Autographa californica Multiple Nucleopolyhedrovirus me53 (ac140) Is a Nonessential Gene Required for Efficient Budded-Virus Production
Autographa californica Multiple Nucleopolyhedrovirus me53 (ac140) Is a Nonessential Gene Required for Efficient Budded-Virus Production
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DOI：
10.1128/jvi.02390-08
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发表时间：
2009-08-01
影响因子：
5.4
通讯作者：
Krell, Peter J.
中科院分区：
文献类型：
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作者：
de Jong, Jondavid;Arif, Basil M.;Krell, Peter J.
me 53是在所有鳞翅目杆状病毒中发现的高度保守的杆状病毒基因，迄今为止已经完全测序。推定的ME 53蛋白含有锌指结构域，并且先前已被描述为主要的早期转录物。我们产生了me 53无效杆粒（Ac Delta me 53 GFP），以及修复病毒（AcRepME 53：HA-GFP）携带me 53与C-末端血凝素（HA）标签，在其天然的早期和晚期启动子元件的控制下。与亲本苜蓿银纹夜蛾多核型多角体病毒和修复细菌相比，用Ac Delta me 53 GFP转染的Sf 9和BTI-Tn-5 b1细胞导致出芽病毒（BV）产生的3-log减少，表明尽管me 53对于复制不是必需的，但在其缺失的情况下复制受到损害。我们的数据还表明me 53不影响DNA复制。细胞分级显示，ME 53被发现在细胞核和细胞质中，早在6小时感染后。早期转录起始位点的缺失导致BV产量减少10至360倍;然而，晚期启动子（ATAAG）的缺失导致BV产量减少160至1，000倍，这表明在BV产生的背景下，ME 53在感染周期的早期和晚期都是必需的。对来自修复病毒的纯化病毒体的另外的蛋白质印迹分析揭示，ME 53：HA与BV和闭塞衍生的病毒体两者相关。总之，这些结果表明，me 53，虽然不是病毒复制所必需的，是需要有效的BV生产。
me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (Ac Delta me53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with Ac Delta me53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.