Stability of the human papillomavirus type 18 E2 protein is regulated by a proteasome degradation pathway through its amino-terminal transactivation domain.
The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G(1) growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.
Bellanger
S
S
Unité des Virus Oncogènes, Département des Biotechnologies, URA 1644 du CNRS, Institut Pasteur, 75724 Paris Cedex 15, France.
Demeret
C
C
Goyat
S
S
Thierry
F
F
D003546:Cysteine Endopeptidases / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000502:physiology*; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000502:physiology*; D027383:Papillomaviridae / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014779:Virus Replication
D009097:Multienzyme Complexes; D009856:Oncogene Proteins, Viral; C069102:oncogene protein E2, Human papillomavirus type 18; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex