Sénéchal Hélène, Poirier Guy G, Coulombe Benoit, Laimins Laimonis A, Archambault Jacques
Laboratory of Molecular Virology, Institut de recherches cliniques de Montréal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.
Virology. 2007 Feb 5;358(1):10-7. doi: 10.1016/j.virol.2006.08.035. Epub 2006 Oct 3.
The E2 protein of papillomaviruses binds to specific sites in the viral genome to regulate its transcription, replication and segregation in mitosis. Amino acid substitutions in the transactivation domain (TAD) of E2, of Arg37 and Ile73, have been shown previously to impair the transcriptional activity of the protein but not its ability to support viral DNA replication. To understand the biochemical basis of this defect, we have used the TADs of a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) as affinity ligands to capture proteins from whole cell extracts that can associate with these domains. The major TAD-binding protein was identified by mass spectrometry and western blotting as the long isoform of Brd4. Binding to Brd4 was also demonstrated for the E2 TADs of other papillomaviruses including cutaneous and animal types. For HPV11, HPV31 and CRPV E2, we found that binding to Brd4 is significantly reduced by substitutions of Arg37 and Ile73. Since these amino acids are located near each other in the 3-dimensional structure of the TAD, we suggest that they define a conserved surface involved in binding Brd4 to regulate viral gene transcription.
乳头瘤病毒的E2蛋白与病毒基因组中的特定位点结合，以调节其在有丝分裂中的转录、复制和分离。先前已表明，E2反式激活结构域（TAD）中的氨基酸取代，即精氨酸37和异亮氨酸73，会损害该蛋白的转录活性，但不影响其支持病毒DNA复制的能力。为了了解这种缺陷的生化基础，我们使用了低风险（HPV11）和高风险（HPV31）人乳头瘤病毒（HPV）的TAD作为亲和配体，从全细胞提取物中捕获能够与这些结构域结合的蛋白质。通过质谱和蛋白质印迹法鉴定出主要的TAD结合蛋白为Brd4的长异构体。其他乳头瘤病毒（包括皮肤型和动物型）的E2 TAD也被证明与Brd4结合。对于HPV11、HPV31和CRPV E2，我们发现精氨酸37和异亮氨酸73的取代会显著降低与Brd4的结合。由于这些氨基酸在TAD的三维结构中彼此靠近，我们认为它们定义了一个保守表面，参与Brd4结合以调节病毒基因转录。
