Modulation of Monocyte Function by Cytomegalovirus IL-10

描述(申请人提供)：巨细胞病毒是一种广泛传播的人类病原体，具有终身潜伏感染的能力。虽然CMV感染通常是亚临床的，但免疫抑制者可能会发生危及生命的疾病。我们的长期目标是确定人类巨细胞病毒用来建立潜伏感染和从潜伏感染中重新激活的机制。假设是单核细胞中CMV编码的白介素10(cmvlL-10)信号触发的事件可能有助于潜伏期的建立或重新激活。这是基于以下几点：1)cmvlL-10与细胞IL-10受体(IL-10R)结合，尽管与人IL-10(hll-10)只有27%的序列同源性；2)cmvlL-10与hll-10一样，具有强大的免疫抑制特性，如下调MHC表达和抑制单核细胞产生细胞因子；3)携带潜伏巨细胞病毒的细胞表达与IL-10同源的转录本，编码交替剪接形式的cmvlL-10(LA-cmvlL10)。此外，编码cmvlL-10的UL111.5A区域对溶血性病毒复制不是必需的，提示cmvlL-10在病毒感染的其他方面起作用。基于这些观察，拟议的研究将更彻底地检查cmvlL-10对单核细胞的影响，单核细胞是病毒持续存在的部位。其具体目标是：1)定义由CMV-1L10参与IL-10R所产生的信号事件。我们将研究a)Stat转录因子的激活，b)参与蛋白激酶，以及c)基因表达的变化。2)探讨cmvlL-10对单核细胞功能的调节机制。1可能的机制包括抑制转录因子NF-kappaB的活性。为了测试这一点，我们将检测a)NF-kappaB激活，b)I-kappaB蛋白水平，以及c)I-kappaB-α磷酸化状态。3)确定LA-cmvlL-10是否具有生物学功能。我们将a)表达LA-cmvlL-10，b)检测与IL-10R的结合，以及c)评估对细胞因子产生的抑制。这些结果将有助于更好地理解cmvlL-10的分子作用，并可能确立cmvlL-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.