Cottontail Rabbit Papillomavirus E8 Protein Is Essential for Wart Formation and Provides New Insights into Viral Pathogenesis

Abstract

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.Papillomaviruses (PVs) are small DNA viruses that induce cutaneous and mucosal epithelial proliferations in animals and humans. These lesions usually regress, but those associated with a subset of PVs may persist and progress into invasive carcinomas. The Shope cottontail rabbit papillomavirus (CRPV) induces skin warts and carcinomas in domestic rabbits (50). Among human PVs (HPVs), HPV5 is associated with flat wartlike lesions and carcinomas of the skin in patients suffering from epidermodysplasia verruciformis (47), and genital highrisk HPVs, mainly HPV16 and -18, cause anogenital intraepithelial lesions and are responsible for the vast majority of carcinomas of the uterine cervix (64). PVs contain two main oncogenes, E6 and E7, that are essential for epithelial proliferation and vegetative viral DNA replication. Both play a central role in the malignant progression of lesions associated with high-risk genotypes (for reviews, see references 10 and 44). High-and low-risk genital HPVs encode also an E5 oncoprotein with weak transforming activity in vitro. HPV E5 are short hydrophobic proteins associated with intracellular membranes that upregulate the mitogen-activated protein kinase (MAPK) signal cascade initiated by the epidermal growth factor (EGF) receptor (reviewed in reference 20) and increase the transcription of jun and fos early respo...

Wounding prior to challenge substantially improves infectivity of cottontail rabbit papillomavirus and allows for standardization of infection

“…This characteristic, therefore, allows papillomavirus DNA mutants to be studied in vivo, something that cannot be done in other animal models to date. However, different CRPV DNA challenge methods have been used in different laboratories (Brandsma et al 1991;Jeckel et al 2002;Kreider et al 1995;Lin et al 1993;Nonnenmacher et al 2006;Salmon et al 1997;Salmon et al 2000). Some delivery methods are more efficient than others and these outcomes complicate the interpretation of results obtained in the different laboratories.…”

Cottontail Rabbit Papillomavirusin Langerhans Cells inSylvilagusspp.

“…22,25 Lesions may regress via immune-mediated processes or may develop into carcinomas. 5,21,22 Notably, a high proportion of these tumors in Oryctolagus spp. undergo conversion to carcinoma.…”

Les papillomavirus humains cutanés oncogènes sont-ils des commensaux défectifs ?

“…Cette étude était d'autant plus justifiée qu'une publication récente avait montré qu'une protéine précoce du PVH16, la protéine E5 (16 E5), était capable d'interagir avec ZnT-1 [7]. Aucune interaction n'a été observée avec les protéines E6 et E7 de PVH oncogènes cutanés ou génitaux.…”

“…Il avait été préalablement montré que la protéine E8 du papillomavirus du lapin cottontail, une protéine possédant une structure et des propriétés analogues à celles de la protéine E5, était également capable d'interagir avec ZnT-1 et, de ce fait, jouer un rôle important dans l'homéostasie du zinc et dans le cycle de réplication du virus [7]. Ainsi, selon le type de papillomavirus, deux protéines virales (E5 et E8) pourraient interférer avec la régulation du zinc et favoriser l'activation de voies de signalisation impliquées dans la synthèse de facteurs de transcription essentiels pour l'expression et la répli-cation du génome viral (Figure 2).…”