Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses.

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Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor ␤1 (TGF-␤1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-␤1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.Epidermodysplasia verruciformis (EV) is a rare genodermatosis (Online Mendelian Inheritance in Man no. 226400) characterized by abnormal susceptibility to a group of related specific human papillomaviruses (HPVs) (EV HPVs), including oncogenic genotypes HPV5 and HPV8 and nononcogenic subtypes such as HPV9. HPV5 is associated with 80% of skin carcinomas developing in EV patients (13,18). This has led to the suspicion that EV HPVs has a role in skin carcinogenesis in the general population (20,24). Numerous studies have focused on the mechanisms exerted by the E6 and E7 proteins of oncogenic genital HPVs in the virus life cycle involving the alteration of specific cellular factors that play a major role in the cell cycle, apoptosis, or some other essential functions that must be activated or inhibited to allow viral replication and transformation (16,17). In contrast, little is known about the cellular pathways altered by EV HPV oncoproteins, in particular, major oncoprotein E6 of HPV5 (5E6). The E6 protein of oncogenic EV HPVs does not bind the cellular p53 protein, whereas the E7 protein interacts poorly with the retinoblastoma protein pRb (2,20). It is worth stressing that the E6 oncoprotein of HPV5 and HPV8 abrogates apoptosis by promoting Bak degradation (7,8).In order to identify cellular partners of the 5E6 oncoprotein and to get some insight into the cellular pathways altered during HPV5 infection, we performed yeast two-hybrid screening and developed ex vivo assays to better characterize the biological properties of 5E6.Yeast two-hybrid screening identified SMAD3 as a 5E6-interacting protein. The 5E6 gene fused to the GAL4 DNA binding domain in pGBKT7 (pGBKT7-5E6 bait plasmid) was used to screen a Matchmaker cDNA library from the human keratinocyte HaCaT cell line (Clontech) (1). This cDNA library was cloned into vector pACT2 (Clontech). The Y187 yeast strain 112 gal4⌬ mel gal80⌬ URA3::GAL1 UAS -GAL1 TATA -lacZ) was transformed with the pACT2-HaCaT cDNA library by standard procedures (4). One aliquot (1.55 ϫ 10 9 yeast cells) was used for screening by mating with yeast strain AH109 gal4⌬ gal80⌬ LYS2::GAL1 UAS -GAL1 TATA -HIS3 GAL2 UAS -GAL2 TATA -ADE2 URA3::MEL1 UAS -MEL1 TATA -lacZ) transformed with the bait plasmid pGBKT7-5E6. Yeast cells were grown on synthetic dex...