Baculovirus lef-12 is not required for viral replication
Baculovirus lef-12 is not required for viral replication
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DOI：
10.1128/jvi.76.23.12032-12043.2002
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发表时间：
2002-12-01
影响因子：
5.4
通讯作者：
Dong, W
中科院分区：
文献类型：
--
作者：
Guarino, LA;Mistretta, TA;Dong, W
杆状病毒lef-12 （orf41）基因是杆状病毒晚期基因瞬时表达所必需的。为了分析LEF-12在感染细胞中的作用，构建了两个突变病毒。这两个突变体在高5型毛多翅虫和长尾夜蛾Sf9细胞中均能存活。然而，单步生长曲线表明，在没有LEF-12的情况下，病毒产量减少了约五倍。感染细胞的脉冲标记显示，LEF-12突变病毒进入晚期，合成晚期蛋白的水平相当于或仅低于野生型病毒感染细胞的两倍。Western blot分析证实，突变病毒感染的细胞中没有合成LEF-12。在野生型病毒感染的细胞中，直到感染后18小时才检测到LEF-12，并且在感染后24至36小时达到积累高峰。引物延伸图谱显示，left -12 mRNA在感染后12 h合成，在感染后18 ~ 24 h达到峰值。此外，添加aphidicolin抑制了lef-12 mRNA和lef-12蛋白的合成，表明lef-12是在DNA复制后表达的。
The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.