Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

Abstract

The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.The p53 protein is a tumor suppressor that can cause cell cycle arrest or apoptosis in response to DNA damage and other forms of stress (19). The critical role played by p53 in regulating normal cell growth is highlighted by the observations that p53-deficient mice are rendered highly susceptible to sporadic cancers (6) and germ line mutations in p53 result in Li-Fraumeni syndrome, in which individuals are predisposed to a variety of cancer types (22). Furthermore, approximately 50% of tumors contain mutated p53 genes (15), while the remaining 50% frequently appear to have compromised p53 function. For example, wild-type p53-containing tumors may overexpress the mdm2 cellular oncogene or the E6 oncogene from high-risk human papillomavirus (HPV) types. Tight regulation of p53 activity involves a variety of posttranslational modifications including ubiquitination, proteasome-mediated degradation, and control of subcellular localization. Mdm2, a cellular ubiquitin ligase (16), is an important negative regulator of p53 activity and mediates the ubiquitin-dependent degradation of p53 by the proteasome (13, 17).High-risk HPV types, such as types 16 and 18, are the etiological agents for the development of cervical cancer (34). The oncogenic potential of these viruses correlates with the in vitro transforming activities of the viral oncogenes E6 and E7. The E7 protein promotes the upregulation of proliferation-related genes (25) thr...

Comparative Analysis of 19 Genital Human Papillomavirus Types with Regard to p53 Degradation, Immortalization, Phylogeny, and Epidemiologic Risk Classification

“…3A, whereas p53 localizes predominantly to the nucleus in normal keratinocytes, the amount of nuclear p53 in all HPVimmortalized cell lines is diminished and only causes a weak cytoplasmic signal. This finding is in line with the observation that HPV18 E6 not only mediates degradation of p53 but also influences its subcellular distribution (34). No differences were observed between the prototype HPV18 and the other HPV types investigated.…”

Selective induction of apoptosis by leptomycin B in keratinocytes expressing HPV oncogenes

“…It has been demonstrated that the HPV 16 E6 protein preferentially localises to the nucleus of cervical cancer cells 34 and that HPV 18 E6 is co-exported with p53 from the nucleus to the cytoplasm. 35 LMB treatment of cells expressing Ôhigh riskÕ HPV E6 results in a block of p53 nuclear export and stabilisation of p53, 36 however this LMB-induced reduction in HPV 18 E6-mediated degradation was shown to be only partial, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. 35 Our data indicate that despite any predicted LMB-induced E6-mediated p53 stabilisation, the level of E6-mediated p53 degradation observed following LMB treatment is still strong enough to allow a significant protective effect to cells against LMB-induced apoptosis.…”

Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells

“…This is suggested by the fact that adenovirus E4orf6 and E1B55K (Blanchette et al, 2004) and HSV-1 regulatory protein ICP0 (Boutell and Everett, 2003) have been reported to be able to interact and ubiquitinate p53 for subsequent nuclear export and proteasomal degradation. It has also been shown that human papillomaviruses (HPV) type 18 E6 can poly-ubiquitinate p53, independent of Mdm2, for nuclear proteasomal degradation or for a nuclear export to cytoplasm through the Crm1 nuclear export pathway for subsequent cytoplasmic proteasomal degradation (Stewart et al, 2005). In addition, HCMV proteins US2 and US11 are known to catalyze the dislocation and transfer of MHC class I heavy chains from the endoplasmic reticulum for ubiquitination and degradation (Shamu et al, 2001;Shamu et al, 1999;Wiertz et al, 1996), providing additional evidence that viral proteins can influence protein trafficking within the cell for ubiquitination and proteasomal degradation.…”