An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID.

Abstract

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.Infection of mammalian cells with picornaviruses results in the inhibition of host cell RNA synthesis (2,3,17). Infection of HeLa cells with poliovirus, for example, causes a dramatic decrease in total cellular transcription within 3 h of infection (7,16,29). Transcription mediated by each of the three polymerase systems (RNA polymerases [pol] I, II, and III) is affected.Early attempts to elucidate the mechanism of picornavirus-induced inhibition of host cell transcription focused on the polymerases. RNA pol I, II, and III solubilized from infected-cell extracts, however, were shown to be fully active when assayed under conditions requiring only nonspecific initiation (27). Furthermore, no differences were observed in the chromatographic properties of partially purified RNA polymerases prepared from infected and uninfected cells (1, 27). Finally, two-dimensional gel electrophoresis revealed no differences in the subunit structure of RNA pol II isolated from infected and uninfected cells (1). These results suggested that a transcriptional component other than the elongating polymerase was inactivated by picornavirus infection.Crawford et al. first showed that the poliovirus-induced inhibition of transcription observed in vivo could be studied in vitro (7). In contrast to mock-infected cell extracts, poliovirus-infected cell extracts prepared 3 h postinfection were unable to support RNA pol II-mediated transcription from the adenovirus type 2 (Ad2) major late promoter (MLP). Addition of purified RNA pol II to poliovirusinfected cell extracts failed to restore transcription. However, pol II transcription in infected-cell extracts was restored by the addition of an S100 extract containing transcription factors. When the S100 extract was fractionated by chromatography on phosphocellulose, the restoring activity eluted ...

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

“…The decrease in Pol II transcription in poliovirusinfected cells is correlated with a specific decrease in the activity of a partially purified fraction which contains TFIID (18). In addition, TFIID and the activity required to specifically restore Pol II transcription in poliovirus-infected cell extracts have been shown to copurify through three columns and to have the same kinetics of heat inactivation (18). However, the mechanism responsible for decreased TFIID activity in poliovirus-infected cells is not known.…”

“…Binding of TFIID to the TATA box sequence present in most Pol II promoters is thought to be the first step in the assembly of an active Pol II transcription complex (3,33). The decrease in Pol II transcription in poliovirusinfected cells is correlated with a specific decrease in the activity of a partially purified fraction which contains TFIID (18). In addition, TFIID and the activity required to specifically restore Pol II transcription in poliovirus-infected cell extracts have been shown to copurify through three columns and to have the same kinetics of heat inactivation (18).…”

“…Using an in vitro transcription assay, Crawford et al showed that highly purified RNA polymerase II (Pol II) was unable to restore transcription of a Pol II gene in poliovirusinfected extracts, but a chromatographic fraction containing Pol II transcription factors did restore transcription (8). Recent studies from our laboratory have shown that poliovirus-induced inhibition of transcription in all three polymerase systems is correlated with the inactivation of specific transcription factors (12,18,29). While we have proposed mechanisms to explain how poliovirus inactivates host cell RNA Pol III-mediated transcription, mechanisms for the inhibition of Pol I-and Pol II-mediated transcription by poliovirus have not yet been elucidated (4,5).…”

“…This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP) Recent studies from our laboratory have shown that poliovirus-induced inhibition of transcription in all three polymerase systems is correlated with the inactivation of specific transcription factors (12,18,29). While we have proposed mechanisms to explain how poliovirus inactivates host cell RNA Pol III-mediated transcription, mechanisms for the inhibition of Pol I-and Pol II-mediated transcription by poliovirus have not yet been elucidated (4,5).…”

Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex

“…Cells infected with poliovirus display a number of phenotypic alterations collectively called the cytopathic effect. Among these alterations are inhibition of transcription by RNA polymerase II (Clark et al, 1993;Crawford et al, 1981;Kliewer and Dasgupta, 1988), inhibition of translation in host cells (Ehrenfeld, 1982;Helentjaris and Ehrenfeld, 1977;Jen et al, 1978;Jen et al, 1980) and the inhibition of protein secretion (Doedens and Kirkegaard, 1995).…”