ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells
ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells
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DOI：
10.1128/jvi.75.13.6143-6153.2001
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发表时间：
2001-07-01
影响因子：
5.4
通讯作者：
Schaffer, PA
中科院分区：
文献类型：
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作者：
Halford, WP;Kemp, CD;Schaffer, PA
在之前的研究中，我们证明感染细胞多肽 0 (ICP0) 对于在潜伏感染的三叉神经节 (TC) 细胞的原代培养物中有效重新激活 1 型单纯疱疹病毒 (HSV-1) 是必需的 (W. P. Halford 和 P. A. Schaffer, J. Virol. 75:3240-3249, 2001)，本研究旨在确定 ICP0 是否足够 触发潜伏感染的 TG 细胞中 HSV-1 的重新激活。为了检验这一假设，构建了在四环素反应元件（TRE）启动子控制下表达野生型和突变型 ICP0 的复制缺陷型腺病毒载体。还构建了编码野生型ICP4、HSV-1起源结合蛋白(OBP)的野生型和突变型以及VP16的野生型和突变型的类似腺病毒载体。 TRE 启动子是通过用测试载体和腺病毒载体共感染 Vero 细胞来诱导的，腺病毒载体在强力霉素存在下表达反向四环素调节的反式激活因子。 Northern印迹分析表明，腺病毒表达载体中OEP基因的转录随多西环素浓度在0.1至10μM范围内的变化而增加。同样，Western印迹分析表明，向腺病毒载体感染的Vero细胞中添加3μM多西环素导致OBP表达增加100倍。由腺病毒载体表达的野生型 ICP0、ICP 1、OBP 和 VP16 之所以具有功能，是因为它们能够补充具有这些基因突变的复制缺陷型 HSV-1 菌株在 Vero 细胞中形成的噬斑。表达野生型 ICP0、ICP4 或 VP16 的腺病毒载体分别在 86% +/- 5%、86% +/- 5% 和 97% +/- 5% 的 TG 细胞培养物中诱导 HSV-1 重新激活（平均值 +/- 标准差）。相反，表达野生型 OBP 或突变型 ICP0、OBP 或 VP16 的载体分别在 5% +/- 5%、8% +/- 0%、0% +/- 0% 和 13% +/- 6% 的 TG 细胞培养物中诱导再激活。在对照感染中，腺病毒载体在TG神经元中有效表达绿色荧光蛋白，但不诱导HSV-1重新激活，因此，ICP0、ICP4或VP16的表达足以在潜伏感染的TG细胞培养物中诱导HSV-1重新激活。我们得出的结论是，该系统提供了一个强大的工具，用于确定哪些细胞酸性病毒蛋白足以诱导 HSV-1 从神经元潜伏期重新激活。
In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TC) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001), The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OEP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 muM, Likewise, Western blot analysis demonstrated that addition of 3 muM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP 1, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4 or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation, Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular acid viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.