Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I Is an Essential Viral Early Gene Transcription Termination Factor

Abstract

Deng and Shuman (J. Biol Chem. 271, 29386 (1996)) reported that an ATPase different from the known viral termination factor, VTF, is required for vaccinia virus early gene transcription termination. Properties of this ATPase were similar to those of a known vaccinia virus enzyme, nucleoside triphosphate phosphohydrolase I (NPH I) the product of gene D11L. Transcription-competent cell-free extracts were prepared from A549 cells infected with wild-type or mutant vaccinia virus harboring ts mutations in gene D11L. These extracts were employed to investigate the role of NPH I in early gene transcription termination. Extracts prepared under nonpermissive conditions from both wild-type virus and ts mutant virus-infected cells exhibited high levels of early and intermediate gene transcription activity but were incapable of supporting late gene transcription. ts mutant extract lacked signal-dependent early gene transcription termination activity, which was restored by the addition of either free NPH I or a GST-NPH I fusion protein. A comparison of the NPH I amino acid sequence to the protein databases revealed the presence of a set of sequences characteristic of nucleic acid helicase superfamily II members. A series of site-specific mutations in the helicase motifs and N-terminal and C-terminal deletion mutations were expressed as GST fusion proteins and their activities assessed. Of the mutations in helicase motifs I to VI, alteration of all but motif III reduced the ATPase activity. Removal of as few as 24 amino acids from the N-terminal end eliminated ATPase activity, while deletion of 68 C-terminal amino acids exhibited only a modest decrease in ATP hydrolysis. Larger C-terminal deletions eliminated ATPase activity. Each deletion mutation, and site-specific mutations other than the motif III mutation, failed to support transcription termination in vitro. Mutations in motifs I, II, V, and VI inhibit wild-type NPH I transcription termination activity. However, deletion of up to 68 amino acids from the C-terminal end eliminates this inhibitory property. This observation is particularly interesting since these C-terminal deletions retain both ATPase activity and single-stranded DNA binding activity. Their failure to inhibit transcription termination suggests that these C-terminal deletion mutations eliminate a site required for a function other than from DNA binding or ATP hydrolysis.

UUUUUNU Oligonucleotide Stimulation of Vaccinia Virus Early Gene Transcription Termination, in Trans

“…Transcription Extracts-Transcription extracts were prepared from virus-infected cells by lysolecithin treatment, as described (23,28). A549 cells were infected with either WT virus or tsC50 mutant virus at a multiplicity of infection of 15, at 37 or 31°C, respectively.…”

“…Both extracts are naturally deficient in VTF (23,33) so maximum termination requires the addition of exogenous VTF, and in the case of C50 extract, both VTF and NPH I must be added. Transcription initiation and elongation to G21 permits the preparation of an isolated ternary complex and effectively separates transcription initiation from termination.…”

“…VTF is also the virion mRNA capping enzyme employed in catalyzing the first three steps in cap formation (6,21). In addition, ATPase activity catalyzed by nucleoside-triphosphate phosphohydrolase I (NPH I), the product of gene D11L, is essential for transcription termination and transcript release (22,23). An interaction between the C-terminal end of NPH I and the N-terminal end of Rap 94 is required for termination (15)(16)(17)24).…”

Vaccinia Virus Gene A18R DNA Helicase Is a Transcript Release Factor

“…NPH-I is postulated to bind to the non-template DNA strand within the transcription bubble. Recognition of the early termination signal by vaccinia termination factor would activate the ATPase activity of NPH-I resulting in the release of the nascent RNA (9,10).…”

“…The model for early termination proposes that vaccinia termination factor (identical to the vaccinia virus capping enzyme) is poised to scan the RNA for the termination signal. Recognition of the signal by vaccinia termination factor activates the ATPase activity of NPH-I, a virus-coded DNA-dependent ATPase, resulting in release of the nascent transcript from the elongation complex (9,10). Termination occurs 20 -50 nt downstream of the termination signal (11,12).…”