カイコ核多角体病ウイルス遺伝子の構造と機能の解析

对家蚕核型多角体病毒（BmSNPV） DNA聚合酶基因进行了克隆和测序。该基因包含一个开放阅读框（ORF），编码988个氨基酸的多肽，预测分子质量为114.65 kDa。BmSNPV pol ORF的氨基酸序列与加利福尼亚Autographa NPV (AcMNPV) pol ORF和Lymantria dispar NPV pol ORF的氨基酸序列总体一致性分别为96%和45%，并且包含在多种真核生物和病毒复制DNA聚合酶中保守的序列。BmSNPV pol缺乏一个典型的TATAA元件，但在转录起始区含有一个富含G+ c的序列。通过Northern blot杂交、RNase保护实验、引物延伸和3‘和5’ RACE （cDNA末端快速扩增）分析表明，BmSNPV pol至少表达了7个大约3.1 kb的不同转录本，它们共享一个共同的3'端。这些转录本的表达在病毒感染过程中受到不同程度的调控。7个物种中有5个的转录开始于基序5‘-GCGTGCT-3’附近和内部。其中一个转录本的起始位点位于motif 5‘-AGAGCGT-3‘，另一个转录本位于motif 5’-GGCGGTGG-3’。5‘-GCGTGCT-3’和5‘-AGAGCGT-3’基序在pol和AcMNPV的其他基因中被鉴定为含有转录起始位点的保守序列，而在BmSNPV pol中作为直接重复序列排列的5‘-GGCGGTGG-3’基序在AcMNPV pol中没有被定义为负责转录起始位点的序列。BmSNPV pol转录本在感染后2小时检测到（p.i）。感染细胞经蛋白质合成抑制剂环己亚胺处理后，BmSNPV pol的表达不受抑制，但明显延迟表达，而DNA聚合酶抑制剂aphidicolin则抑制BmSNPV pol的转录。这些结果提示BmSNPV pol表达调控机制复杂而独特。少

A gene encoding a putative DNA polymerase (pol) of Bombyx mori nuclear polyhedrosis virus (BmSNPV) was cloned and sequenced. The gene included an open reading frame (ORF) encoding a polypeptide of 988 amino acids with a predicted molecular mass of 114.65 kDa. The deduced amino acid sequence of the BmSNPV pol ORF showed an overall identity of 96 and 45% to those of the Autographa californica NPV (AcMNPV) pol ORF and the Lymantria dispar NPV pol ORF,respectively, and contained sequences conserved in a variety of eukaryotic and viral replicative DNA polymerases. The BmSNPV pol lacked a canonical TATAA element but contained a G+C-rich sequence in the transcriptional initiation region. Analyzes by Northern blot hybridization, RNase protection assay, primer extension, and 3' and 5' RACE (rapid amplification of cDNA ends) showed that at least seven different transcripts of approximately 3.1 kb that shared a common 3' end were expressed from the BmSNPV pol. The expression of these transcripts … More from BmSNPV pol was regulated differentially during virus infection. Transcription of five of the seven species initiated in the close vicinity of and within the motif 5'-GCGTGCT-3'. One transcript placed its initiation site within the motif 5'-AGAGCGT-3' and the remaining one within the motif 5'-GGCGGTGG-3'. The motifs 5'-GCGTGCT-3' and 5'-AGAGCGT-3' have been identified in pol and other genes of AcMNPV as conserved sequences containing transcriptional initiation sites, whereas the motif 5'-GGCGGTGG-3', which is arranged as a direct repeat in BmSNPV pol but not in AcMNPV pol, has not been defined as the sequence responsible for transcriptional initiation sites. The BmSNPV pol transcripts were detectable at 2 hr postinfection (p.i.) , peaked at 10 hr p.i., and declined to a low level by 18 hr p.i. The expression of BmSNPV pol was not inhibited but delayd dramatically by the protein synthesis inhibitor cycloheximide upon treatment of infected cells, whereas aphidicolin, an inhibitor of DNA polymerase, inhibited BmSNPV pol transcription. These results suggest a complicated and unique mechanism for the regulation of BmSNPV pol expression. Less