A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells
A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells
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DOI：
10.1128/jvi.74.24.11608-11618.2000
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发表时间：
2000-12-01
影响因子：
5.4
通讯作者：
Desai, PJ
中科院分区：
文献类型：
--
作者：
Desai, PJ
UL36开放阅读框（ORF）编码最大的单纯疱疹病毒1型（HSV-1）蛋白，这是一个270 kda的多肽，命名为VP1/2，它也是病毒粒子被膜的一个组成部分。在UL36基因中产生了一个零突变，以阐明其在病毒生命周期中的作用。由于UL36基因指定了一个基本功能，因此产生了编码UL36 ORF序列的互补细胞系。一种被命名为K δ UL36的突变病毒编码UL36基因的零突变，并在这些细胞系中进行了分离和繁殖。当分析感染K Delta UL36的非互补细胞时，检测到末端基因组DNA片段和含DNA的衣壳（C衣壳）；因此，DNA的切割或包装不需要UL36。对突变感染细胞的裂解物进行沉降分析，发现存在具有C衣壳物理特征的颗粒。与此一致的是，突变颗粒的多肽谱显示缺少主要的包膜和被皮成分。超微结构分析显示，K Delta ul36感染细胞的细胞质中存在大量含有衣壳的无包膜DNA。用vp26绿色荧光蛋白标记UL36突变体颗粒，并在活细胞中监测其运动。在K Delta ul36感染的细胞中，在整个细胞质中观察到与衣壳颗粒相对应的广泛颗粒荧光。在野生型感染的细胞中，在质膜上观察到荧光的积累，这表明病毒粒子的成熟和出口，但在K Delta ul36感染的细胞中没有。在缺乏UL36功能的情况下，产生充满dna的衣壳；这些衣壳穿过核膜后进入细胞质，并不成熟为包膜病毒。UL36突变体颗粒的成熟和出口被取消，可能是由于这种复杂多肽的功能较晚，即将衣壳靶向到正确的成熟途径。
The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated K Delta UL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with K Delta UL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of K Delta UL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In K Delta UL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in K Delta UL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.