LOCATION, SEQUENCE, TRANSCRIPTIONAL MAPPING, AND TEMPORAL EXPRESSION OF THE GP64 ENVELOPE GLYCOPROTEIN GENE OF THE ORGYIA-PSEUDOTSUGATA MULTICAPSID NUCLEAR POLYHEDROSIS-VIRUS
LOCATION, SEQUENCE, TRANSCRIPTIONAL MAPPING, AND TEMPORAL EXPRESSION OF THE GP64 ENVELOPE GLYCOPROTEIN GENE OF THE ORGYIA-PSEUDOTSUGATA MULTICAPSID NUCLEAR POLYHEDROSIS-VIRUS
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DOI：
10.1016/0042-6822(89)90445-5
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发表时间：
1989-06-01
期刊：
影响因子：
3.7
通讯作者：
ROHRMANN, GF
中科院分区：
文献类型：
--
作者：
BLISSARD, GW;ROHRMANN, GF
编码gp 64的基因，包膜糖蛋白的出芽病毒（BV）的Orgyia pseudotsugata多壳核型多角体病毒（OpMNPV），被映射到HindIII-E片段的病毒基因组和gp 64基因的表达进行了检查，在不同的时间感染后。为了定位gp 64基因，使用交叉反应单克隆抗体（AcV 5）（A. W. Hohmann和P. Faulkner，1983，Virology，125，432-444）用于筛选苜蓿银纹夜蛾多衣壳核型多角体病毒（AcMNPV）的gp 64蛋白。利用OpMNPV gt 11表达文库和免疫阳性重组子的插入DNA进行Southern杂交。对gp 64基因进行测序，并通过北方印迹、S1核酸酶和引物延伸分析来检查gp 64基因的转录。在感染过程中检测到两组gp 64转录物：一个早期转录物起始于-43 nt，四个晚期转录物起始于相对于gp 64开放阅读框起始的-152、-167、-174和-175 nt。gp 64早期转录起始位点与其他几个早期杆状病毒基因的比较揭示了一个四核苷酸的共识序列（CAGT），这是保守的IE-1和39 K基因的早期转录起始位点。四个晚期gp 64转录本起始于四个上游ATAAG基序中的两个。所有gp 64 mRNA似乎在3“末端共末端。对gp 64基因核苷酸序列的分析表明，晚期gp 64 mRNA是双顺反子，由位于509个氨基酸gp 64开放阅读框上游70 nt的三个氨基酸的小顺反子组成。早期的抄本不包含小顺反子。gp 64开放阅读框为1527个核苷酸，编码509个氨基酸，分子量为58 kDa。预测的gp 64蛋白含有7个潜在的N-连接的糖基化位点和疏水性的N-和C-末端特征的信号和膜上发现的包膜糖蛋白的锚序列。通过蛋白质印迹分析和间接免疫荧光显微镜，我们发现，gp 64蛋白存在于早期（6小时）感染后，并在感染的细胞中积累，移动到周边在感染后的时间。与p39衣壳蛋白的gp 64蛋白的时间表达的Western印迹比较显示，这两个病毒体结构蛋白基因在其初始表达的时间上显著不同。比较了OpMNPV和AcMNPV gp 64基因的上游调控区、开放阅读框和预测蛋白。在上游调控区，三个序列是高度保守的：（a）上游晚期启动子（ATAAG）基序周围的序列，（B）包括早期启动子的推定TATA盒的序列，和（c）包含早期转录起始位点的序列。两个预测的氨基酸序列显示78%的氨基酸同一性。两个基因之间的保守性的进化意义进行了讨论。
The gene encoding gp64, the envelope glycoprotein of the budded virus (BV) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV), was mapped to the HindIII-E fragment of the viral genome and expression of the gp64 gene was examined at various times postinfection. To locate the gp64 gene, a cross-reacting monoclonal antibody (AcV5) (A.W. Hohmann and P. Faulkner, 1983, Virology, 125, 432-444) directed against the gp64 protein of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to screen a .lambda. gt11 expression library of OpMNPV and insert DNAs from immunopositive recombinants were used for Southern hybridization mapping. The gp64 gene was sequenced and transcription of the gp64 gene was examined by Northern blot, S1 nuclease, and primer extension analysis. Two sets of gp64 transcripts were detected during infection: a single early transcript which initiated at -43 nt and four late transcripts which initiated at -152, -167, -174, and -175 nt relative to the start of the gp64 open reading frame. Comparison of the gp64 early transcription initiation site with several other early baculovirus genes revealed a four-nucleotide consensus sequence (CAGT) which is conserved at the early transcription initiation sites of the IE-1 and 39K genes. The four late gp64 transcripts initiated at two of the four upstream ATAAG motifs. All gp64 mRNAs appear to be coterminal at the 3'' end. Analysis of the nucleotide sequence of the gp64 gene revealed that the late gp64 mRNAs are bicistronic, consisting of a three amino acid minicistron located 70 nt upstream of the 509 amino acid gp64 open reading frame. Early transcripts do not contain the minicistron. The 1527-nt gp64 open reading frame encodes a predicted protein of 509 amino acids with a molecular weight of 58 kDa. The predicted gp64 protein contains seven potential N-linked glycosylation sites and hydrophobic N- and C-termini characteristic of signal and membrane anchor sequences found on envelope glycoproteins. By western blot analyses and indirect immunofluorescence microscopy, we show that the gp64 protein is present at early times (6 hr) postinfection and accumulates in the infected cell, moving to the periphery at later times postinfection. Western blot comparisons of the temporal expression of the gp64 protein with the p39 capsid protein revealed that these two virion structural protein genes differ significantly in the timing of their initial expression. The upstream regulatory regions, open reading frames, and predicted proteins from the OpMNPV and AcMNPV gp64 genes were compared. In the upstream regulatory region, three sequences were highly conserved: (a) sequences surrounding the upstream late promoter (ATAAG) motif, (b) sequences including a putative TATA box of an early promoter, and (c) sequences containing the early transcription initiation site. The two predicted amino acid sequences show 78% amino acid identity. Evolutionary implications of the conservation between the two genes are discussed.