Karadeniz Technical University, Faculty of Sciences, Department of Biology, 61080 Trabzon, Turkey.
Virus Res. 2012 Aug;167(2):353-7. doi: 10.1016/j.virusres.2012.05.025. Epub 2012 Jun 12.
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 4h 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-l 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.
虹彩病毒（CIV）是一种昆虫病毒，属于虹彩病毒科。其 DNA 基因组（212482 个碱基对）已完全测序，但关于病毒基因调控、表达和功能的了解甚少。本研究调查了 CIV 012L 基因的结构和转录调控。在存在 Ara-C（抑制 DNA 复制）或环己酰亚胺（抑制蛋白质合成）的情况下感染家蚕 SPC-BM-36 细胞，然后对分离的总 RNA 进行 RT-PCR，结果表明 CIV 012L 作为早期基因转录。在感染早期检测到 CIV 012L 的 RNA 转录物，证实了用抑制剂获得的关于该基因时间类别的数据。该基因的时间过程转录表明，转录在感染后立即开始，并在 4 小时达到最高水平。从感染 CIV 的 BM 细胞中分离的 RNA 进行 5'RACE 分析表明，转录起始位点位于翻译起始密码子上游 30 个核苷酸处。为了绘制该基因假定启动子的界限，将不同长度的上游序列克隆在萤火虫荧光素酶报告基因的前面。将所得质粒构建体在转染测定中进行测试，其中杆状病毒 IE-1 启动子与 Renilla 荧光素酶融合作为转染效率的内部对照。相对于转录起始位点，随着缺失从-200 延伸至-10，荧光素酶表达逐渐减少。显然，相对于转录起始位点在-20 到-10 之间的序列对于 CIV 012L 基因具有关键启动子活性。然而，在家蚕其他潜在的早期基因的上游区域未发现该关键序列。
