INHIBITION OF HOST RNA-POLYMERASE II-DEPENDENT TRANSCRIPTION BY VESICULAR STOMATITIS-VIRUS RESULTS FROM INACTIVATION OF TFIID

During infection with vesicular stomatitis virus (VSV), host-cell mRNA synthesis is inhibited due to shut off of host-cell transcription. Th e transcriptional activity of nuclear extracts prepared from VSV-infec ted cells was compared to the activity of nuclear extracts from uninfe cted cells. An exogenous DNA template was used which contained an aden ovirus major late promoter (AdMLP) but lacked upstream activating sequ ences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuc lear extracts from infected cells as early as 3 h p.i. and by >90% by 6 h p.i. Mixing nuclear extracts from uninfected and VSV-infected cell s revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the pres ence of an excess of inhibitory factor. To determine which transcripti on factors were lacking from nuclear extracts of infected cells, host: transcription initiation factors isolated from uninfected cells by io n-exchange chromatography were added separately to nuclear extracts in activated by VSV infection. A phosphocellulose column fraction from un infected cells eluted with 0.8 M KCl, which contained transcription fa ctor IID (TFIID), overcame the inhibition. The corresponding fraction from infected cells had no detectable activity in a TFIID-dependent in vitro transcription assay. TATA-binding protein (TBP) is the DNA-bind ing subunit of TFIID and has been shown previously to substitute for T FIID in basal transcription. Purified recombinant TBP also reconstitut ed the transcription activity of nuclear extracts from infected cells, supporting the idea that TFIID is the target of virus-induced inhibit ion. Western blot analysis showed that the level of TBP in nuclear ext racts or in the 0.8 M KCl column fraction was not changed by VSV infec tion. These results indicated that VSV infection leads to an inhibitio n of host transcription by inactivation of TFIID rather than reduction in the level of TFIID. (C) 1998 Academic Press.