Differential transcription of baculovirus late and very late promoters: fractionation of nuclear extracts by phosphocellulose chromatography

Abstract

An in vitro transcription system based on a cytidine-free cassette was developed for the late 39k gene and the very late polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). Optimization of transcription conditions revealed that a preincubation step was not required for transcription of late and very late promoters, although preincubation was essential for efficient transcription from an early promoter. The 39k and polyhedrin constructs were actively transcribed by nuclear extracts prepared from AcNPV-infected Spodoptera frugiperda cells at 12 or 36 h postinfection but not by nuclear extracts prepared from uninfected or infected cells harvested during the early phase of infection. Transcription from the very late polyhedrin promoter was fivefold higher than that from the 39k late promoter with the nuclear extract prepared at 36 h postinfection. The 36-h extract was fractionated by phosphocellulose chromatography. Transcription activity eluted in two fractions, at 0.3 and 0.5 M KCl. Both the 39k and polyhedrin constructs were transcribed by these fractions; however, the patterns of late and very late transcription were distinctly different. With the 0.3 M fraction, incorporation into the 39k transcript was approximately 10-fold higher than incorporation into the polyhedrin transcript. Alternatively, with the 0.5 M fraction, transcription of the polyhedrin construct was twofold higher than transcription of the 39k construct. These results indicate that this in vitro system will be useful for purification and identification of factors that discriminate between late and very late promoters.

Guanylyltransferase Activity of the LEF-4 Subunit of Baculovirus RNA Polymerase

“…AcNPV RNA polymerase was purified from S. frugiperda cells infected with a recombinant baculovirus that overexpresses all four RNA polymerase subunits as described previ-ously (11). The purified RNA polymerase was filtered through a Superose 6 size exclusion column in 2 M KCl, and individual fractions were tested for RNA polymerase activity in our standard in vitro transcription assay (31). This assay uses two nucleoside-free templates that are separately linked to the late 39k gene and the very late polyhedrin gene.…”

“…Much of our understanding of the mechanisms that control the temporal expression of baculovirus genes is derived from in vitro transcription systems. We and others have shown that in vitro extracts faithfully reproduce the temporal progression of viral gene expression (12,17,31). Recently, we used an in vitro transcription assay to purify the RNA polymerase that transcribed late and very late baculovirus genes.…”

The LEF-4 Subunit of Baculovirus RNA Polymerase Has RNA 5′-Triphosphatase and ATPase Activities

“…RNA polymerase was purified from Spodoptera frugiperda cells infected with vBAC-RNA pol as previously described (13). In vitro transcription assays were performed as described by Xu et al (41).…”

The Host Factor Polyhedrin Promoter Binding Protein (PPBP) Is Involved in Transcription from the Baculovirus Polyhedrin Gene Promoter

“…The cells were lysed in 5 ml of lysis buffer, and the nuclei were pelleted as described previously (2). Further treatment of the nuclei was carried out as described by Xu et al (58). The pelleted nuclei were resuspended and lysed, and globular nuclear proteins were precipitated by the dropwise addition of ammonium sulfate (0.33 g per ml of supernatant).…”

“…In vitro transcription assay. In vitro transcription was carried out essentially as described earlier (58), with minor modifications. The transcription reaction was carried out in a 50-l reaction volume with 1.5 g of the plasmid construct pPolh/CFS containing a C-free cassette (58) as the template and 50 g of protein prepared as described by Xu et al from AcNPV-infected Sf21 cells 36 h p.i.…”