カイコ核多角体病ウイルス遺伝子の構造と機能の解析

克隆了家蚕核型多角体病毒（BmSNPV）DNA聚合酶（pol）基因，并测定了其序列。该基因包含一个开放阅读框（ORF），编码988个氨基酸，预测分子量为114.65 kDa。推导的氨基酸序列的BmSNPV波尔ORF显示出96和45%的苜蓿银纹夜蛾核型多角体病毒（AcMNPV）波尔ORF和舞毒蛾核型多角体病毒ORF，分别的整体同一性，并包含在各种真核生物和病毒复制型DNA聚合酶的保守序列。BmSNPV pol缺乏典型的TATAA元件，但在转录起始区含有富含G+ C的序列。通过北方印迹杂交、RNA酶保护试验、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种约3.1kb的不同转录物，它们共享一个共同的3'末端。 ...更多信息 BmSNPV pol在病毒感染过程中受到不同程度的调控。7个物种中有5个的转录起始于基序5 '-GCGTGCT-3'附近和基序内。一个转录物将其起始位点置于基序5 '-AGAGCGT-3'内，而另一个转录物将其起始位点置于基序5 '-GGCGGTGG-3'内。基序5 '-GCGTGCT-3'和5 '-AGAGCGT-3'在AcMNPV的pol和其它基因中被鉴定为含有转录起始位点的保守序列，而基序5 '-GGCGGTGG-3'在BmSNPV pol中排列为直接重复序列，但在AcMNPV pol中没有，尚未被定义为负责转录起始位点的序列。在感染后2小时（p.i.）在注射后10小时达到峰值，并且在感染后18小时下降到低水平。蛋白质合成抑制剂放线菌酮处理感染的细胞后，BmSNPV pol的表达不受抑制，但显着延迟，而DNA聚合酶抑制剂aphidicolin则抑制BmSNPV pol的转录。这些结果表明BmSNPV pol表达的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