A Conserved Glycine Residue Is Required for Proper Functioning of a Baculovirus VP39 Protein
A Conserved Glycine Residue Is Required for Proper Functioning of a Baculovirus VP39 Protein
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DOI：
10.1128/jvi.02253-16
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发表时间：
2017-03-01
影响因子：
5.4
通讯作者：
Kokusho, Ryuhei
中科院分区：
文献类型：
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作者：
Katsuma, Susumu;Kokusho, Ryuhei
杆状病毒VP39蛋白是病毒繁殖所必需的主要核衣壳蛋白。然而，VP39蛋白的关键结构域或残基尚未确定。在这里，我们用5-溴-2‘-脱氧尿苷对家蚕核型多角体病毒(BmNPV)进行了诱变实验，并分离到了一个比野生型病毒产生更少包涵体的BmNPV突变体。该突变体在培养细胞和家蚕幼虫中产生的传染性萌芽病毒(BV)也比野生型病毒少。利用基因组文库进行的标记挽救实验确定了vp39基因中的一个单核苷酸突变。该突变导致甘氨酸276(Gly-276)处的氨基酸替换为丝氨酸，这是在突变体中观察到的所有缺陷表型所必需的。序列比较表明，该残基在已测序的甲型杆状病毒、贝塔型杆状病毒和伽马杆状病毒的VP39蛋白中完全保守。虽然早期病毒基因的表达没有受到明显影响，但晚期基因vcath的表达水平降低了。此外，在感染该突变的细胞中，两个非常晚的基因显著下调。Western印迹和定量PCR分析表明，与野生型病毒感染细胞相比，感染该突变体的细胞产生的BV含有较少的VP39蛋白和病毒基因组DNA。结合透射电子显微镜的结果，VP39 Gly-276对正确的核衣壳组装、病毒DNA包装和病毒基因的表达，特别是对非常晚的基因的表达是必不可少的。虽然已经鉴定了几种与VP39蛋白相互作用的病毒和宿主蛋白，但该蛋白的重要功能结构域或残基仍不清楚。本研究揭示了276位甘氨酸残基对于VP39的功能，即核衣壳的结构组装和病毒DNA的包装，是非常重要的，该残基在已测序的甲型杆状病毒、β-杆状病毒和伽马杆状病毒中完全保守。此外，我们的结果为核衣壳的形成和病毒非常晚期基因的转录之间的联系提供了证据。
The baculovirus VP39 protein is a major nucleocapsid protein essential for viral propagation. However, the critical domains or residues of the VP39 protein have not yet been identified. Here, we performed mutagenesis experiments with Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2'-deoxyuridine and isolated a BmNPV mutant that produced fewer occlusion bodies than the wild-type virus. This mutant also produced fewer infectious budded viruses (BVs) than the wildtype virus in both cultured cells and B. mori larvae. Marker rescue experiments using genomic libraries identified a single nucleotide mutation in the vp39 gene. This mutation resulted in an amino acid substitution at glycine 276 (Gly-276) to serine, which was required for all the defective phenotypes observed in the mutant. Sequence comparison revealed that this residue is completely conserved among the VP39 proteins of the sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. Although early viral gene expression was not significantly affected, the level of expression of a late gene, vcath, was reduced. In addition, two of the very late genes were markedly downregulated in cells infected with this mutant. Western blot and quantitative PCR analyses revealed that the BVs produced from cells infected with this mutant contained smaller amounts of the VP39 protein and viral genomic DNA than those produced from wild-type virus-infected cells. Combined with the results of transmission electron microscopy, VP39 Gly-276 can be concluded to be essential for correct nucleocapsid assembly, viral DNA packaging, and viral gene expression, especially of very late genes.IMPORTANCE The major nucleocapsid protein gene vp39 is one of the most wellknown baculovirus genes. Although several viral and host proteins that interact with the VP39 protein have been identified, the functionally important domains or residues of this protein remain unknown. The present study revealed that the glycine residue at residue 276, which is completely conserved among sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses, is important for the VP39 function, i.e., structural assembly of nucleocapsids and viral DNA packaging. Moreover, our results provide evidence for the link between nucleocapsid formation and the transcription of viral very late genes.