The protein encoded by the Us3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown
The protein encoded by the Us3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown
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DOI：
10.1128/jvi.79.7.3987-3997.2005
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发表时间：
2005-04-01
影响因子：
5.4
通讯作者：
Osterrieder, N
中科院分区：
文献类型：
--
作者：
Schumacher, D;Tischer, BK;Osterrieder, N
马立克氏病病毒（MDV）编码的蛋白质与单纯疱疹病毒1型的U（S）3蛋白和水痘-带状疱疹病毒的基因66产物具有高度的氨基酸相似性。在感染性细菌人工染色体克隆BAC 20中，用卡那霉素抗性基因替换MDV U（S）3直向同源物。转染Us 3阴性BAC 20 DNA（20 Delta U（S）3）后，所得重组20 Delta U（S）3病毒显示出显著降低的生长动力学。与亲本BAC 20病毒相比，鸡胚细胞上的病毒滴度降低约10倍，并且噬斑大小显著更小（降低65%）。U（S）3阴性MDV的缺陷在表达具有羧基末端FIAG标签的U（S）3蛋白的回复突变病毒（20 U（S）3*）中完全恢复。电子显微镜研究表明，20 Delta U（S）3突变体在细胞间有效传播的缺陷是伴随着主要有包膜病毒粒子在核周空间中积聚在含有几个病毒颗粒的特征囊泡中，这导致颗粒数量减少。在细胞质中。在用亲本BAC 20病毒或20 U（S）3* 回复突变体病毒感染的细胞中未观察到这些囊泡的形成。在感染或转染U（S）3表达质粒后，通过用鬼笔环肽-Alexa 488观察肌动蛋白来研究MDV U（S）3蛋白在肌动蛋白应力纤维断裂中的作用。将肌动蛋白解聚药物细胞松弛素D添加到转染或感染的细胞中，用低至50 nM的药物就能完全抑制噬斑形成，而浓度高达50 μ M的诺考达唑对MDV噬斑形成的影响相对较小。结果表明，MDV U（S）3丝氨酸-苏氨酸蛋白激酶瞬时参与MDV介导的应激纤维断裂，肌动蛋白的聚合，而不是微管，在MDV细胞间传播中起重要作用。
Marek's disease virus (MDV) encodes a protein exhibiting high amino acid similarity to the U(S)3 protein of herpes simplex virus type 1 and the gene 66 product of varicella-zoster virus. The MDV U(S)3 orthologue was replaced with a kanamycin resistance gene in the infectious bacterial artificial chromosome clone BAC20. After transfection of Us3-negative BAC20 DNA (20 Delta U(S)3), the resulting recombinant 20 Delta U(S)3 virus exhibited markedly reduced growth kinetics. Virus titers on chicken embryo cells were reduced by approximately 10-fold, and plaque sizes were significantly smaller (65% reduction) compared to parental BAC20 virus. The defect of the U(S)3-negative MDV was completely restored in a revertant virus (20U(S)3*) expressing a U(S)3 protein with a carboxy-terminal FIAG tag. Electron microscopical studies revealed that the defect of the 20 Delta U(S)3 mutant to efficiently spread from cell to cell was concomitant with an accumulation in the perinuclear space of primarily enveloped virions in characteristic vesicles containing several virus particles, which resulted in reduced numbers of particles in the cytoplasm. The formation of these vesicles was not observed in cells infected with either parental BAC20 virus or the 20U(S)3* revertant virus. The role of the MDV U(S)3 protein in actin stress fiber breakdown was investigated by visualizing actin with phalloidin-Alexa 488 after infection or transfection of a U(S)3 expression plasmid. Addition of the actin-depolymerizing drug cytochalasin D to cells transfected or infected w:ith BAC20 resulted in complete inhibition of plaque formation with as little as 50 nM of the drug, while concentrations of nocodazole as high as 50 mu M only had a relatively minor effect on MDV plaque formation. The results indicated that the MDV U(S)3 serine-threonine protein kinase is transiently involved in MDV-mediated stress fiber breakdown and that polymerization of actin, but not microtubulles, plays an important role in MDV cell-to-cell spread.