ROLE OF FV2 IN ERYTHROPOIESIS

感染Friend病毒复合体的小鼠会发生急性红细胞增生症，并迅速发展为红白血病。Friend病毒复合体的复制缺陷成分SFFV编码一个突变的逆转录病毒包膜蛋白(Gp55)，它具有致病性。对Friend病的易感性受到许多宿主因素的影响。其中之一是Friend病毒易感性2基因(FV2)。大多数近交系小鼠对FV2敏感，但C57BL6及相关品系对FV2抗性除外。对FV2的抗药性不能阻止逆转录病毒的感染或复制。相反，FV2似乎决定了受感染的红细胞是否会对gp55做出反应而增殖。有证据表明，FV2、gp55和促红细胞生成素受体(EPOR)是有丝分裂复合体的一部分，该复合体具有结构性活性，并导致红细胞增殖。为了验证这一假设，并确定FV2在红系增殖和转化中的作用，我们进行了FV2的定位克隆。在我们的初步实验中，我们绘制了FV2区间，并克隆了横跨该区间的5个细菌人工染色体(BAC)的重叠群。我们已经将10个基因定位到这一区间，包括干细胞激酶受体(STK)。该受体是散布因子家族的成员，包括禽类致癌基因v-SEA，它能引起鸟类的红细胞增多症。我们发现该受体的截短形式(SF-STK)在FV2耐药小鼠中特异性不表达，并且在T细胞耗竭的情况下，SF-STK的表达使FV2耐药小鼠对Friend病毒敏感。因此，我们提出了以下实验：在具体目标1中，我们建议确定SF-STK是否是FV2。我们将通过在FV2抗性背景下生产SF-STK转基因小鼠，并通过敲除FV2敏感小鼠中SF-STK的表达来做到这一点。在具体目标2中，我们采取了广泛的方法来识别FV2。这包括：在体内与FV2间期的BAC互补；生成FV2间期的转录图谱；从分子上和转基因小鼠中评估候选基因。在具体目标3中，我们建议确定FV2的作用机制，以及FV2在红系增殖和转化中的作用。我们将通过在小鼠中敲除STK(两种形式的转录本)，通过EPOR复合体的生化研究，以及旨在检查FV2和EPOR下游的信号转导通路在Friend病毒诱导的增殖中的作用的遗传学研究来实现这一点。这些研究将提高我们对正常造血和白血病转化的理解。

Mice infected with Friend virus complex develop acute erythroblastosis, which rapidly progresses to erythroleukemia. The replication defective component of Friend virus complex, SFFV, encodes a mutant retroviral envelope protein (gp55), which confers pathogenicity. Susceptibility to Friend disease is influenced by a number of host factors. One of these, is the Friend virus susceptibility-2 gene (Fv2). Most inbred strains of mice are Fv2 sensitive, except for C57BL6 and related strains which are Fv2 resistant. Fv2 resistance does not prevent retroviral infection or replication. Rather, Fv2 appears to determine whether infected erythroblasts will proliferate in response to gp55. There is evidence that Fv2, gp55, and the erythropoietin receptor (EPOR) are part of a mitogenic complex that is constitutively active and leads to erythroblast proliferation. To test this hypothesis, and to determine the role of Fv2 in erythroid proliferation and transformation, we undertook the positional cloning of Fv2. In our preliminary experiments, we have mapped the Fv2 interval and cloned a contig of 5 bacterial artificial chromosomes (BACs) spanning that interval. We have mapped 10 genes to that interval, including the stem cell kinase receptor (STK). This receptor is a member of the scatter factor family and includes the avian oncogene, v-sea, which causes erythroblastosis in birds. We found that a truncated form of that receptor (SF-STK) is specifically not expressed in Fv2 resistant mice and that expression of SF-STK makes Fv2 resistant mice sensitive to Friend virus, in the setting of T-cell depletion. Therefore, the following experiments are proposed: In specific aim 1, we propose to determine if SF-STK is Fv2. We will do this by producing SF-STK transgenic mice on an Fv2 resistant background, and by knocking out SF-STK expression in Fv2 sensitive mice. In specific aim 2, we take a broad-based approach to identify Fv2. This includes; in vivo complementation with BACs in the Fv2 interval; generation of a transcription map of the Fv2 interval; and evaluation candidate genes molecularly, and in transgenic mice. In specific aim 3, we propose to determine the mechanism of action of Fv2, and the role of Fv2 in erythroid proliferation and transformation. We will accomplish this by knocking out STK (both forms of the transcript) in mice; with biochemical studies of the EPOR complex; and with genetic studies designed to examine the role of signal transduction pathways, downstream of Fv2 and the EPOR, in Friend virus induced proliferation. These studies should improve our understanding of normal hematopoiesis and leukemic transformation.