Auerbach Marcy R, Shu Chang, Kaplan Artem, Singh Ila R
Department of Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11678-83. doi: 10.1073/pnas.2034020100. Epub 2003 Sep 22.
Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of gag remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the gag region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus gag, some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral gag gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.
逆转录病毒Gag蛋白在病毒组装中发挥重要作用，但也参与病毒生命周期的其他步骤。传统的突变分析已经产生了关于这些功能所必需结构域的大量信息，但gag的许多区域仍未得到表征。我们使用遗传足迹法（一种允许生成和同时分析大量突变的技术）对莫洛尼鼠白血病病毒gag区域的639个核苷酸进行了近饱和诱变和功能分析。我们在此报告由八个足迹定义的功能图谱，这些足迹代表莫洛尼鼠白血病病毒gag中对复制至关重要的区域，其中一些区域以前未被表征。我们发现，与衣壳蛋白的N端一半不同，基质蛋白和p12蛋白的大部分区域对插入具有耐受性。我们通过传统方法分析了文库中的30个突变体，以验证这些足迹。对其中六个突变体进行了详细表征，确定了它们复制受阻的精确阶段。除了提供逆转录病毒gag基因最全面的功能图谱外，我们的研究还证明了通过对病毒序列进行遗传足迹分析可以收集到丰富的信息。
