Analysis of the function of paramyxovaus V protein

1.我鉴定了一种宿主蛋白AIPI/阿利克斯，其作为hPIV 2 NP和V蛋白的相互作用伴侣参与几种包膜病毒的细胞凋亡和有效出芽。为了研究AIPI在hPIV 2病毒生长中的可能参与，我建立了细胞系，其中通过使用添加Dox诱导的短发夹RNA耗尽细胞内AIP 1。wt hPIV 2在建立的细胞中的生长被抑制10至100倍，但具有不能与AIP I结合的突变V蛋白的rPIV 2的生长速率相似。这些结果表明AIPI/阿利克斯和V蛋白之间的关联对于病毒的生长是重要的.我研究了V蛋白在hPIV 2复制中的作用，使用无牛痘病毒的微型基因组系统。我发现V蛋白的C-末端对于抑制和与L蛋白的相互作用是必需的，但对于与NP蛋白的相互作用不是必需的。这些数据表明，hPIV 2 V蛋白的抑制作用是L蛋白结合的结果，而不是NP的结果。我发现一组均一的PIV 2或PIV 5的NP、P和L蛋白能够驱动hPIV 2微型基因组系统的报告基因表达，但所有异质组合都是无活性的.利用酵母双杂交技术，以hPIV 2 V为诱饵，以目的cDNA文库为模板，筛选出了一些阳性克隆。我想研究这些克隆体和V之间的相互作用。

1. I identified a host protein, AIPI/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of hPIV2 NP and V proteins. To investigate possible involvement of AIPI in hPIV2 virus growth, I established cell lines in which were depleted the intracellular AIP 1 by using short hairpin RNA induced by adding Dox. The wt hPIV2 growth in the established cells was inhibited by 10- to 100-fold, but the growth rates of rPIV2s that have mutant V proteins of inability to bind with AIP I were similar. These results have shown that the association between AIPI/Alix and V proteins is important for virus growth.2. I investigated the role of the V protein in hPIV2 replication, using a mini-genome system free of vaccinia virus. I found that the C-terminal of the V protein was essential for the inhibition and for interaction with the L protein, but not for interaction with the NP protein. These data suggest that the inhibitory effect of the hPIV2 V protein is the result of L protein binding, and not that of NP.3. I found that a homogeneous set of NP, P and L proteins of PIV2 or PIV5 was able to drive reporter gene expression from the hPIV2 mini-genome system, but all heterogeneous combinations were inactive.4. By using a yeast two-hybrid-based screening assay with hPIV2 V as bait and a target cDNA library, I got some positive clones. I would like to investigate the interaction between these clones and V.