Lin Guangyun, Blissard Gary W
Boyce Thompson Institute at Cornell University, Ithaca, New York 14853-1801, USA.
J Virol. 2002 Jun;76(11):5503-14. doi: 10.1128/jvi.76.11.5503-5514.2002.
The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAc(lef6KO)) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two "repair" AcMNPV BACmids (vAc(lef6KO-REP-P) and vAc(lef6KO-REP-ie1P)) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc(lef6KO) BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAc(lef6KO)) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.
苜蓿银纹夜蛾多核衣壳核型多角体病毒（AcMNPV）的lef - 6基因先前在瞬时晚期表达试验中已表明对于从AcMNPV晚期启动子进行最佳转录是必需的。在本研究中，我们检测了lef - 6在AcMNPV感染周期中的表达及细胞定位，并构建了一个lef - 6缺失病毒以研究lef - 6在感染周期中的作用。在感染后4至48小时检测到lef - 6的转录，并且在受感染细胞核的致密区域鉴定出LEF - 6蛋白，这一发现与其作为晚期转录因子的潜在作用一致。为了在AcMNPV感染周期的背景下研究lef - 6，我们从在大肠杆菌中作为感染性BACmid进行繁殖的AcMNPV基因组中删除了lef - 6基因。出乎意料的是，所得的AcMNPV lef - 6缺失BACmid（vAc(lef6KO)）能够在细胞培养物中繁殖，尽管病毒产量大幅降低。因此，lef - 6基因对于Sf9细胞中的病毒复制不是必需的。通过将lef - 6基因转座到vAc(lef6KO) BACmid的多角体蛋白基因座中，产生了两个“修复”AcMNPV BACmid（vAc(lef6KO - REP - P)和vAc(lef6KO - REP - ie1P)）。这两种修复病毒产生的病毒产量与野生型AcMNPV或对照（BACmid衍生）病毒产生的产量相似。进一步检测lef - 6缺失BACmid（vAc(lef6KO)），以确定lef - 6的缺失是否在感染背景下影响DNA复制或晚期基因转录。lef - 6的缺失似乎不影响病毒DNA复制。使用Northern印迹分析，我们发现尽管早期转录明显未受影响，但在感染lef - 6缺失BACmid的细胞中，晚期和极晚期转录均延迟。在含有重新插入多角体蛋白基因座的lef - 6基因的病毒中，这种表型得到了挽救。因此，lef - 6基因对于病毒DNA复制或晚期基因转录都不是必需的，但lef - 6的缺失导致晚期转录的起始出现实质性延迟。因此，lef - 6似乎加速了AcMNPV的感染周期。
