Ragaa Mohamed

Interaction between nucleoside triphosphate phosphohydrolase I and the H4L subunit of the viral RNA polymerase is required for vaccinia virus early gene transcript release.

Signal-dependent termination is restricted to early poxvirus genes whose transcription is catalyzed by the virion form of RNA polymerase. Two termination factors have been identified. Vaccinia termination factor/capping enzyme is a multifunctional heterodimer that also catalyzes the first three steps of mRNA cap formation and is an essential intermediate gene transcription initiation factor. Nucleoside triphosphate phosphohydrolase I (NPH I) is a single-stranded DNA-dependent ATPase. COOH-terminal deletion mutations of NPH I retain both ATPase and DNA binding activities but are unable either to terminate transcription or to act as dominant negative mutants in vitro. One appealing model posits that the COOH-terminal region of NPH I binds to one or more components in the termination complex. In an attempt to identify NPH I-related protein/protein interactions involved in transcription termination, a series of pull-down experiments were done. Among several vaccinia virus proteins tested, the H4L subunit, unique to the virion form of RNA polymerase, was shown to bind glutathione S-transferase (GST)-NPH I. To further confirm this interaction in virus-infected cells, we constructed recombinant vaccinia virus, vNPHINGST, that expresses GST-tagged NPH I. The H4L subunit of virion RNA polymerase specifically co-purified with GST-NPH I, consistent with a physical interaction. Through the analysis of a series of NH2- and COOH-terminal truncation mutations of H4L, the NPH I interaction site was localized to the NH2-terminal 195 amino acids of the H4L protein. The H4L binding site on NPH I was mapped to the COOH-terminal region between 457 and 631. Furthermore, COOH-terminal deletion mutations of NPH I failed to bind the NH2-terminal region of H4L, explaining their inability to support transcription termination. The COOH-terminal end of NPH I was also shown to be required for transcript release activity and for dominant negative inhibition of release. The requirement for an essential interaction between NPH I and H4L provides an explanation for the observed restriction of transcription termination to early viral genes.

The Viral RNA Polymerase H4L Subunit Is Required for Vaccinia Virus Early Gene Transcription Termination

Vaccinia virus early gene transcription is catalyzed by a multisubunit virion form of RNA polymerase that possesses a unique subunit, H4L. Prior studies from this laboratory showed that the NH2-terminal domain of H4L, containing amino acids 1–195, interacts with the COOH-terminal end of nucleoside triphosphate phosphohydrolase I (NPH I), an ATPase that is employed in early gene transcription termination. Carboxyl-terminal deletion mutations of NPH I lose both the ability to mediate transcription termination and binding to H4L, providing evidence that the interaction between NPH I and H4L is required for termination. In order to test this model further, antibodies raised against segments of H4L were tested for their ability to inhibit transcription terminationin vitro. A bead-bound template was employed in these studies, which permitted us to separate transcription initiation from elongation and termination. Antibodies raised against H4L amino acids 1–256 inhibited termination in an in vitro assay using virus-infected cell extracts lacking NPH I, but antibodies raised against H4L amino acids 568–795 did not. Preincubation of anti-H4L1–256 antibodies with H4L fragments 1–256 or 1–195 prevented antibody inhibition of termination, demonstrating that inhibition was mediated by antibody binding to one or more epitopes in the NH2-terminal end of H4L. Antibody inhibition of termination is reduced in wild type virus-infected cell extracts containing NPH I. Furthermore, preincubation of a NPH I minus cell extract with NPH I prior to antibody addition, or readdition of NPH I to isolated ternary complexes prepared in the absence of NPH I, prevented antibody inhibition of transcription termination. These data show that NPH I and the inhibitory antibodies compete for a binding site(s) on H4L, providing further evidence that the H4L subunit of the vaccinia virus RNA polymerase plays a direct role in transcription termination.

Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro.

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) is an essential early gene transcription termination factor. The C-terminal end of NPH I binds to the N-terminal end of the H4L subunit (RAP94) of the virion RNA polymerase. This interaction is required for transcription termination and transcript release. To refine our understanding of the specific amino acids in the C-terminal end of NPH I involved in binding to H4L, and to develop a collection of mutations exhibiting various degrees of activity to be employed in in vivo studies, we prepared a set of short deletions, and clustered substitutions of charged amino acids to alanine, or bulky hydrophobic amino acids to alanine mutations. These NPH I mutant proteins were expressed, purified, and tested for ATPase activity, binding to H4L, and transcription termination activity. Most mutations in amino acids 609 to 631 exhibited reduced activity. Deletion of the terminal five amino acids (627-631), or substitution of Y(629) with alanine or glutamic acid, dramatically reduced NPH I mediated transcription termination. Deletion of the terminal F(631), or substitution of F(631) with alanine, reduced binding to H4L and eliminated termination activity. These observations demonstrate that the terminal five amino acids directly participate in binding to RNA polymerase and in early gene transcription termination.

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

Vaccinia virus early gene transcription termination requires the vaccinia termination factor (VTF), NPH I, a single stranded DNA-dependent ATPase, the virion form of RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU, which resides in the nascent mRNA, located 30 to 50 bases upstream from the poly(A) addition site. Evidence indicates that a required termination factor acts through binding to the UUUUUNU signal. To further investigate the function of UUUUUNU, the ability of UUUUUNU containing oligonucleotides to inhibit transcription termination was tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature transcription termination and transcript release, in trans. Activation of premature termination required VTF, NPH I, Rap 94, and ATP, demonstrating that the normal termination machinery was employed. Premature termination was not stimulated by RNA harboring a mutant UUUUUNU, demonstrating specificity. These data are consistent with a model in which a required termination factor is converted from an inactive to an active form by binding to a UUUUUNU containing oligonucleotide. The active termination factor then interacts with the ternary complex stimulating transcription termination through the normal mechanism, independent of the nascent mRNA sequence.

Determinants of vaccinia virus early gene transcription termination

Vaccinia virus early gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia early gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.

UUUUUNU Stimulation of Vaccinia Virus Early Gene Transcription Termination OLIGONUCLEOTIDE SEQUENCE AND STRUCTURAL REQUIREMENTS FOR STIMULATION OF PREMATURE TERMINATION IN VITRO

Vaccinia virus early genes are unique in that transcription terminates in a signal- and factor-dependent manner. Recent results from this laboratory demonstrated that a 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence- and concentration-dependent stimulation of premature transcription termination and transcript release in trans. In an effort to better understand the different aspects of the U5NU stimulation of premature termination, we evaluated the activity of various oligonucleotides in vitro. Neither RNA containing a mutant U5NU signal nor single-stranded DNA containing T5NT was able to stimulate premature termination, demonstrating both sequence specificity and a requirement for ribose. Furthermore, neither oligonucleotide was able to compete with U5NU, demonstrating that each failed to bind to the U5NU recognition factor. Substitution of the U9 signal with either BrU9 or BrdU9 inhibited normal termination but did not stimulate premature termination. The addition of BrdU5NdU inhibited U5NU stimulation of premature termination, demonstrating that both oligonucleotides bind to the same site on the U5NU recognition factor. Finally, U5NU containing RNA as short as nine bases served as an effective stimulator of premature termination. These observations impact directly on the development of oligonucleotide based anti-poxvirus therapeutic agents.