Bowman J Jason, Orlando Joseph S, Davido David J, Kushnir Anna S, Schaffer Priscilla A
Department of Medicine, Harvard Medical School at the Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
J Virol. 2009 Sep;83(17):8733-43. doi: 10.1128/JVI.00810-09. Epub 2009 Jun 17.
Of the five herpes simplex virus type 1 immediate early (IE) proteins, the least is known about the function of ICP22 during productive infection and latency. Research characterizing the physical and functional properties of the protein has been limited because ICP22 has proven to be difficult to express in transient assays. In addition, genetic analysis of ICP22 has been complicated by the fact that the C terminus of ICP22 is expressed as a discrete protein product. In order to characterize properties of mutant and wild-type ICP22, we developed a transient expression system. We found that ICP22 can be expressed at detectable levels when placed under the control of the cytomegalovirus IE promoter, confirming recent observations by K. A. Fraser and S. A. Rice (J. Virol. 81:5091-5101, 2007). We extended this analysis to show that ICP22 can also be expressed from its own promoter in the presence of other viral factors, either by coexpression with ICP0 or by infection with an ICP22 null virus. Notably, infection of cells transfected with an ICP22 expression vector yielded ICP22 protein that was modified in a manner similar to that of ICP22 protein detected in wild-type-infected cells. We go on to demonstrate that the failure of ICP22 protein to be expressed in transiently transfected cells was not due to inactivity of the ICP22 promoter, but rather to the ability of ICP22 to inhibit expression of reporter gene activity, including its own, in transient assays. Of special note was the observation that expression of ICP22 was sufficient to prevent transactivation of reporter genes by ICP0. Finally, transient expression of ICP22 was sufficient to complement replication of an ICP22 null virus, demonstrating that this system can be used to study functional properties of ICP22. Collectively, this transient expression system facilitates tests of the physical and functional properties of ICP22 and ICP22 mutants prior to introduction of mutant genes into the viral genome.
在单纯疱疹病毒1型的5种立即早期（IE）蛋白中，人们对感染性感染和潜伏期间ICP22的功能了解最少。由于已证明ICP22在瞬时分析中难以表达，因此对该蛋白的物理和功能特性进行表征的研究一直很有限。此外，ICP22的遗传分析也因ICP22的C末端表达为一种离散的蛋白产物这一事实而变得复杂。为了表征突变型和野生型ICP22的特性，我们开发了一种瞬时表达系统。我们发现，当置于巨细胞病毒IE启动子的控制下时，ICP22可以在可检测的水平上表达，这证实了K. A. Fraser和S. A. Rice最近的观察结果（《病毒学杂志》81:5091 - 5101, 2007）。我们扩展了这一分析，以表明在存在其他病毒因子的情况下，ICP22也可以从其自身的启动子表达，要么通过与ICP0共表达，要么通过感染ICP22缺失病毒。值得注意的是，用ICP22表达载体转染的细胞感染后产生的ICP22蛋白，其修饰方式与在野生型感染细胞中检测到的ICP22蛋白相似。我们继续证明，ICP22蛋白在瞬时转染细胞中未能表达，不是由于ICP22启动子无活性，而是由于ICP22在瞬时分析中抑制报告基因活性表达的能力，包括其自身的报告基因。特别值得注意的是，观察到ICP22的表达足以阻止ICP0对报告基因的反式激活。最后，ICP22的瞬时表达足以补充ICP22缺失病毒的复制，这表明该系统可用于研究ICP22的功能特性。总的来说，这种瞬时表达系统便于在将突变基因引入病毒基因组之前，对ICP22和ICP22突变体的物理和功能特性进行测试。
