Functional analysis of viral immediate early gene promoters

Description

Immediate early viral proteins are the first viral proteins to be expressed, and they rely completely on the host’s transcriptional mechanisms for their expression. To assay viral IE gene promoter activity, we developed two plasmids that used either EGFP or luciferase is a repoter.

Brief protocol

Promoter activity assay (EGFP as a reporter): The plasmid used as a starting point in this assay, pIZΔIE/V5-His, was modified from the plasmid pIZ/V5-His (Invitrogen) by deleting the OpIE2 (OpMNPV ie2) promoter located in front of the MCS (multiple cloning sites). To construct the derived plasmids, the EGFP gene was first inserted into the MCS to produce the plasmid pIZΔIE/V5-EGFP-His. Next, each of the IE gene upstream of the ATG was amplified containing the appropriate restriction endonuclease recognition sites. Then, each of the PCR products digested with restriction endonucleases and inserted into the pIZΔIE/V5-EGFP-His MCS in front of the EGFP gene. For DNA transfection, Sf-9 insect cells were seeded into a 24-well plate (1.2´10⁵ cells/well) and grown in Sf-900 II SFM serum-free medium (Invitrogen) overnight at 27 °C. Plasmid DNA was diluted to 1 μg/μl in TE buffer (pH 8.0), and liposome-mediated transfection of the Sf-9 cells (1μg of plasmid DNA per well) was carried out by using the Cellfectin Reagent (Invitrogen) according to the manufacturer’s instructions. At 72 h after transfection, EGFP fluorescence signals were observed under an Olympus IX71 inverted fluorescence microscope and photographically recorded using an Olympus DP50 digital microscope camera.

Promoter activity assay (Luciferase as a reporter): To analyze the immediate early gene promoter activity, the DNA fragments, which contained promoter region and had appropreciate restriction enzyme cutting sites, were then cloned into the pGL3-Basic firefly luciferase reporter vector (Promega). The internal control plasmid, phRL/AcMNPVie1, which contained the Renilla luciferase reporter gene, was constructed by cloning the AcMNPV ie1 promoter into the phRL-null vector (Promega), and it was used to monitor and normalize transfection efficiency. Transient transfections and dual luciferase reporter assays were then conducted. For DNA transfection, the Sf-9 insect cells were seeded onto a 24-well plate and grown in Sf-900 II SFM serum-free medium overnight at 27°C. Plasmids containing the firefly luciferase gene (including the pGL3-Basic firefly luciferase reporter vector, which was used as a negative control) were transfected into the Sf-9 cells by using the Cellfectin reagent (1 μg of plasmid DNA per well), and this was followed by cotransfection with 100 ng of the Renilla luciferase plasmid phRL/AcMNPVie1. Cells were collected at 48 h after transfection, and cell lysates were prepared according to the Promega instruction manual for the dual luciferase assay system. Luciferase activity was measured with a luminometer (Labsystems). Firefly luciferase activity values were then normalized against the activities of the Renilla luciferase to correct for transfection efficiency, and data were expressed as relative luciferase activity. Independent triplicate experiments were performed for each plasmid,and the mean and standard deviation (SD) were calculated.

References

1. Liqun Lu, Hai Wang, Ivanus Manopo, Li Yu and Jimmy Kwang. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene. Virology Journal. 23 August 2005.

Some of our publications that have used this platform

1. Liu, W.J., Chang, Y.S., Wang, C.H., Kou, G.H., and Lo, C.F. (2005). Microarray and RT-PCR screening for white spot syndrome virus immediate early genes in cycloheximide-treated shrimp. Virology 334, 327-41.
2. Liu, W.J., Chang, Y.S., Wang, Andrew.H.J., Kou, G.H., and Lo, C.F. (2007). White spot syndrome virus annexes a shrimp STAT to enhance the expression of the immediate-early gene ie1. J. Virol. 81, 1461-1471.