A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells.
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.
Department of Microbiology and Immunology, University of California, School of Medicine, Los Angeles 90024.
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Clark
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Dasgupta
D000135:Acid Phosphatase / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000502:physiology; D010766:Phosphorylation; D011051:Poliomyelitis / Q000235:genetics*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D004247:DNA; D000135:Acid Phosphatase; D010447:Peptide Hydrolases