Transcriptional activity of baculovirus very late factor 1

Abstract

The product of the vlf-1 (very late factor 1) gene is required for expression of very late genes during the final phase of infection. To determine whether VLF-1 functions as a transcriptional activator, VLF-1 was overexpressed and purified by affinity and cation exchange chromatography. The addition of purified protein to transcription assays containing baculovirus RNA polymerase stimulated transcription of the very late polyhedrin promoter but not the late 39k promoter. Furthermore, construction and analysis of chimeric templates identified sequences within the polyhedrin promoter that were necessary for enhancement.

FIG. 1.

Purification of VLF-1. Baculovirus expression. Nuclear extracts (NE) were prepared at 60 h postinfection from S. frugiperda cells infected with wild-type (WT) AcNPV (lane 2) or the recombinant Ac-VLF1 (lane 3). Nuclear extracts were purified on a heparin (Hep) column, and peak fractions (lane 4) were further purified on a Mono S column (S) (lane 5). Each lane contains 2 μg of protein. Lane 1, crude extract; lane 2, protein eluted from Talon matrix with 100 mM imidazole; lane 3, peak fraction from a Mono S column. The position of VLF-1 is indicated by an arrow on the right, and the positions of relevant molecular markers are indicated on the left.

FIG. 2.

VLF-1 enhances transcription from the polyhedrin promoter. (A) Schematic diagram of the transcriptional templates pPolh/CFS (polh/CFS) and 39kL/CFS. The open box represents the C-free cassette. The darkly shaded box represents the polyhedrin (polh) promoter, the lightly shaded box represents the 39k late promoter, and the black box represents the burst sequence. (B) Transcription assay. Lane 1, φX174/HinfI marker; lanes 2 to 6, transcripts produced from reaction mixtures containing 0.04 μM purified RNA polymerase; 0.4 pmol of each template in the absence of VLF-1 (lane 2) or increasing amounts of purified VLF-1 (0.16 to 1.28 μM in twofold increments [lanes 3 to 6]) were incubated under standard transcription reaction conditions at 30°C for 12 min. The RNA transcripts were resolved on 6% polyacrylamide-8 M urea gel and exposed on PhosphorImager plates. The positions of the transcripts are indicated on the right. The positions of relevant molecular size markers are indicated in nucleotides on the left. (C) Transcripts were quantitated by using ImageQuant software and plotted as the ratio of the polh transcript to the 39k transcript as a function of input VLF-1 concentration.

FIG. 3.

Transcriptional enhancement by VLF-1 requires a burst sequence. (A) Transcription from the polyhedrin promoter in the presence and absence of a burst sequence. Transcripts were produced from in vitro transcription reaction mixtures containing pPolh/CFS and pPolhΔburst/CFS. Transcripts were quantified by using ImageQuant software and calculated as the ratio of pPolh/CFS to pPolhΔburst/CFS transcript. (B) Transcription from the chimeric p39kLburst/CFS template is stimulated by VLF-1. Reactions contained equimolar amounts of p39kL/CFS and pp39kLburst/CFS. Transcripts were quantified by using ImageQuant software.