Modulation of Monocyte Function by Cytomegalovirus IL-10

描述（由申请方提供）：巨细胞病毒是一种广泛存在的人类病原体，能够作为终身潜伏感染持续存在。虽然CMV感染通常是亚临床的，但危及生命的疾病可能发生在免疫抑制者中。我们的长期目标是确定人类CMV建立潜伏感染和从潜伏期重新激活的机制。假设是单核细胞中CMV编码的白细胞介素-10（cmvIL-10）信号传导触发可促进潜伏期的建立或从潜伏期再激活的事件。其依据如下：1）cmvIL-10结合细胞IL-10受体（IL-10 R），尽管与人IL-10（hIL-10）仅具有27%的序列同一性，2）cmvIL-10，像hIL-10一样，具有有效的免疫抑制特性，例如下调MHC表达和抑制单核细胞产生细胞因子，和3）携带潜伏CMV的细胞表达与编码cmvIL-10的交替剪接形式（LA-cmvIL-10）的IL-10同源的转录物。此外，编码cmvIL-10的UL111.5A区域对于裂解性病毒复制是非必需的，表明cmvIL-10在病毒感染的其它方面中的作用。基于这些观察结果，所提出的研究将更彻底地检查cmvIL-10对单核细胞（病毒持续存在的部位）的作用。具体目标是：1）定义由cmv-ILlO接合IL-10 R引起的信号传导事件。我们将检查a）Stat转录因子的激活，B）参与蛋白激酶，和c）基因表达的变化。2)检查cmvIL-10诱导的单核细胞功能调节的机制。1可能的机制涉及抑制转录因子NF-κ B活性。为了检验这一点，我们将检查a）NF-κ B激活，B）I-κ B蛋白水平，和c）I-κ B-α磷酸化状态。3)确定LA-cmvIL-10是否具有生物学功能。我们将a）表达LA-cmvIL-10，B）检查与IL-10 R的结合，和c）评价细胞因子产生的抑制。结果将提供对cmvIL-10的分子作用的更好理解，并且可能建立cmvIL-10在调节病毒潜伏期中的作用。

DESCRIPTION (provided by applicant): Cytomegalovirus is a widespread human pathogen with the ability to persist as a lifelong latent infection. Although CMV infection is typically subclinical, life-threatening disease may occur in the immunosuppressed. Our long-term goal is to identify mechanisms used by human CMV to establish latent infection and reactivate from latency. The hypothesis is that CMV-encoded interleukin-10 (cmvlL-10) signaling in monocytes triggers events that may facilitate establishment of or reactivation from latency. This is based on the following: 1) cmvlL-10 binds to the cellular IL-10 receptor (IL-10R), despite having only 27% sequence identity to human IL-10 (hlL-10), 2) cmvlL-10, like hlL-10, has potent immunosuppressive properties, such as down-regulation of MHC expression and inhibition of cytokine production by monocytes, and 3) cells harboring latent CMV express a transcript with homology to IL-10 encoding an alternately spliced form of cmvlL-10 (LA-cmvlL10). Furthermore, the UL111.5A region encoding cmvlL-10 is non-essential for lytic virus replication, suggesting a role for cmvlL-10 in other aspects of virus infection. Based on these observations, the proposed study will more thoroughly examine the effect of cmvlL-10 on monocytes, a site of virus persistence. The specific aims are: 1) Define signaling events that result from IL-10R engagement by cmv-lL10. We will examine a) activation of Stat transcription factors, b) participating protein kinases, and c) changes in gene expression. 2) Examine the mechanism for cmvlL-10-induced modulation of monocyte function. 1 possible mechanism involves inhibition of transcription factor NF-kappaB activity. To test this, we will examine a) Nf-kappaB activation, b) I-kappaB protein, levels, and c) I-kappaB-alpha phosphorylation state. 3) Determine whether LA-cmvlL-10 has biological function. We will a) express LA-cmvlL-10, b) examine binding to IL- 10R, and c) evaluate inhibition of cytokine production. The results will provide a better understanding of the molecular action of cmvlL-10, and possibly establish a role for cmvlL-10 in regulation of virus latency.