Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells
Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells
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DOI：
10.1128/jvi.00311-08
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发表时间：
2008-08-01
影响因子：
5.4
通讯作者：
Kinchington, Paul R.
中科院分区：
文献类型：
--
作者：
Erazo, Angela;Yee, Michael B.;Kinchington, Paul R.
水痘-带状疱疹病毒(VZV)开放阅读框66(ORF66)编码一种丝氨酸/苏氨酸蛋白激酶，在大多数细胞类型中不是水痘病毒生长所必需的，但在T细胞中高效生长是必需的。ORF66激酶影响主要调节蛋白IE62的核输入和病毒包装，并调节T细胞的凋亡。在这里，我们使用表达绿色荧光蛋白(GFP)标记的功能性和激酶阴性的ORF66蛋白的VZV重组体进一步研究了ORF66的重要性。在MRC-5成纤维细胞中，带有截短或激酶失活的ORF66蛋白的VZV病毒粒子对生长和后代产量有轻微的抑制作用，但在低传代原代人角膜基质成纤维细胞(PCF)中生长和复制严重受损。为了确定生长障碍是否是由于ORF66激酶对IE62核进口的调节，我们制备了表达IE62的重组VZV，其丙氨酸残基位于S686，可能是ORF66激酶阻断IE62核进口的靶点。VZV重组表达的IE62 S686A在感染过程中始终保持核状态，不被包装成病毒粒子。然而，突变病毒仍然在PCF细胞中高效复制。我们还表明，ORF66激酶的失活仅导致PCF细胞凋亡水平的轻微增加，这不能完全解释ORF66激酶的细胞特异性生长需求。因此，独特的短区VZV激酶具有重要的细胞类型特异性功能，与那些影响IE62和细胞凋亡的功能是分开的。
Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.