Human Papillomavirus Type 5 E6 Oncoprotein Represses the Transforming Growth Factor β Signaling Pathway by Binding to SMAD3

Abstract

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...

Proteomic analysis of the gamma human papillomavirus type 197 E6 and E7 associated cellular proteins

“…Similarly, HPV5 and 8 E6 proteins only weakly interact with UBE3A, do not detectably bind TP53 (Rozenblatt-Rosen et al, 2012; White et al, 2012a), and they lack C-terminal PDZ binding sequences. These E6 proteins, however, inhibit the NOTCH and TGFß tumor suppressor pathways through association with MAML1 and SMAD3 proteins, respectively (Brimer et al, 2012; Mendoza et al, 2006; Meyers et al, 2013; Rozenblatt-Rosen et al, 2012; Tan et al, 2012; White et al, 2012a). HPV5 E6, E7 and E2 score as oncogenic when expressed in basal epithelial cells of transgenic mice particularly in combination with UV (Marcuzzi et al, 2009; Pfefferle et al, 2008; Schaper et al, 2005), further linking these viruses to initiation of non-melanoma skin cancers at least in EV and immune suppressed patients (McLaughlin-Drubin, 2015).…”

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

“…ORFs encoding nsP2 from CHIKV or SFV were transferred by in vitro recombination (LR cloning reaction; Gateway Technology, Invitrogen) from pDONR207 into the pCI-neo-3ϫFLAG vector to be expressed in human cells in a fusion with a 3ϫFLAG tag (35). HEK-293T cells were plated in 24-well plates (2 ϫ 10 5 cells per well).…”

The EVER Proteins as a Natural Barrier against Papillomaviruses: a New Insight into the Pathogenesis of Human Papillomavirus Infections

“…Nevertheless, it can be speculated that beta-HPV could actively participate in the hyperproliferation of keratinocytes during wound healing and, under certain conditions, implement symbiosis with human beings. The involvement of beta-HPV in the regulation of keratinocyte proliferation is poorly understood, but early proteins of beta-HPV have been shown to inhibit TGF-beta-triggered signaling and are expected to stimulate the proliferation of keratinocytes (137). Furthermore, the HPV8 E2 protein was suggested to promote the transition of keratinocytes into the transiently amplifying compartment (150).…”