Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication
Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication
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DOI：
10.1128/jvi.78.15.8026-8035.2004
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发表时间：
2004-08-01
影响因子：
5.4
通讯作者：
Koszinowski, UH
中科院分区：
文献类型：
--
作者：
Bubeck, A;Wagner, M;Koszinowski, UH
基本病毒蛋白通过与其他病毒和细胞基因产物的复杂相互作用，在形态发生过程中发挥重要功能。在这里，我们提出了一种新的方法，全面诱变必需的巨细胞病毒基因和在230kBP基因组背景下的生物学分析。通过FLP/FLP识别靶点系统，将单基因水平的随机Tn7突变与定点重组相结合，用于病毒基因组重组。我们展示了来自更大的M50/p35文库的100多个突变体的功能，M50/p35是一种参与衣壳从细胞核外出的蛋白质。这种蛋白质将其他病毒蛋白和细胞酶招募到核内膜上。我们的方法使我们能够快速区分编码序列中的必要和非必要区域。根据对筛选结果的预测，我们能够在氨基酸水平上定位病毒蛋白-蛋白相互作用所必需的位置。
Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.