The human cytomegalovirus (HCMV) immediate early 1 (IE1) and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the posttranscriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5′ untranslated region (5′ UTR) plays an important role in regulating mRNA translation. Therefore, to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared 5′ UTR of the IE1 and IE2 mRNAs (MIE 5′ UTR) in regulating translation. In a cell-free system, the MIE 5′ UTR repressed translation, as predicted based on its length and sequence composition. However, in transfected cells we found that the MIE 5′ UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5′ UTR has a positive role in translation control. We also found that the MIE 5′ UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5′ UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild-type MIE 5′-UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5′ UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.

IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5′ UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date, most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore, this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation and thus inhibit lytic replication or prevent HCMV reactivation.

人巨细胞病毒（HCMV）立即早期1（IE1）和IE2蛋白是病毒复制的关键调节器。这两种蛋白都是有效建立裂解感染所必需的，而IE1和IE2的新生表达对于从潜伏期重新激活至关重要。因此，IE1和IE2蛋白表达的调节是HCMV感染结果的重要事件。编码IE1和IE2的主要转录本的转录已得到很好的研究，但对控制IE1和IE2蛋白质合成的转录后机制的了解相对较少。mRNA 5'非翻译区（5'UTR）在调节mRNA翻译中起重要作用。因此，为了更好地理解IE1和IE2 mRNA翻译的控制，我们检查了IE1和IE2 mRNA的5'UTR共享（MIE 5'UTR）在调节翻译中的作用。在无细胞系统中，MIE 5'UTR会抑制翻译，这是根据其长度和序列组成预测的。然而，在转染的细胞中，我们发现MIE 5'UTR增加了报告基因的表达并增强了其与多核糖体的结合，表明MIE 5'UTR在翻译控制中具有积极作用。我们还发现，MIE 5'UTR对于感染期间有效的IE1和IE2翻译是必需的。尽管IE1和IE2 mRNA的水平相似，但用相同长度的非结构化序列代替MIE 5'UTR会降低IE1和IE2蛋白的表达，并降低IE1和IE2 mRNA与多核糖体的结合。野生型MIE 5'-UTR序列对于有效的HCMV复制也是必需的。

重要性HCMV IE1和IE2蛋白是HCMV复制的关键调节剂，无论是在原发感染期间还是病毒潜伏期重新激活期间。因此，定义调节IE1和IE2表达的因素对于理解控制HCMV复制周期的分子事件很重要。在这里，我们确定了MIE 5'UTR在介导IE1和IE2 mRNA的有效翻译中的积极作用。由于几个原因，该结果是重要的进步。迄今为止，大多数对IE1和IE2调节的研究都集中在定义调节IE1和IE2转录的事件上。我们的工作表明，除了转录的调控外，IE1和IE2还受翻译水平的调控。所以，这项研究很重要，因为它确定了控制IE1和IE2表达并进而控制HCMV发病机理的另一层调控。这些翻译调节事件可能会受到限制IE1和IE2 mRNA翻译从而抑制裂解复制或阻止HCMV激活的新型抗病毒治疗药物的靶向。