A Hsp40 chaperone protein interacts with and modulates the cellular distribution of the primase protein of human cytomegalovirus.
A Hsp40 chaperone protein interacts with and modulates the cellular distribution of the primase protein of human cytomegalovirus.
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DOI：
10.1371/journal.ppat.1002968
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Liu F
中科院分区：
文献类型：
--
作者：
Pei Y;Fu W;Yang E;Shen A;Chen YC;Gong H;Chen J;Huang J;Xiao G;Liu F
基因组DNA复制是包括人巨细胞病毒（HCMV）在内的所有疱疹病毒普遍存在的基本过程。HCMV UL 70蛋白被认为编码病毒DNA复制机制的引发酶活性，并且在疱疹病毒中高度保守，需要定位于病毒DNA合成的核中。没有宿主因子促进UL 70的核输入的报道。在这项研究中，我们提供了第一个直接的证据表明，UL 70特异性相互作用的高度保守和普遍表达的热休克蛋白Hsp 40/DNAJ家族的成员，DNAJB 6，这是表达为两种异构体，a和B，作为选择性剪接的结果。通过酵母中的双杂交筛选和人细胞中的免疫共沉淀鉴定了UL 70与DNAJB 6a和B的共同区域的相互作用。在转染的细胞中，UL 70主要分别与DNAJB 6a共定位在细胞核中，与DNAJB 6 b共定位在细胞质中。在DNAJB 6a上调或DNAJB 6 b下调的细胞中，UL 70的核输入增加，而在DNAJB 6a下调或DNAJB 6 b上调的细胞中，UL 70的核输入减少。此外，在DNAJB 6a上调或DNAJB 6 b下调的细胞中，病毒DNA合成和子代产生的水平增加，而在DNAJB 6a下调或DNAJB 6 b上调的细胞中，病毒DNA合成和子代产生的水平降低。因此，DNAJB 6a和B似乎分别增强了UL 70的核输入和细胞质积累。我们的研究结果还表明，DNAJB 6亚型的相对表达水平可能在调节UL 70的细胞定位中起关键作用，从而导致HCMV DNA合成和裂解感染的调制。基因组DNA复制在包括人巨细胞病毒（HCMV）在内的所有疱疹病毒中高度保守，并且是目前大多数FDA批准的抗疱疹治疗剂的靶标。很少有人知道UL 70，这被认为是编码的引物酶活性的病毒DNA复制机器和基因组复制是必不可少的，是如何输入到细胞核，病毒DNA合成的网站。在这项研究中，我们证明了HCMV引发酶与属于热休克蛋白40（Hsp 40）家族的高度保守和普遍表达的伴侣蛋白DNAJB 6相互作用。作为选择性剪接的结果，DNAJB 6表达为两种同种型a和B。虽然DNAJB 6 b促进病毒引发酶的细胞质积累，但DNAJB 6a增强其核分布，代表了参与促进疱疹病毒引发酶核输入的细胞因子的第一个例子。我们的研究表明，DNAJB 6亚型的相对表达水平可能代表了一种新的机制，通过调节病毒引发酶的细胞定位来调节HCMV裂解性复制。此外，我们的研究结果提出了开发新的策略来治疗疱疹病毒复制的可能性，通过改变细胞蛋白质的表达来调节引发酶的细胞分布。
Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV). HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection. Genomic DNA replication is highly conserved across all herpesviruses including human cytomegalovirus (HCMV) and is the target for most of the current FDA-approved anti-herpes therapeutic agents. Little is known about how UL70, which is believed to encode the primase activity of the viral DNA replication machinery and is essential for genomic replication, is imported to the nuclei, the site of viral DNA synthesis. In this study, we demonstrated that the HCMV primase interacts with a highly conserved and ubiquitously expressed chaperone protein DNAJB6 that belongs to the heat shock protein 40 (Hsp40) family. As a result of alternative splicing, DNAJB6 is expressed as two isoforms, a and b. While DNAJB6b promotes cytoplasmic accumulation of the viral primase, DNAJB6a enhances its nuclear distribution, representing the first example of a cellular factor involved in facilitating nuclear import of a herpesvirus primase. Our study suggests that the relative expression level of DNAJB6 isoforms may represent a novel mechanism for modulating HCMV lytic replication by regulating the cellular localization of the viral primase. Furthermore, our results raise the possibility of developing new strategies for treating herpesvirus replication by modulating the cellular distribution of the primase with altered expression of a cellular protein.
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