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.

The vaccinia virus core contains a 195 kb double stranded DNA genome, a multi-subunit RNA polymerase, transcription initiation and termination factors and mRNA processing enzymes. Upon infection, vaccinia virus early gene transcription takes place in the virus core. Transcription initiates at early promoters and terminates in response to a termination motif, UUUUUNU, in the nascent mRNA. Early gene transcription termination requires the vaccinia virus termination factor, VTF, a single stranded DNA-dependent ATPase, and NPH I, the Rap94 subunit of the virion RNA polymerase, as well as the presence of the UUUUUNU motif in the nascent RNA. The position of UUUUUNU in the ternary complex suggests that it serves as a site of interaction with one or more components of the transcription termination complex. In order to identify the factor(s) that interact with UUUUUNU a series of direct UV photo crosslinking and ribonuclease A protection studies were undertaken. Through these analyses both VTF and Rap94 were shown to interact with UUUUUNU in the isolated ternary complex. Evidence indicates that the interaction is not mutually exclusive. VTF was shown to bind to UUUUUNU through the N-terminal domain of the large D1 subunit. Furthermore, VTF protects from RNAse A digestion both the 5' region of the nascent transcript as well as a large central component containing UUUUUNU. The addition of an oligonucleotide containing the (5Br)U9 sequence both directly inhibits transcription termination, in vitro and inhibits UV photo crosslinking of VTF to the nascent RNA in the ternary complex. These results support a model in which the availability of the UUUUUNU motif outside of the transcribing RNA polymerase permits binding of both transcription termination factors, VTF and Rap94, to UUUUUNU. The assembly of this termination complex initiates the transcription termination sequence.

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.

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.

The vaccinia virus virion RNA polymerase that is active in early gene transcription contains a unique subunit encoded by the H4L gene. Prior studies demonstrated that this protein is required both for early gene transcription initiation and for transcription termination. Polyclonal antibodies raised against H4L amino acids 1 to 256 prevent both initiation and termination of transcription, in vitro. Pretreatment of the anti-H4L antibody with a H4L fragment containing amino acids 1 to 99 prevents antibody inhibition of both steps, mapping the inhibitory antibody-binding site to this region. A combination of immunoprecipitation and competition studies of antibody binding to wild-type and site-specific mutations of H4L(1-195) mapped the strong epitope to a site that includes Y18. H4L fragments containing an Y18A mutation exhibit diminished ability to block antibody inhibition of transcription initiation and termination. Antibodies inhibit preinitiation complex (PIC) formation but not the activity of preformed PICs, indicating that this region of H4L interacts with one or more factors during active PIC formation. Furthermore, isolated H4L(1-195) directly inhibits PIC activity, supporting this model. Anti-H4L antibody inhibition of transcription termination is only observed in the absence of the essential termination cofactor NPH I. In contrast, antibody inhibition of PIC formation is unaffected by NPH I, demonstrating that the inhibitory antibody and NPH I can bind to H4L at the same time.