High-risk human papillomaviruses (HPVs) are etiologic agents of cervical cancer, a leading cause of cancer death in young women, worldwide. Due to HPV genome integration during malignant progression, cervical cancers consistently express two viral proteins E6 and E7. Ectopic HPV E6/E7 expression in human cell culture and transgenic mouse models recapitulates key steps of cervical neoplasia and cancer, and sustained E6/E7 expression is necessary for maintenance of the transformed state of cervical cancer lines. High-risk HPV E7 proteins induce proteasome-mediated degradation of the retinoblastoma tumor suppressor pRB. To determine the biochemical basis for this activity, we used an immunoaffinity chromatography based procedure to purify HPV-16 E7-associated cellular protein complexes. Individual components were identified by mass spectrometry. We discovered that HPV-16 E7 is associated with a Cullin-2 ubiquitin ligase complex and we hypothesize that E7 may target associated proteins for ubiquitin-mediated proteasomal degradation through interaction with the Cullin-2 ubiquitin ligase complex. We will investigate whether pRB is degraded through the E7/Cullin-2 complex, and perform additional biochemical experiments to determine the molecular composition of E7/Cullin-2 complexes. With these experiments we will also identify additional E7-associated cellular proteins that may be degraded through the E7/Cullin-2 ubiquitin ligase. Using RNA interference and dominant negative mutants we will determine the biological relevance of the E7/Cullin-2 interaction. We will perform studies aimed to determine whether association of E7 with the Cullin-2 ubiquitin ligase complex may perturb von Hippel Lindau tumor suppressor activity, which functions as a physiological substrate specificity adapter for the Cullin-2 ubiquitin ligase complex. Based on our discovery that the HPV-16 E7 oncoprotein can uncouple centrosome duplication from the cell division cycle and induce centrosome-associated mitotic errors in normal human cells, we will determine the biochemical basis for this activity.
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