You Jianxin, Srinivasan Viswanathan, Denis Gerald V, Harrington William J, Ballestas Mary E, Kaye Kenneth M, Howley Peter M
Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
J Virol. 2006 Sep;80(18):8909-19. doi: 10.1128/JVI.00502-06.
The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.
卡波西肉瘤相关疱疹病毒（KSHV）的潜伏相关核抗原（LANA）是潜伏感染期间宿主细胞中病毒附加体维持所必需的。该蛋白的两个区域参与将LANA/病毒附加体拴系到宿主有丝分裂染色体上，并且LANA染色体结合位点备受关注。因为先前的研究已确定溴结构域蛋白Brd4是牛乳头瘤病毒E2蛋白的有丝分裂染色体锚定蛋白，该蛋白将病毒附加体拴系到宿主有丝分裂染色体上（J. You、J. L. Croyle、A. Nishimura、K. Ozato和P. M. Howley，《细胞》117:349 - 360，2004年，以及J. You、M. R. Schweiger和P. M. Howley，《病毒学杂志》79:14956 - 14961，2005年），所以我们研究了KSHV LANA是否与Brd4相互作用。我们发现LANA在体内和体外均与Brd4结合，并且这种结合是由Brd4的ET（末端外）结构域与LANA先前涉及染色体结合的羧基末端区域之间的直接蛋白质 - 蛋白质相互作用介导的。Brd4在整个有丝分裂过程中与有丝分裂染色体相关联，并在宿主有丝分裂染色体上与LANA和KSHV附加体表现出强烈的共定位。尽管另一种溴结构域蛋白RING3/Brd2在体外以类似方式与LANA结合，但在未感染KSHV的细胞中它基本被排除在有丝分裂染色体之外，而在感染KSHV的细胞中它部分被募集到染色体上。这些数据确定Brd4是有丝分裂染色体上LANA羧基末端的相互作用蛋白，并暗示两种溴结构域蛋白RING3/Brd2和Brd4在LANA结合中具有不同的功能作用。此外，因为最近已表明Brd4在转录中起作用，所以我们研究了Brd4是否可以调节可被LANA反式激活的CDK2启动子。
