Insertion of an EYFP-pp71 (UL82) Coding Sequence into the Human Cytomegalovirus Genome Results in a Recombinant Virus with Enhanced Viral Growth
Insertion of an EYFP-pp71 (UL82) Coding Sequence into the Human Cytomegalovirus Genome Results in a Recombinant Virus with Enhanced Viral Growth
复制标题
将 EYFP-pp71 (UL82) 编码序列插入人类巨细胞病毒基因组中可产生具有增强病毒生长的重组病毒
DOI：
10.1128/jvi.01006-08
复制
发表时间：
2008
影响因子：
5.4
通讯作者：
T. Stamminger
中科院分区：
文献类型：
--
作者：
Nina Tavalai;M. Kraiger;Nina Kaiser;T. Stamminger
摘要：人类巨细胞病毒 (HCMV) UL82 编码的被膜蛋白 pp71 最近被证明可以通过中和 ND10 蛋白 hDaxx 建立的细胞内在免疫防御来激活病毒立即早期 (IE) 基因表达。 Pp71 在感染后定位于 ND10 并诱导 hDaxx 降解。在这里，我们报告成功生成了表达增强型黄色荧光蛋白（EYFP）的重组 HCMV，该蛋白融合到 pp71 的 N 末端。有趣的是，将 EYFP-UL82 编码序列插入 HCMV AD169 基因组中产生了一种重组病毒，称为 AD169/EYFP-pp71，其复制滴度明显高于野生型 AD169。特别是，我们注意到 AD169/EYFP-pp71 接种后 pp71 的蛋白质水平强烈增加。尽管高丰度的 pp71 导致外皮蛋白更多地包装到病毒颗粒中，但在 AD169/EYFP-pp71 感染后未检测到 hDaxx 降解增加。相反，进一步的研究表明，与野生型 AD169 相比，病毒 DNA 复制显着增强。因此，我们假设 pp71 的尚未鉴定的功能有助于增强 AD169/EYFP-pp71 的感染性。 AD169/EYFP-pp71 感染后早期和晚期基因表达的增加与 IE 蛋白水平升高无关，这一观察结果还支持了这一假设。最后，免疫荧光分析证实，hDaxx 决定了感染后 pp71 的 ND10 定位，因为 pp71 在没有 hDaxx 的情况下表现出核仁分布。总之，我们生成了重组 HCMV，它构成了一个有用的工具，不仅可以更精确地剖析 pp71 亚核定位的体内动力学，而且还可以探索该病毒反式激活因子的新特征。
ABSTRACT The human cytomegalovirus (HCMV) UL82-encoded tegument protein pp71 has recently been shown to activate viral immediate-early (IE) gene expression by neutralizing a cellular intrinsic immune defense instituted by the ND10 protein hDaxx. Pp71 localizes to ND10 upon infection and induces the degradation of hDaxx. Here, we report the successful generation of a recombinant HCMV expressing enhanced yellow fluorescent protein (EYFP) fused to the N terminus of pp71. Intriguingly, insertion of the EYFP-UL82 coding sequence into the HCMV AD169 genome gave rise to a recombinant virus, termed AD169/EYFP-pp71, that replicates to significantly higher titers than wild-type AD169. In particular, we noticed strongly increased protein levels of pp71 after AD169/EYFP-pp71 inoculation. Although the high abundance of pp71 resulted in augmented packaging of the tegument protein into viral particles, no increased hDaxx degradation was detectable upon AD169/EYFP-pp71 infection. In contrast, further investigation revealed a significantly enhanced viral DNA replication compared to wild-type AD169. Thus, we hypothesize that an as-yet-unidentified function of pp71 contributes to the enhanced infectivity of AD169/EYFP-pp71. This assumption is additionally supported by the observation that increased early and late gene expression after AD169/EYFP-pp71 infection occurs independent of elevated IE protein levels. Finally, immunofluorescence analyses confirmed that hDaxx determines the ND10-localization of pp71 upon infection, since pp71 exhibited a nucleolar distribution in the absence of hDaxx. Taken together, we generated a recombinant HCMV that constitutes a useful tool not only to dissect the in vivo dynamics of pp71 subnuclear localization more precisely but also to explore new features of this viral transactivator.
影响因子：
3.7
作者：
J. Demarchi
通讯作者：
J. Demarchi
影响因子：
4
作者：
A. Hollenbach;C. McPherson;E. Mientjes;R. Iyengar;G. Grosveld
通讯作者：
A. Hollenbach;C. McPherson;E. Mientjes;R. Iyengar;G. Grosveld
DOI：
10.1073/pnas.97.26.14506
发表时间：
2000-12-19
影响因子：
11.1
作者：
Bresnahan, WA;Shenk, TE
通讯作者：
Shenk, TE