Human Cytomegalovirus UL28 and UL29 Open Reading Frames Encode a Spliced mRNA and Stimulate Accumulation of Immediate-Early RNAs
Human Cytomegalovirus UL28 and UL29 Open Reading Frames Encode a Spliced mRNA and Stimulate Accumulation of Immediate-Early RNAs
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DOI：
10.1128/jvi.00396-09
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发表时间：
2009-10-01
影响因子：
5.4
通讯作者：
Terhune, Scott S.
中科院分区：
文献类型：
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作者：
Mitchell, Dora P.;Savaryn, John P.;Terhune, Scott S.
我们已经鉴定了一种剪接的转录本，其含有来自HCMV UL29和UL28开放阅读框的序列。它含有氨基末端UL 29序列，随后是UL 28序列，并且它包括源自UL 26开放阅读框后的3 '非翻译区的聚腺苷酸信号。UL29/28 RNA表达具有早期动力学，并且在UL29的氨基末端插入有FLAG表位的病毒表达了类似于79-kDa蛋白质的标记的pUL29/28，其在感染后6小时被检测到。该病毒还表达了一种丰度较低的带标签的41-kDa蛋白，其大小对应于可以通过翻译未剪接的UL29/28转录物产生的蛋白。与这一预测一致，从多核糖体分离的RNA中存在未剪接和剪接的UL 29/28转录本。来自UL29/28位点的FLAG标记蛋白在感染的早期阶段在核病毒复制中心内积累。感染后晚期，它也存在于细胞质中，并且该蛋白存在于部分纯化的病毒颗粒制剂中，并且对蛋白酶处理具有抗性。通过突变破坏UL 29/28基因座导致DNA复制水平降低10倍，沿着病毒产量的类似降低。定量逆转录-PCR分析显示，与野生型病毒相比，在感染后4至10小时立即早期基因表达减少了2倍，pUL 29/28的瞬时表达激活了主要的立即早期启动子。我们的研究结果表明，UL29/28位点有助于激活即刻早期基因表达。
We have identified a spliced transcript that contains sequences from the HCMV UL29 and UL28 open reading frames. It contains amino-terminal UL29 sequences followed by UL28 sequences, and it includes a poly(A) signal derived from the 3'-untranslated region following the UL26 open reading frame. UL29/28 RNA is expressed with early kinetics, and a virus containing a FLAG epitope inserted at the amino terminus of UL29 expressed a tagged similar to 79-kDa protein, pUL29/28, that was detected at 6 h postinfection. The virus also expressed a less-abundant tagged 41-kDa protein, which corresponds in size to a protein that could be produced by translation of an unspliced UL29/28 transcript. Consistent with this prediction, both unspliced and spliced UL29/28 transcript was present in RNA isolated from polysomes. FLAG-tagged protein from the UL29/28 locus accumulated within nuclear viral replication centers during the early phase of infection. Late after infection it was present in the cytoplasm as well, and the protein was present and resistant to proteinase treatment in partially purified preparations of viral particles. Disruption of the UL29/28 locus by mutation resulted in a 10-fold decrease in the levels of DNA replication along with a similar reduction in virus yield. Quantitative reverse transcription-PCR analysis revealed an similar to 2-fold decrease in immediate-early gene expression at 4 to 10 h postinfection compared to the wild-type virus, and transient expression of pUL29/28 activated the major immediate-early promoter. Our results argue that the UL29/28 locus contributes to activation of immediate-early gene expression.