ERK1/2 and MEK1/2 induced by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) early during infection of target cells are essential for expression of viral genes and for establishment of infection
ERK1/2 and MEK1/2 induced by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) early during infection of target cells are essential for expression of viral genes and for establishment of infection
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DOI：
10.1128/jvi.79.16.10308-10329.2005
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发表时间：
2005-08-01
影响因子：
5.4
通讯作者：
Chandran, B
中科院分区：
文献类型：
--
作者：
Sharma-Walia, N;Krishnan, HH;Chandran, B
Kaposi在体外靶靶细胞感染中与肉瘤相关的疱疹病毒（KSHV）的特征是与潜伏期相关的基因ORF 73（LANA-1），ORF 72和K13的表达，以及由极有限数量的lytic基因的短暂表达例如裂解周期开关基因ORF 50（RTA）和立即（IE）裂解K5，K8和V-IRF2基因。在感染的早期阶段，几个重叠的多步复合事件先于病毒基因表达的启动。 KSHV包膜糖蛋白GB诱导FAK-SRC-PI3K-RBOGTPase（FAK是局灶性粘附激酶）信号传导途径。感染后5分钟（P.I.），KSRV通过PI3K-PKC Zeta-Mek途径诱导细胞外信号调节的激酶1和2（ERK1/2）。此外，KSHV调节了原代人皮肤微血管内皮细胞（HMVEC-D）和成纤维细胞（HFF）细胞的几个宿主基因的转录2小时和4小时。 PI-3K和其他细胞酪氨酸激酶抑制剂对病毒进入和感染的中和化感染表明，信号分子在靶细胞KSHV感染中起着关键作用。在这里，我们研究了KSHV和KSHV包膜糖蛋白GB和GPK8.1A的ERK1/2诱导，以及诱导ERK在病毒和宿主基因表达中的作用。感染期间的早期，即使在血清饥饿的细胞中以及血清存在下的活体和紫外线灭活的KSHV感染的多样性下，也观察到显着的ERK1/2诱导。紫外线灭活病毒的进入和缺乏病毒基因表达表明ERK1/2诱导是由KSHV结合和进入引起的初始信号级联介导的。纯化的可溶性GPK8.1A诱导了HMVEC-D和HFF中的MEK1/2依赖性ERK1/2，但不诱导ERK1/2和P38丝裂原激活的蛋白激酶（MAPK）。仅在HMVEC-D中看到具有可溶性GB的中等ERK诱导。 GPK8.1A与肝素或抗GPK8.1A抗体的预孵育抑制了ERK诱导。 MEK/ERK的选择性抑制剂U0126阻止了GPK8.1A-和KSHV诱导的ERK激活。 ERK1/2抑制没有阻止病毒DNA内在化，并且对从头感染期间KSHV DNA的核递送没有显着影响。通过定量的实时反transciptase PCR对病毒基因表达的分析表明，用U0126对细胞进行1小时的预处理，在2-H感染期间，KSRV显着抑制了ORF 73，ORF 50（RTA）和IE--- K8和V-IRF2基因。但是，裂解IE-K5基因的表达没有显着影响。通过与U0126预孵育，在BCBL-1细胞中ORF 73的表达也显着抑制。 ERK1/2的抑制也抑制了某些重要宿主基因（例如DUSP5）（双重特异性磷酸酶5），ICAM-1（细胞间粘附分子1），肝素结合表皮生长因子和血管内皮生长因子的转录 - 在KSHV感染期间进行调节。在感染期间早期诱导了几种MAPK调节的宿主转录因子，例如C-JUN，STAT1 Alpha，MEF2，C-MYC，ATF-2和C-FOS，ERK抑制显着阻止了C-FOS，C-JUN，C-JUN，C。 -MYC和STAT1α激活在被感染的细胞中。在感染的细胞核提取物中很容易检测到与RTA启动子结合的AP1转录因子与RTA启动子结合，而ERK抑制作用显着降低了。调节病毒基因表达和宿主细胞GNE的启动，这进一步支持了我们的假设，即病毒DNA递送的导管旁边在细胞质中，KSHV与宿主细胞受体的相互作用会产生适当的细胞内环境促进感染。
Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RboGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSRV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKC zeta-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transciptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSRV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1 alpha, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1 alpha activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition.Together, these results suggest that very early during de novo, infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell gnes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection.