Proteasomal degradation of the papillomavirus E2 protein is inhibited by overexpression of bromodomain-containing protein 4.
The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.
Gagnon
David
D
Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada.
Joubert
Simon
S
Sénéchal
Hélène
H
Fradet-Turcotte
Amélie
A
Torre
Sabrina
S
Archambault
Jacques
J
D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D054875:Ubiquitination; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C117355:E2 protein, Human papillomavirus type 11; C098597:E2 protein, Human papillomavirus type 31