MUTATIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS E4 PROTEINS - IDENTIFICATION OF STRUCTURAL FEATURES IMPORTANT IN THE FORMATION OF CYTOPLASMIC E4 CYTOKERATIN NETWORKS IN EPITHELIAL-CELLS

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks w hich specifically colocalize with cytokeratin intermediate-filament (I F) networks when expressed in simian virus 40-transformed keratinocyte s. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of t he cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode o f interaction of E4 with the cytokeratin Ifs is unknown. To identify E 4 sequences important in mediating this interaction, we have construct ed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expre ssed them by using the same simian virus 40-epithelial expression syst em. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the forma tion of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 protein s, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved m otif is likely to play a central role in the association between E4 an d the cytokeratins. An HPV 1 E4 mutant protein containing a deletion o f residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDD FC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residu es 42 to 80, which are rich in charged amino acids, appeared to be imp ortant in the cytoplasmic localization of E4. In addition, we have map ped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypepti des expressed by using the baculovirus system and shown that they begi n at tyrosine 16 and alanine 59, respectively. Similar-sized E4 protei ns are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filame ntous networks. These findings support the postulate that N-terminal p roteolytic processing of the E1^ E4 protein may modulate its function in vivo.