Inhibition of Host Transcription by Vesicular Stomatitis Virus Involves a Novel Mechanism That Is Independent of Phosphorylation of TATA-Binding Protein (TBP) or Association of TBP with TBP-Associated Factor Subunits

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.

水泡性口炎病毒（vesicular stomatitis virus, VSV）的基质（matrix, M）蛋白是一类可在体内强效抑制宿主三类RNA聚合酶（RNA polymerases, RNAP）转录活性的蛋白。就宿主RNA聚合酶II（RNA polymerase II, RNAPII）而言，其转录受抑制的根源在于TATA盒结合蛋白（TATA-binding protein, TBP）活性缺失——而TBP正是基础转录因子TFIID的核心亚基。尽管M蛋白诱导的体内抑制作用极强，但本文呈现的实验结果显示，M蛋白无法在体外直接灭活TFIID。将M蛋白添加至未感染细胞的细胞核提取物中，并未对转录活性产生抑制，这表明该抑制作用并非直接介导，而是通过宿主因子完成的。目前已知可调控TBP活性的宿主因子包括宿主激酶介导的磷酸化修饰，以及与不同TBP相关因子（TBP-associated factor, TAF）亚基的结合。不过，经离子交换高效液相色谱法与沉降速度分析法检测证实，VSV感染的细胞内TBP仍可与其TAF亚基正常组装。此外，通过pH梯度凝胶电泳也观察到，VSV感染细胞中TBP的磷酸化模式并无异常。综合上述实验数据可知，现有已知的调控TBP活性的机制均无法解释该抑制现象，因此M蛋白是通过一种全新的机制，实现对RNA聚合酶II依赖型转录过程中TBP活性的灭活。