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. 2013 Dec;447(1-2):213-20.
doi: 10.1016/j.virol.2013.09.007. Epub 2013 Oct 3.

Cascade Regulation of Vaccinia Virus Gene Expression Is Modulated by Multistage Promoters

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Free PMC article

Cascade Regulation of Vaccinia Virus Gene Expression Is Modulated by Multistage Promoters

Zhilong Yang et al. Virology. .
Free PMC article

Abstract

Vaccinia virus contains ~200 genes classified temporally as early, intermediate or late. We analyzed 53 intermediate promoters to determine whether any have dual late promoter activity. Our strategy involved (i) construction of a cell line that stably expressed the three late transcription factors, (ii) infection with a vaccinia virus mutant that expresses RNA polymerase but neither intermediate nor late transcription factors, and (iii) transfection with plasmids containing a luciferase reporter regulated by an intermediate promoter. After confirming the specificity of the system for late promoters, we found that many intermediate promoters had late promoter activity, the strength of which correlated with a TAAAT at the initiator site and T-content from positions -12 to -8 of the coding strand. In contrast, intermediate promoter activity correlated with the A-content from positions -22 to -14. The sequence correlations were confirmed by altering the specificities of strict intermediate and late promoters.

Keywords: Consensus promoter sequence; Poxvirus transcription; Regulation of gene expression; mRNA Synthesis.

Figures

Fig. 1
Fig. 1
Construction and characterization of the RK-G8-A1-A2Flag cell line. (A) Diagram depicting the assay to determine dual late promoter activity of intermediate promoters. A cell that expresses the three late transcription factors G8, A1 and A2, is infected with a VACV mutant with a deletion of the A23 intermediate transcription factor and is transfected with a plasmid containing LUC regulated by a strict intermediate (I) promoter or by a dual intermediate/late (I/L) promoter. Black circle indicates no LUC expression; yellow circle indicates LUC expression. (B) Diagram of a tricistronic cassette regulated by a CMV promoter. The picornavirus 2A-like sequences F2A and T2A separate the G8R and A1L ORFs and the A1L and A2L ORFs, respectively. The A2L ORF has a C-terminal Flag tag. (C) Western blots showing G8, A1 and A2 expression. A2 was detected with antibody to the Flag epitope; G8 and A1 were detected with antibody to the 2A peptide. The positions of protein markers in kDa are shown on the left and the A2, G8 and A1 bands on the right. (D) Expression of a late and intermediate gene. RK-13, RK-G8-A1-A2Flag, or RK13-A8/A23 cells were infected with vA23Δ and transfected with plasmids encoding the late F17R and intermediate G8R ORFs with 3XFlag tags regulated by their natural promoters. After 18 h, the cells were lysed and analyzed by Western blotting with anti-Flag antibody. The single flag epitope on the A2 protein was not detected in the small amount of extract used for analysis of the triple flag on the F17 and G8 proteins.
Fig. 2
Fig. 2
Intracellular localization of transcription factors in RK-G8-A1-A2Flag cells. Uninfected RK-G8-A1-A2Flag cells (upper panel) and RK-G8-A1-A2 cells infected with VACV for 7 h (lower panels) were fixed and permeabilized. The samples were stained with a rabbit polyclonal primary antibody to the picornavirus 2A peptide, which remains attached to the G8 and A1 proteins, a mouse anti-Flag MAb to detect FLAG attached to the A2 ORF and the mouse MAb to A14, followed by donkey anti rabbit IgG and goat anti-mouse IgG coupled to Alexa Fluor 488 and Alexa Fluor 594, respectively. The nuclei and cytoplasmic viral factories (arrows) were stained with DAPI.
Fig. 3
Fig. 3
Late activities of intermediate promoters. RK-A8-A1-A2Flag cells were infected with vA23Δ nd transfected with plasmids encoding Firefly LUC regulated by an intermediate promoter. After 16 to 18 h, LUC activity was determined and normalized as described in Methods.
Fig. 4
Fig. 4
Mutational analyses of an intermediate and late promoter. (A) Intermediate promoter mutations. The sequences of the WT G8R promoter and mutated G8R promoters attached to GFP are shown. Plasmids containing these constructs were transfected into cells that had been infected with VACV in the absence (−) or presence (+) of AraC. After 16 h, the cells were lysed and analyzed by Western blotting with fluorescent antibody to GFP. Relative band intensities shown below the blot were determined using ImageJ. (B) Late promoter mutations. The WT A14L promoter and mutated A14L promoters were attached to the A14L ORF with a Flag tag. The sequences of the promoters are shown. Infection, transfection and Western blotting were similar to that of panel A except that antibody to the Flag epitope was used.
Fig. 5
Fig. 5
Gene functions of intermediate promoters with low and high late promoter activities. Intermediate promoters were divided into those with low <5% (striped) and high >5% (black) of the activity of the late F17R promoter in the late promoter-specific transfection assay. The numbers of genes in each category are indicated.

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