The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid
The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid
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DOI：
10.1128/jvi.75.22.10923-10932.2001
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发表时间：
2001-11-01
影响因子：
5.4
通讯作者：
Brown, JC
中科院分区：
文献类型：
--
作者：
Newcomb, WW;Juhas, RM;Brown, JC
在 1 型单纯疱疹病毒 (HSV-1) 复制过程中，病毒 DNA 在受感染的细胞核中合成，不含 DNA 的衣壳也在此处组装。然后，从更大的多基因组串联体中切下基因组长度的 DNA 分子，并将其包装到衣壳中。在此，我们报告了实验结果，以测试 HSV-1 UL6 基因产物 (pUL6) 形成病毒 DNA 进入衣壳时所通过的门户这一观点。由于 DNA 必须进入独特的位点，因此进行了免疫电子显微镜实验以确定 pUL6 的位置。对 HSV-1 B 衣壳进行特异性免疫金染色后，通过其附着的金标记，在 12 个衣壳顶点之一发现了 pUL6。在多个顶点、非顶点位点或缺乏 pUL6 的衣壳中未观察到标记。在免疫印迹实验中，发现纯化的 B 衣壳中的 pUL6 拷贝数为 14.8 +/- 2.6。进行了分离 pUL6 的生化实验，从感染表达 UL6 基因的重组杆状病毒的昆虫细胞开始。纯化后，发现pUL6呈环状，在电子显微照片中观察到其外径和内径分别为16.4+/-1.1和5.0+/-0.7nm，高度为19.5+/-1.9nm。通过扫描透射电子显微镜测定的各个环的颗粒重量显示，大多数群体的质量对应于寡聚状态 12。结果被解释为支持 pUL6 形成 DNA 进入门户的观点，因为它存在于衣壳中的独特位点，并形成 DNA 可以通过的通道。 HSV-1 门户是在感染真核生物的病毒中首次发现的。就其尺寸和寡聚状态而言，pUL6 门户类似于双链 DNA 噬菌体（例如 phi 29、T4 和 P22）中用于 DNA 衣壳化的连接器或门户复合物。这种相似性支持了疱疹病毒和双链 DNA 噬菌体之间所提出的进化关系，并表明 DNA 包装的基本机制是保守的。
During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi 29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.