Crawford A M, Miller L K
Department of Genetics, University of Georgia, Athens 30602.
J Virol. 1988 Aug;62(8):2773-81. doi: 10.1128/JVI.62.8.2773-2781.1988.
The region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) encompassing the EcoRI T fragment (29.0 to 30.1 map units) was characterized by DNA sequencing, transcriptional mapping, and site-directed mutagenesis. The largest transcript from this region, an early 1.7-kilobase (kb) poly(A)+ RNA, encompassed three tandem, nonoverlapping open reading frames (ORFs). The largest of these ORFs, ETL, was proximal to the 5' end of the transcript and had the capacity to encode a 28-kilodalton (kDa) polypeptide. A recombinant virus, vETL beta gal, containing the Escherichia coli beta-galactosidase (beta gal) gene fused to the N-terminal two-thirds of the ETL ORF, produced blue plaques in the presence of a chromogenic indicator of beta gal and wild-type levels of polyhedra in cell culture. This recombinant was also infectious in insect larvae by oral administration of occluded virus. Comparison of vETL beta gal and wild-type viral proteins pulse-labeled at various times postinfection (p.i.) revealed (i) absence of a virus-induced 28-kDa polypeptide, (ii) early expression of a large (approximately 130-kDa) polypeptide which may be the ETL-beta gal fusion protein, (iii) a delay in expression of early 35 and 40-kDa polypeptides, and (iv) a 4- to 6-h delay in the expression of late proteins in vETL beta gal-infected cells. Cycloheximide did not inhibit synthesis of the 1.7-kb RNA but did inhibit its shutoff, which occurs at 12 h p.i. in the absence of inhibitors. Thus, the ETL gene product is apparently an early 28-kDa protein which is necessary, directly or indirectly, for timely expression of many other AcMNPV genes. The promoter-leader regions of the 1.7-kDa transcript showed significant sequence similarities to the leader of the AcMNPV IE-1 gene. The middle ORF within the 1.7-kb transcript, ETM, would encode a hydrophobic polypeptide of 113 amino acid residues. ETS, a small ORF within and proximal to the 3' end of the 1.7-kb transcript, was also transcribed as a set of smaller (approximately 0.5-kb) RNAs initiated heterogeneously in the region between ETL and ETS and persisting throughout infection.
对苜蓿银纹夜蛾核型多角体病毒（AcMNPV）包含EcoRI T片段（29.0至30.1个图谱单位）的区域进行了DNA测序、转录图谱分析和定点诱变。该区域最大的转录本是一个早期的1.7千碱基（kb）多聚腺苷酸化（poly(A)+）RNA，包含三个串联、不重叠的开放阅读框（ORF）。其中最大的ORF，即ETL，靠近转录本的5'端，有能力编码一个28千道尔顿（kDa）的多肽。一种重组病毒vETLβgal，含有与ETL ORF的N端三分之二融合的大肠杆菌β-半乳糖苷酶（βgal）基因，在βgal的显色指示剂存在下产生蓝色噬菌斑，并且在细胞培养中产生野生型水平的多角体。这种重组病毒通过口服感染性病毒在昆虫幼虫中也具有感染性。对感染后不同时间（p.i.)进行脉冲标记的vETLβgal和野生型病毒蛋白进行比较，发现（i）没有病毒诱导的28-kDa多肽，（ii）一种大的（约130-kDa）多肽早期表达，可能是ETL-βgal融合蛋白，（iii）早期35-kDa和40-kDa多肽的表达延迟，以及（iv）在vETLβgal感染的细胞中晚期蛋白的表达延迟4至6小时。放线菌酮不抑制1.7-kb RNA的合成，但抑制其在没有抑制剂时在感染后12小时发生的关闭。因此，ETL基因产物显然是一种早期的28-kDa蛋白，它直接或间接地对许多其他AcMNPV基因的及时表达是必需的。1.7-kDa转录本的启动子-前导区与AcMNPV IE-1基因的前导区显示出显著的序列相似性。1.7-kb转录本中的中间ORF，即ETM，将编码一个113个氨基酸残基的疏水多肽。ETS是1.7-kb转录本3'端内且靠近3'端的一个小ORF，也被转录为一组较小的（约0.5-kb）RNA，在ETL和ETS之间的区域异源起始，并在整个感染过程中持续存在。
