The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-asscciated nuclear bodies at very early times in infected permissive cells
The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-asscciated nuclear bodies at very early times in infected permissive cells
复制标题
DOI：
10.1128/jvi.71.6.4599-4613.1997
复制
发表时间：
1997-06-01
影响因子：
5.4
通讯作者：
Hayward, GS
中科院分区：
文献类型：
--
作者：
Ahn, JH;Hayward, GS
人巨细胞病毒(HCMV)的主要即刻早期(MIE)基因产物是核磷蛋白，被认为在启动裂解周期基因调控途径中起关键作用。我们研究了IE1和IE2蛋白在病毒感染和DNA转基因细胞中的核内定位模式。人巨细胞病毒感染的人二倍体成纤维细胞(HF)经特异性单抗染色后，早期(2 H)IE1定位为核弥散和点状分布的混合型，以后则转变为核扩散型。相反，IE2在感染后2~12h内持续分布于核斑点结构。这些点状结构类似于先前存在的PML相关核体(ND10或PML致癌结构域[PODS])，作为单纯疱疹病毒(HSV)感染的非常早期事件，IE110蛋白破坏和分散了这些核体。然而，与HSV不同的是，HCMV导致PML和SP100蛋白的分布从点状小体转变为均匀的弥漫模式，这一过程在感染后2小时50%的细胞和4h后90%的细胞完成。共聚焦双标记间接免疫荧光分析证实，IE1和IE2在感染后很早的时间内与PML在斑点体内瞬时共存。在瞬时表达分析中，单独将IE1编码的质粒DNA导入Vero或HP细胞产生典型的PML与IE1蛋白一起进入均匀的核扩散模式，而单独导入IE2编码的质粒DNA导致IE2蛋白与PML在豆荚中稳定地共存。IE1的截短突变形式提供了大的核聚集并未能重新分布PML，类似地，IE2的缺失突变形式未能与点状PML小体共定位，证实了这些效应的特异性。此外，表达IE1的Vero和U373细胞株也表现出完全的PML重定位，与IE1蛋白一起进入核扩散模式，尽管不表达IE1的细胞中有很小一部分恢复为点状PML模式，最后，单独表达IE1或IE2的E1a阴性重组腺病毒载体感染证实了IE1的PML再分布活性和IE2与PML点状小体的直接关联。这些结果证实，IE1对PML相关核体的瞬时共定位、VITH和破坏以及IE2对PML相关核体的持续靶向是这两种MIE调控蛋白的固有特性，我们认为这可能是HCMV有效裂解周期感染的关键初始事件。
The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HP cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern, Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization,vith and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.