Fig 1.
Schematic diagram of viral constructs with and without SV40 polyA signals for egfp gene expression under the AcMNPV polh promoter control at the polh, egt and gp37 loci of AcMNPV genome to test the functions of SV40 polyA in the baculovirus expression vector system.
The putative polyA signal in Acgp37SV40+ and Acgp37SV40- is a prediction but not verified since no 3’ RACE products were obtained.
Fig 2.
EGFP expression yield comparison of Sf21 cells infected with viral constructs with or without the SV40 polyA signal at the polh, egt and gp37 loci of the AcMNPV genome.
A. Quantitative analysis of EGFP yields to test the effects of SV40 polyA on EGFP expression in the baculovirus expression vector system. Sf21 cells were infected by different viral constructs at an m. o. i. of 10 p.f.u./cell and the cells were harvested at 48 h post infection for fluorescence emission measurement in three independent cell infections. * indicates significant difference at P = 0.05. NS, not significant. Error bars denote SD. B. Validation of the fluorescence measurement to ascertain fluorescence values falling in the linear range. EGFP produced by the Sf21 cells infected by AcegtSV40- were diluted and fluorescence emission values were plotted against the number of cells. C. Western blotting analysis of EGFP yields by viral constructs with or without the SV40 polyA signal. Equal amounts of total proteins from Sf21 cells infected with viruses were used for SDS-PAGE. The separated proteins from the gel were transferred onto a nitrocellulose membrane and probed with an anti-GFP antibody to detect the amounts of EGFP production from different viral infections. A separate membrane identical to that for the EGFP detection was probed with an anti-VP39 antibody. D. Comparison of polyhedra production of Sf21 cells infected with either AcBacPolh-SV40UTR or AcBacPolh-PolhUTR. E. Quantitative comparison of polyhedra production of Sf21 cells infected with either AcBacPolh-SV40UTR or AcBacPolh-PolhUTR. * indicates significant difference at P = 0.05.
Fig 3.
Transcription analysis of egfp from different viral infections.
Real-time quantitative reverse transcription PCR analysis of egfp. Viral constructs with or without the SV40 polyA signal were used to infect Sf21 cells and the infected cells were harvested at 48 h p. i. for total RNA extraction. Equal amounts of RNA from different viral cell infections were used as templates for amplification of a 153 bp amplicon of the egfp gene using SYBR green dye RT-PCR kit from Bio-Rad for real-time quantification of the egfp transcripts. Sf21 cellular 28S rRNA gene was used as a house keeping gene in the real-time qPCR to normalize the reaction. Error bars represent SD from three independent cell infections. B. Dot-blot analysis of egfp transcripts. Total RNA (2 μg) from the Sf21 cells infected with AcMNPV with or without the SV40 polyA signal as well as the wt AcMNPV (negative control) and a plasmid pBlueGFP (positive control) were blotted to a nylon membrane and probed with either a egfp or 28S rDNA fragment labeled with biotin.
Fig 4.
Analysis of untranslated regions of transcripts of viral constructs with or without the SV40 polyA signal in Sf21 cell infection.
A. 3’ RACE analysis of egfp gene transcripts. An oligo (dT) 3’ RACE adapter primer was used to synthesize cDNA from total RNA isolated from the Sf21 cells infected with different viruses at 48 h p. i. A pair of primers (GFP-486F/3’ RACE outer reverse primer) was used to amplify the 3’ ends of the egfp transcripts by PCR. The PCR products were separated by agarose gel electrophoresis. B. RT-PCR confirmation of the presence of polyadenylated egfp mRNA initiated from the polh promoter at the gp37 locus of AcMNPV. NTC, no-template control. M, DNA size marker. C. Schematic of egfp gene 3’ RACE analysis. Dotted lines indicate 3’ RACE products depicted from A.
Fig 5.
Mapping of the 3’ ends of the egfp transcripts from the Sf21 cells infected with different viral constructs with or without the SV40 polyA signal showing the polyA signal (AATAAA) upstream of the 3’ end of transcripts (not drawn to scale).
* indicates alternative 3’ ends. // indicates sequences omitted.
Fig 6.
Polyadenylation signal analysis of the gp37 UTR of AcMNPV.
A. Comparison of the 3’ UTRs of polh, egt, gp37 and SV40. UTR sequences were retrieved from GenBank and searched for the polyadenylation AATAAA and GU-rich motifs. UTR sequences start right after the gene translation stop codon, except SV40 polyA that starts with the 5’ end of the insert. The putative polyadenylation signal AAUAAA or AATAAA is in bold letters. Putative GU-rich motifs are in underlined italic letters. The primary 3’ end nucleotides are in capital letters. Underlines sequences are reverse primers for PCR. B. PCR analysis using GFP486F/gp37 reverse primers (R1, R2, R3 and R4) and plasmid DNA template (pAcgp37SV40-, Fig 1). NTC, no-template control. M, DNA size marker. C. PCR same as B except that cDNA from mRNA isolated from the Sf21 cells infected with Acgp37SV40- (Fig 1). Outer, 3’ RACE outer reverse primer (S1 Table).
Table 1.
Comparison of polyadenylation signals of AcMNPV late gene 3’ UTRs with SV40 polyA.