Clark M E, Dasgupta A
Department of Microbiology and Immunology, University of California, School of Medicine, Los Angeles 90024.
Mol Cell Biol. 1990 Oct;10(10):5106-13. doi: 10.1128/mcb.10.10.5106-5113.1990.
In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This inhibition is due primarily to the reduction in transcriptional activity of the pol III transcription factor TFIIIC in poliovirus-infected cells. However, the specific binding of TFIIIC to the VAI gene B-box sequence, as assayed by DNase I footprinting, is not altered by poliovirus infection. We have used gel retardation analysis to analyze TFIIIC-DNA complexes formed in nuclear extracts prepared from mock- and poliovirus-infected cells. In mock-infected cell extracts, two closely migrating TFIIIC-containing complexes, complexes I and II, were detected in the gel retardation assay. The slower migrating complex, complex I, was absent in poliovirus-infected cell extracts, and an increase occurred in the intensity of the faster-migrating complex (complex II). Also, in poliovirus-infected cell extracts, a new, rapidly migrating complex, complex III, was formed. Complex III may have been the result of limited proteolysis of complex I or II. These changes in TFIIIC-containing complexes in poliovirus-infected cell extracts correlated kinetically with the decrease in TFIIIC transcriptional activity. Complexes I, II, and III were chromatographically separated; only complex I was transcriptionally active and specifically restored pol III transcription when added to poliovirus-infected cell extracts. Acid phosphatase treatment partially converted complex I to complex II but did not affect the binding of complex II or III. Dephosphorylation and limited proteolysis of TFIIIC are discussed as possible mechanisms for the inhibition of pol III-mediated transcription by poliovirus.
在HeLa细胞中，脊髓灰质炎病毒感染会严重抑制RNA聚合酶III（pol III）介导的转录。这种抑制主要是由于脊髓灰质炎病毒感染细胞中pol III转录因子TFIIIC的转录活性降低。然而，通过DNA酶I足迹法检测，TFIIIC与VAI基因B盒序列的特异性结合并未因脊髓灰质炎病毒感染而改变。我们使用凝胶阻滞分析来分析从模拟感染和脊髓灰质炎病毒感染细胞制备的核提取物中形成的TFIIIC-DNA复合物。在模拟感染细胞提取物的凝胶阻滞试验中，检测到两种迁移速度相近的含TFIIIC的复合物，即复合物I和复合物II。迁移速度较慢的复合物I在脊髓灰质炎病毒感染的细胞提取物中不存在，而迁移速度较快的复合物（复合物II）的强度增加。此外，在脊髓灰质炎病毒感染的细胞提取物中，形成了一种新的快速迁移复合物，即复合物III。复合物III可能是复合物I或II有限蛋白水解的结果。脊髓灰质炎病毒感染细胞提取物中含TFIIIC复合物的这些变化在动力学上与TFIIIC转录活性的降低相关。复合物I、II和III通过色谱法分离；只有复合物I具有转录活性，当添加到脊髓灰质炎病毒感染的细胞提取物中时能特异性恢复pol III转录。酸性磷酸酶处理部分将复合物I转化为复合物II，但不影响复合物II或III的结合。去磷酸化和TFIIIC的有限蛋白水解被讨论为脊髓灰质炎病毒抑制pol III介导转录的可能机制。
