Kamil Jeremy P, Coen Donald M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., SGMB 304A, Boston, MA 02115, USA.
J Virol. 2007 Oct;81(19):10659-68. doi: 10.1128/JVI.00497-07. Epub 2007 Jul 18.
UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.
UL97是由人巨细胞病毒（HCMV）编码的一种蛋白激酶，是针对这种普遍存在的疱疹病毒的抗病毒药物的重要靶点，该病毒是免疫功能低下宿主中危及生命的机会性感染的主要原因。为了更好地理解UL97在HCMV复制过程中的功能，一种重组HCMV，即NTAP97，它在UL97的氨基末端表达串联亲和纯化（TAP）标签，被用于从感染细胞中获得UL97蛋白复合物。如质谱和蛋白质印迹分析所示，65 kDa的病毒体被膜磷蛋白pp65（也称为UL83）在TAP过程中与UL97特异性共纯化。使用感染细胞裂解物进行的相互免疫共沉淀实验也表明UL97与pp65之间存在相互作用。此外，在谷胱甘肽S-转移酶（GST）下拉实验中，纯化的GST-pp65融合蛋白特异性结合体外翻译的UL97，表明UL97和pp65不需要其他病毒蛋白即可形成复合物，并且可能直接相互作用。值得注意的是，先前有报道称，当UL97受到药理抑制或基因敲除时，pp65在病毒复制过程中会形成异常聚集体，并且观察到一个pp-65缺失突变体对UL97抑制剂表现出适度抗性（M. N. Prichard、W. J. Britt、S. L. Daily、C. B. Hartline和E. R. Kern，《病毒学杂志》79:15494 - 15502，2005）。pp65与UL97之间稳定的蛋白质-蛋白质相互作用可能与这些蛋白质在病毒体形态发生过程中掺入HCMV颗粒有关，对HCMV的免疫调节具有潜在影响，并且也可能是HCMV复制过程中UL97受到负调控的一种机制。
