Hou Wangheng, Torres Lilith, Cruz-Cosme Ruth, Arroyo Fernando, Irizarry Luis, Luciano Dalia, Márquez Arturo, Rivera Leslie L, Sala Antonio L, Luo Min-Hua, Tang Qiyi
Department of Microbiology, Howard University College of Medicine, Washington, DC, USA.
Department of Microbiology/RCMI Program, Ponce School of Medicine and Health Sciences, Ponce, Puerto Rico.
J Virol. 2016 Jul 27;90(16):7339-7349. doi: 10.1128/JVI.00837-16. Print 2016 Aug 15.
The human cytomegalovirus (HCMV) major immediate early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), the first 3 exons, and the first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one is essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's fourth intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a chromatin immunoprecipitation (ChIP) assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target.
CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than two proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified two Pys in intron 4 and found that the first Py is important and the second is required for the splicing and expression of IE2. We further investigated the mechanisms of gene switching from IE1 to IE2 and found that the two Pys are responsible for U2AF65's binding with intron 4. Therefore, the Pys in intron 4 are the cis elements that determine the fate of IE2 splicing. Furthermore, we found that a small RNA that is complementary to intron 4 repressed IE2 expression. Hence, we provide the first piece of evidence for a unique mechanism of MIE gene regulation at the splicing level.
人巨细胞病毒（HCMV）主要立即早期（MIE）基因对病毒复制至关重要。MIE基因编码的最丰富产物包括IE1和IE2。IE1和IE2基因共享MIE启动子（MIEP）、前3个外显子和前2个内含子。在CMV感染或MIE基因转染后，IE1比IE2表达更早。在本研究中，我们在第4内含子（外显子4和5之间）中鉴定出2个聚嘧啶（Py）序列，它们负责转录从IE1切换到IE2。第一个Py很重要，第二个Py对IE2的剪接和表达至关重要。在寻找MIE基因从IE1切换到IE2的机制时，我们发现第二个Py是IE2的第4内含子与剪接因子如U2AF65结合所必需的，这通过RNA电泳迁移率变动分析和染色质免疫沉淀（ChIP）分析确定，而第一个Py增强了U2AF65与内含子的结合。第二个Py发生突变的HCMV BACmid无法产生任何病毒，而第一个Py发生突变的HCMV以缺陷表型进行复制。此外，我们设计了一种与覆盖两个Py的内含子RNA互补的小RNA（scRNAPy）。scRNAPy干扰了U2AF65与内含子的相互作用并抑制了IE2表达。因此，我们的研究表明IE2基因剪接可能是一个抗CMV靶点。
CMV是一种普遍存在的疱疹病毒，是免疫受损者和老年人疾病和死亡的重要原因。对其基因调控的深入了解将为设计抗CMV策略提供线索。MIE基因是CMV最早的基因之一，对CMV复制至关重要。已知MIE基因需要剪接以产生两种以上的蛋白质；然而，MIE基因剪接如何被调控仍然不清楚。在本研究中，我们在第4内含子中鉴定出两个Py序列，发现第一个Py很重要，第二个Py是IE2剪接和表达所必需的。我们进一步研究了从IE1到IE2的基因切换机制，发现这两个Py负责U2AF65与第4内含子的结合。因此，第4内含子中的Py是决定IE2剪接命运的顺式元件。此外，我们发现与第4内含子互补的小RNA抑制了IE2表达。因此，我们提供了第一个证据，证明了MIE基因在剪接水平上独特的调控机制。
