Prins C, Frisque R J
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
J Neurovirol. 2001 Jun;7(3):250-64. doi: 10.1080/13550280152403290.
Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.
JC病毒（JCV）前体早期mRNA的可变剪接产生了五种转录本，这些转录本编码调节这种人类多瘤病毒生命周期的蛋白质。大T蛋白（TAg）介导病毒DNA复制和致癌活性，小t蛋白在某些条件下影响这些功能。最近，发现了三种新的早期蛋白T'(135)、T'(136)和T'(165)，它们包含与氨基末端TAg功能域重叠的序列。对T'蛋白的初步研究表明，它们有助于病毒DNA复制和转化。所有三种T' mRNA使用的一个供体剪接位点发生突变，产生了一个突变体，与野生型JCV相比，其病毒DNA复制减少了10倍。为了评估单个T'蛋白对复制过程的影响，创建了一组T'受体位点突变体，其中每个T' mRNA独特的第二个受体剪接位点被改变，以消除一种、两种或所有三种T' mRNA的产生。检查了这七个突变体中早期mRNA和蛋白质表达的模式，发现T'(135)受体位点的突变导致了隐蔽剪接位点的利用和新T'物种的产生。在测量突变体的DNA复制潜力之前，进行了额外的突变以防止这些异常剪接反应。DpnI分析表明，每个T'蛋白都有助于TAg介导的DNA复制活性。表达两种T'蛋白的三个单突变体和仅产生T'(136)的双突变体，其复制水平与野生型病毒相当，而两个不表达T'(136)的双突变体的复制效率比野生型JCV低约两倍。三受体位点突变体的复制活性，与早期研究中的T'供体位点突变体一样，受到显著损害。
