results of the analysis were as follows : (i) similar to findings for the HPV-1 protein, no mutation of HPV-16 E4 sequences (other than the N-terminal leucine motif) results in a m utant protein which fails to colocalize to the keratin IFs; (ii) the C -terminal domain (residues 61 to 92) is not essential for association with the cytoskeleton; and (iii) deletion of C-terminal sequences (res idues 84 to 92; LTVIVTLHP) corresponding to part of a domain conserved between mucosal E4 proteins affects the ability of the mutant protein to induce cytoskeletal collapse, despite colocalization with the kera tin Ifs. Further analysis of this region showed that conserved hydroph obic residues valines 86 and 88 are important, In addition, we show th at the HPV-16 E4 protein is detergent insoluble and exists as several disulfide-linked, high-molecular-weight complexes which could represen t homo-oligomers. The C-terminal sequences (residues 84 to 92), in par ticular valines 86 and 88, are important in the formation of these ins oluble complexes, The results of this study support our postulate that the E4 proteins include functional domains at the N terminus and the C terminus, with the intervening sequences possibly acting as a flexib le hinge.