Abstract
The mRNAs encoding the vaccinia virus F17 protein and the cowpox A-type inclusion protein are known to possess sequence-homogeneous 3' ends, generated by a post-transcriptional cleavage event. By using partially purified extracts, we have previously shown that the same factor probably cleaves both the F17 and A-type inclusion protein transcripts and that the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. In this study, we have purified the cleavage factor from vaccinia-infected HeLa cells using column chromatography and gel filtration. The factor eluted from the gel filtration column with an apparent molecular mass of approximately 440 kDa. Mass spectrometric analyses of the proteins present in the peak active fractions revealed the presence of at least one vaccinia protein with a high degree of certainty, the H5R gene product. To extend this finding, extracts were prepared from HeLa cells infected with vaccinia virus overexpressing His-tagged H5, chromatographed on a nickel affinity column, and eluted using an imidazole gradient. Cleavage activity eluted with the peak of His-tagged H5. Gel filtration of the affinity-purified material further demonstrated that cleavage activity and His-tagged H5 co-chromatographed with an apparent molecular mass of 463 kDa. We therefore conclude that H5 is specifically associated with post-transcriptional cleavage of F17R transcripts. In addition, we show that dephosphorylation of a cleavage competent extract with a nonspecific phosphatase abolishes cleavage activity implying a role for phosphorylation in cleavage activity.
Highlights
Vaccinia virus employs different mechanisms to mediate 3Ј end formation of early and post-replicative viral mRNAs (2)
Existing evidence suggests that A18 is a transcription termination factor; A18 catalyzes release of nascent RNA from transcription elongation complexes in vitro, and mutants in the A18R gene produce longer than normal post-replicative mRNAs in vivo (12)
Purification of the Cleavage Factor—Our preliminary analyses indicated that the bulk of cleavage activity is present at late times during vaccinia infection and fraction
Summary
Vaccinia virus employs different mechanisms to mediate 3Ј end formation of early and post-replicative viral mRNAs (2). The purification scheme for vaccinia His-tagged proteins (hisH5 and hisA18) was as follows: HeLa NE (200 mg, 12 liters) was applied to a 5-ml DEAE-cellulose column equilibrated with Buffer A containing 400 mM NaCl. The flow-through was collected; the column washed with 10 ml of the starting buffer, and the two fractions were pooled (DEAE pool).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
