Effect of UTP sugar and base modifications on vaccinia virus early gene transcription

Abstract

Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus early gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on early gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2' position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus early gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2'-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2'-amino-U, 2'-azido-U and 2'-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2'-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral early gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in early gene transcription elongation in addition to its known function in mRNA cap formation, early gene transcription termination and intermediate gene transcription initiation.

Determinants of vaccinia virus early gene transcription termination

“…Mutations in this sequence reduce or eliminate transcription termination (Yuen and Moss, 1987). Chemical modification of the base, the 2' position of the sugar or the phosphodiester bond impedes termination, as well, demonstrating the remarkable specificity for a U-rich sequence (Shuman and Moss, 1988;1989;Deng and Shuman, 1997a;Mohamed et al, 2006). UV photo crosslinking studies demonstrated that the UUUUUNU motif is capable of interacting with both Rap94 (Christen et al, 2008), an early gene specific RNA polymerase subunit that is required for both early gene transcription initiation (Ahn et al, 1994;Kane and Shuman, 1992;Ahn and Moss, 1992;Deng and Shuman, 1994) and termination Niles, 2000, 2001;Mohamed et al, 2002), and with VTF, the vaccinia virus early gene transcription termination factor (Hagler et al, 1994;Christen et al, 2008).…”