Mutational analysis of the herpes simplex virus triplex protein VP19C
Mutational analysis of the herpes simplex virus triplex protein VP19C
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DOI：
10.1128/jvi.80.3.1537-1548.2006
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发表时间：
2006-02-01
影响因子：
5.4
通讯作者：
Rixon, FJ
中科院分区：
文献类型：
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作者：
Adamson, WE;McNab, D;Rixon, FJ
单纯疱疹病毒1型(HSV-1)衣壳具有二十面体结构，衣壳由主要衣壳蛋白VP5形成，通过称为三联体的独特结构将其连接在一起。三联体是由两种蛋白质以1：2的化学计量比形成的异三聚体。单拷贝蛋白称为VP19C，二聚体蛋白称为VP23。我们对VP19C进行了插入和缺失突变，并检测了突变对病毒生长和衣壳组装的影响。插入突变表明，该蛋白质的N末端类似于100个氨基酸，对应于疱疹病毒中保守程度较低的区域，对干扰不敏感，插入蛋白质其余部分对病毒生长有不同的影响。一些(但不是全部)严重残疾的突变体在结合VP23或VP5的能力上受到了损害。对缺失突变体的分析表明，在VP19C的N端附近存在一个核定位信号(NLS)，并通过将特定序列融合到绿色荧光蛋白标记上定位到一个33个氨基酸的区域。通过用猴病毒40大T抗原的NLS替换内源性NLS，我们能够证明VP19C的前45个氨基酸不是组装功能性衣壳和感染性病毒颗粒所必需的。然而，去除前63个氨基酸会导致异常衣壳的形成并阻止病毒的生长，这表明保守程度较低的N-末端序列具有一些尚未确定的功能。
Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal similar to 100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.