p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

Abstract

Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high-and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC 50 ]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC 50 s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some highrisk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants.

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…38 Mesplede et al showed that E6 of HPV26, 70 and 82 and to a lesser degree E6 of HPV66, are able to degrade p53 with the same efficiency as HR-HPV types, while LR-HPV11 is not. 39 In addition to pHR-HPV types, our findings of p16 INK4a overexpression in cervical cancer cases with single transcription of the HR-HPV types 39, 51 and 56 that also occur in low frequency in cervical cancer, supports their classification as Group 1 carcinogens.…”

“…34,35 The multiplex type-specific E7 PCR utilizes HPV type-specific primers targeting the E7 region for the detection of 19 HR-/pHR (HPV16, 18,26,31,33,35,39,45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73, 82) and 2 LRtypes (HPV6, 11), with detection limits ranging from 10 to 1,000 copies of the viral genome. 34 Two primers for the amplification of b-globin were also included to provide a positive control for the quality of the template DNA.…”

“…1,2 Epidemiological studies have demonstrated that 20 mucosal HPV types from five phylogenetically related species of the genus alpha (a5, a6, a7, a9 and a11-termed as ''high-risk clade'') are consistently found as single HPV infections in CxCa worldwide. [3][4][5][6] Based on their frequency in CxCa and available biological data, 12 of these types, i.e., types 16,18,31,33,35,39,45, 51, 52, 56, 58 and 59 have been defined as carcinogens (IARC Group 1A)-hereafter referred to as high-risk (HR)-HPV. For eight other types, in the high-risk clade, i.e., types 26, 53, 66, 67, 68, 70, 73 and 82, the combination of their low frequency, lack of data on their active transcription and their transforming potential in model systems, has led them to be classified as only probable/possible carcinogens (IARC Groups 2A and 2B)-hereafter referred to as possible highrisk (pHR)-HPV types.…”

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

“…Mesplède and colleagues examined the ability of 29 different E6s, most from genus alpha, to target p53 for degradation and characterized their subcellular localization patterns (43). A separate study by Cornet and coworkers characterized the ability of various beta HPV E6 proteins to increase the life span of human keratinocytes and to alter p53 functions in beta HPV E6-expressing cells (14).…”

Cigarette Smoking Promotes Infection of Cervical Cells by High-Risk Human Papillomaviruses, but not Subsequent E7 Oncoprotein Expression

“…The detection strategy for hrHPVE7 was based on five different assays: "recomWell HPV 16/18/45 KJhigh", "recomWell HPV 16/18/45 KJlow", "recomWell HPV 39/51/56/59", "recomWell HPV 16/31/33/35/52/58" and "recomWell HPV HR screen" (for 16,18,31,33,35,39,45, 51, 52, 56, 58 and 59 E7). Each assay refers to different combinations of hrHPVE7 or different concentrations of the relevant ELISA antibodies and yields a measurement of the optical density, a continuous variable.…”