Analysis of HPV1 E4 complexes and their association with keratins in vivo

The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (> 95%) exists as complexes which do not contain keratins. The identification of discrete species of 34 K , 58 K, 70 K, 88 K, and 105 K suggests that these are simple multimers of the 17 K monomer, with very little of the soluble E4 being present in complexes larger than 140 K. The truncated 10/11 K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140 K, and these were found to be insoluble. The 34 K (dimer) and 58 K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105 K E4 complex was abolished (and formation of the 58 K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1^E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 54 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers. (C) 1996 Academic Press, Inc.