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.

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Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998) Genes Dev. 12, 538-546; Christen, L. M., et al. (1998) Virology 245, 360-371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure-function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparent Km for ATP is independent of DNA concentration, demonstrating that ssDNA binding alters the kcat for the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, the Kact decreases and the Vmax increases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.

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