Molecular study of mouse viral resistance mechanisms.

我们实验室的长期目标是在分子水平上了解先天性免疫系统如何识别和响应病原体入侵，特别是这种防御可能由基因组元件控制。特别是，由于小鼠巨细胞病毒(MCMV)受NKO连锁的Cmy1基因控制，因此本研究将集中于NK细胞介导的病毒免疫。特别值得注意的是，NK细胞是宿主对包括病毒、细菌和原生动物寄生虫在内的一系列病原体的天然免疫防御的重要贡献者，通过释放细胞因子(如干扰素-γ)和直接的细胞介导的细胞毒机制。此外，在小鼠巨细胞病毒(MCMV)感染期间，NK细胞直接限制病毒在小鼠体内的复制，这种能力直接关系到MCMV攻击时宿主的存活，而NK细胞介导的免疫直接受Cmv1基因的调控。Cmv1最近被定位在NKC的远端，然而Cmv1候选序列尚未报道。在初步数据中，我们报告了一个额外的NKC编码的NK受体(Ly-49H)可能与MCMV抗性有关，但该NKC基因在物理和遗传上与CMv1基因相分离。因此，我们将通过评估新型NKC内重组小鼠和Cmv1缺失/缺失小鼠的MCMV抗性来确定是否有多个NKC基因座需要MCMV抗性(特定目标1)。新的Cmv1编码序列将从C57BL/6小鼠和MCMV敏感品系的小鼠中挑选出来。这些序列将在结构和生化上进行比较，以确定最佳的Cmv1候选序列。Cmv1候选序列的确认将使用转基因方法(特定目标2)。最后，是否应该使用转基因方法获得与MCMV抗性免疫有关的Ly49h的遗传数据。此外，该基因将在C57BL/6和几个对MCMV敏感的小鼠品系中得到彻底的鉴定(特定目标3)。因此，这些研究应该澄清我们对NK细胞识别病毒感染和Cmv1调节的免疫的理解，因为这些目前还不是机械性的理解。

The long-term objective of our laboratory is to understand how the innate immune system recognizes and responds to pathogen invasion at the molecular level, especially since such defenses may be controlled by genomic elements. In particular, this study will focus on NK cell- mediated viral immunity to murine cytomegalovirus (MCMV) as it is controlled by the NKO linked Cmy1 locus. Notably NK cells are important contributors of host innate immune defenses to a range of pathogens including, viruses, bacteria, and protozoan parasites, through cytokines released (e.g. IFN-gamma) and direct cell-mediated cytotoxicity mechanisms. Moreover, NK cells directly limit viral replication in mice during murine cytomegalovirus (MCMV) infection, this capacity directly correlates with host survival during MCMV challenge, and NK cell-mediated immunity is directly regulated by the Cmv1 locus. Cmv1 was recently mapped within the distal NKC, however Cmv1 candidate sequences were not reported. In preliminary data, we report that an additional NKC encoded NK receptor (Ly- 49H) may be involved in MCMV resistance, but this NKC gene is physically and genetically separate from the CMv1 locus. Hence, we will determine whether more than one NKC locus is required MCMV resistance by assessing MCMV resistance in novel intra-NKC recombinant mice and Cmv1 minus/minus mice (Specific Aim 1). Novel Cmv1 coding sequences will be selected form C57BL/6 mice and from MCMV susceptible strains of mice. These sequences will be compared structurally and biochemically to identify a best Cmv1 candidate sequence. Confirmation of Cmv1 candidate sequences will be performed using a transgenic approach (Specific Aim 2). Finally, should genetic data be obtained implicating Ly49h in MCMV resistance immunity using a transgenic approach. Furthermore, this gene will be thoroughly characterized in C57BL/6 and several MCMV susceptible mouse strains (Specific Aim 3). Thus, these studies should clarify our understanding of NK cell recognition of viral infection and Cmv1-regulated immunity, as these are not currently understood mechanistically.