Early vaccinia virus gene transcription takes place in the virion core and is the only class that demonstrates signal dependent termination. Early gene transcription termination requires the signal UUUUUNU in the nascent RNA, VTF, the vaccinia termination factor that is also the viral mRNA capping enzyme, NPH I a single stranded DNA dependent ATPase to provide energy and the virion form of the multi-subunit RNA polymerase containing the additional H4L subunit. The goal of this research is to define the mechanism of early gene transcription termination. In this submission, we propose three specific aims.
In Aim 1, we will focus on the role of NPH I in termination. We will determine whether NPH I binds to the nontemplate strand of the DNA to activate the ATPase activity. In addition, we will investigate the conformational change in NPH I induced upon ssDNA binding. Mutants in NPH I known to affect termination, in vitro, will be constructed in virus and their phenotypes investigated in vivo. Finally, the role of NPH I in transcription elongation will be evaluated.
In Aim 2, our attention switches to VTF, the vaccinia termination factor. We will determine whether VTF is the factor that senses UUUUUNU in the nascent RNA. In addition, we will identify additional VTF interacting partners. Mutagenesis of VTF will permit identification of the termination specific functions. Finally, our novel observation of UUUUUNU specific activation of premature termination in trans will be fully explored.
In Aim 3, we will address the role of the H4L subunit of the virion RNA polymerase in both early gene transcription initiation and termination. We propose to locate the NPH I binding site on H4L. We will map the region of H4L that is required for pre-initiation complex formation. We will search for H4L interacting proteins to both identify the factors required for PIC formation and additional proteins that may function in termination. Finally, we will construct mutations in virus that exhibit altered NPH I binding and evaluate their phenotypes in vivo. Through these studies we will define aspects of the mechanism of early gene transcription. These results will provide important insights into this essential step in gene expression and define new sites for the development of specific poxvirus antiviral agents.
|Christen, Linda A; Piacente, Sarah; Mohamed, Mohamed R et al. (2008) Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex. Virology 376:225-35|
|Piacente, Sarah; Christen, Linda; Dickerman, Benjamin et al. (2008) Determinants of vaccinia virus early gene transcription termination. Virology 376:211-24|
|Mohamed, Mohamed Ragaa; Piacente, Sarah C; Dickerman, Benjamin et al. (2006) Effect of UTP sugar and base modifications on vaccinia virus early gene transcription. Virology 349:359-70|
|Mohamed, Mohamed Ragaa; Niles, Edward G (2004) UUUUUNU oligonucleotide inhibition of RNA synthesis in vaccinia virus cores. Virology 324:493-500|
|Mohamed, Mohamed Ragaa; Niles, Edward G (2003) UUUUUNU stimulation of vaccinia virus early gene transcription termination. Oligonucleotide sequence and structural requirements for stimulation of premature termination in vitro. J Biol Chem 278:39534-41|
|Mohamed, Mohamed Ragaa; Niles, Edward G (2003) UUUUUNU oligonucleotide stimulation of vaccinia virus early gene transcription termination, in trans. J Biol Chem 278:11794-801|
|Piacente, Sarah C; Christen, Linda A; Mohamed, Mohamed Ragaa et al. (2003) 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. Virology 310:109-17|
|Shalaby, Kamal A; Yin, Lei; Thakur, Arvind et al. (2003) Protection against Schistosoma mansoni utilizing DNA vaccination with genes encoding Cu/Zn cytosolic superoxide dismutase, signal peptide-containing superoxide dismutase and glutathione peroxidase enzymes. Vaccine 22:130-6|
|Mohamed, Mohamed R; Christen, Linda A; Niles, Edward G (2002) Antibodies directed against an epitope in the N-terminal region of the H4L subunit of the vaccinia virus RNA polymerase inhibit both transcription initiation and transcription termination, in vitro. Virology 299:142-53|
|Mohamed, M R; Latner, D R; Condit, R C et al. (2001) Interaction between the J3R subunit of vaccinia virus poly(A) polymerase and the H4L subunit of the viral RNA polymerase. Virology 280:143-52|
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