ICP27 interacts with the C-terminal domain of RNA polymerase II and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection
ICP27 interacts with the C-terminal domain of RNA polymerase II and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection
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DOI：
10.1128/jvi.80.7.3567-3581.2006
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发表时间：
2006-04-01
影响因子：
5.4
通讯作者：
Sandri-Goldin, RM
中科院分区：
文献类型：
--
作者：
Dai-Ju, JQ;Li, L;Sandri-Goldin, RM
单纯疱疹病毒1型（HSV-1）ICP 27已显示与RNA聚合酶II（RNAP II）全酶相互作用。在这里，我们表明，ICP 27与RNA-P II的C-末端结构域（CTD）相互作用，并且不能相互作用的ICP 27突变体不能将RNAP H重新定位到病毒转录位点，这表明ICP 27在RNAP II招募中的作用。使用特异于RNAP H CTD的不同磷酸化形式的单克隆抗体，我们发现丝氨酸-2磷酸化形式，这主要是在延长复合物中发现的，没有被招募到病毒转录位点。此外，磷酸丝氨酸-2染色总体减少。蛋白质印迹分析显示，有一个显着的减少磷酸丝氨酸-2形式和整体RNAP II水平在裂解物从细胞感染野生型HSV-1。cdk 9水平没有明显差异，表明发生的是蛋白质降解而不是去磷酸化。用蛋白酶体抑制剂MG-132和lactacystin处理感染的细胞防止了磷酸丝氨酸-2形式和总体RNAP II水平的降低;然而，伴随着几种HSV-1晚期蛋白水平和病毒产量的降低。蛋白酶体降解已显示在DNA损伤位点分解停滞的RNAP II复合物以允许转录物的3'加工。因此，我们提出，在感染后期，当强大的转录和DNA复制发生时，延伸复合物可能会发生碰撞，蛋白酶体降解可能需要解决。
Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNA-P II and that ICP27 mutants that cannot interact fail to relocalize RNAP H to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP H CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3' processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.