During
infection with
vesicular stomatitis virus (VSV), host-cell
mRNA synthesis is inhibited due to shut off of host-cell transcription. The transcriptional activity of nuclear extracts prepared from VSV-infected cells was compared to the activity of nuclear extracts from uninfected cells. An exogenous
DNA template was used which contained an adenovirus major late promoter (AdMLP) but lacked upstream activating sequences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuclear 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 cells revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the presence of an excess of inhibitory factor. To determine which
transcription factors were lacking from nuclear extracts of infected cells, host transcription
initiation factors isolated from uninfected cells by ion-exchange chromatography were added separately to nuclear extracts inactivated by VSV
infection. A
phosphocellulose column fraction from uninfected cells eluted with 0. 8 M KCl, which contained
transcription factor 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-binding subunit of
TFIID and has been shown previously to substitute for
TFIID in basal transcription. Purified recombinant
TBP also reconstituted the transcription activity of nuclear extracts from infected cells, supporting the idea that
TFIID is the target of virus-induced inhibition. Western blot analysis showed that the level of
TBP in nuclear extracts or in the 0.8 M KCl column fraction was not changed by VSV
infection. These results indicated that VSV
infection leads to an inhibition of host transcription by inactivation of
TFIID rather than reduction in the level of
TFIID.