Knebel-Mörsdorf D, Flipsen J T, Roncarati R, Jahnel F, Kleefsman A W, Vlak J M
Institute of Genetics, University of Cologne, Germany.
J Gen Virol. 1996 May;77 ( Pt 5):815-24. doi: 10.1099/0022-1317-77-5-815.
To follow the progression of infection of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) within tissues of its larval host, we have constructed AcMNPV recombinants carrying lacZ reporter genes under the control of the early virus promoters pe38 and me53, in addition to the authentic genes. The early promoter-lacZ gene cassettes were located upstream of the very late polyhedrin gene. In infected insect cell lines, pe38 transcription is initiated at an early promoter, while me53 transcripts start from both early and late sites. Transcriptional mapping of the duplicated me53 and pe38 promoters driving lacZ expression showed that they initiated at the same start sites as in the authentic genes. Expression of lacZ by these recombinants was compared to a recombinant driving beta-glucuronidase expression from the very late p10 promoter and lacZ expression from the constitutive heat shock protein 70 promoter of Drosophila melanogaster. After infection of Spodoptera exigua larvae with the different recombinants, we followed reporter gene expression and polyhedron formation in different tissues using immunohistochemistry and electron microscopy. LacZ expression, indicative of early viral transcriptional activity, was detected in nearly all larval tissues during the course of infection. In most tissues these early events were followed by pathophysiological changes associated with late and very late gene expression. However, p10 transcription and polyhedron formation were not observed in midgut goblet cells, Malpighian tubules and salivary glands. These results suggest that expression of early virus genes, such as me53 and pe38, is not restricted to larval tissues that are permissive for AcMNPV replication.
为追踪苜蓿银纹夜蛾多粒包埋核型多角体病毒(AcMNPV)在其幼虫宿主组织内的感染进程,我们构建了AcMNPV重组体,除了其自身基因外,还携带在早期病毒启动子pe38和me53控制下的lacZ报告基因。早期启动子-lacZ基因盒位于极晚期多角体蛋白基因的上游。在受感染的昆虫细胞系中,pe38转录在早期启动子处起始,而me53转录本则从早期和晚期位点起始。对驱动lacZ表达的重复me53和pe38启动子进行转录图谱分析表明,它们的起始位点与自身基因相同。将这些重组体的lacZ表达与一个从极晚期p10启动子驱动β-葡萄糖醛酸酶表达以及从黑腹果蝇组成型热休克蛋白70启动子驱动lacZ表达的重组体进行了比较。用不同的重组体感染甜菜夜蛾幼虫后,我们通过免疫组织化学和电子显微镜观察了不同组织中报告基因的表达和多面体的形成。在感染过程中,几乎在所有幼虫组织中都检测到了指示早期病毒转录活性的lacZ表达。在大多数组织中,这些早期事件之后伴随着与晚期和极晚期基因表达相关的病理生理变化。然而,在中肠杯状细胞、马氏管和唾液腺中未观察到p10转录和多面体形成。这些结果表明,早期病毒基因如me53和pe38的表达并不局限于对AcMNPV复制允许的幼虫组织。