Transcriptomic analysis of Chilo iridescent virus immediate early promoter

Abstract

Chilo iridescent virus (CIV) is an insect virus belonging to the Iridoviridae. the DNA genome (212,482 base pairs) is entirely sequenced, however very little is known about viral gene regulation, expression and function. the structure and transcriptional regulation of the CIV 012L gene is investigated in this study. Infection of Bombyx mori SPC-BM-36 cells in the presence of Ara-C (inhibits DNA replication) or cycloheximide (inhibits protein synthesis), followed by RT-PCR on isolated total RNA, showed that CIV 012L is transcribed as an immediate-early gene. Detecting the RNA transcript of the CIV 012L early in infection confirmed the data about the temporal class of the gene obtained with the inhibitors. Time course transcription of the gene showed that the transcription starts immediately after infection and reach up to maximum level at 4 h p.i. 5' RACE analysis on RNA isolated from CIV-infected BM cells showed that the transcription initiation site is located 30 nucleotides upstream of the translational start codon. To map the limits of the putative promoter of this gene, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. the resulting plasmid constructs were tested in a transfection assay, in which the baculovirus IE-1 promoter fused to Renilla luciferase was used as an internal control for transfection efficiency. A gradual reduction in luciferase expression occurred as the deletions extended from -200 to -10, relative to the transcription start site. It is clearly shown that sequences between -20 and -10 relative to the transcription start site have key promoter activity for CIV 012L gene. However this key sequence could not be found at the upstream region of CIV's other potential immediate early genes. (c) 2012 Elsevier B.V. All rights reserved.