Malik A K, Martinez R, Muncy L, Carmichael E P, Weller S K
Department of Microbiology, University of Connecticut Health Center, Farmington 06030.
Virology. 1992 Oct;190(2):702-15. doi: 10.1016/0042-6822(92)90908-8.
HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91-99). These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9. In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame. This region lies within the putative helicase domain of the UL9 protein. The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines. These results confirm that the UL9 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed. We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct. Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus. Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.
1-36互补组（hr27和hr156）中的单纯疱疹病毒1型（HSV-1）宿主范围突变体，其突变位于编码起始结合蛋白的UL9基因中，当在非允许性Vero细胞中生长时，无法形成噬斑或合成病毒DNA或晚期病毒蛋白（Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91 - 99）。在允许性细胞系S22中生长可有效弥补这些缺陷，S22包含包括UL9在内的几个HSV基因的野生型版本。在本报告中，通过DNA测序确定了宿主范围突变体hr27和hr156中损伤的精确性质和位置；发现这两个突变体在UL9开放阅读框的第309和311密码子处含有相同的单碱基对替换。该区域位于UL9蛋白的假定解旋酶结构域内。通过插入插入诱变剂ICP6::lacZ破坏了UL9基因，其中大肠杆菌lacZ基因在病毒ICP6启动子的控制下表达。含有这种插入的病毒突变体Hr94在Vero细胞中生长时不形成噬斑或合成病毒DNA，尽管在允许性细胞系上这两个缺陷都能得到有效弥补。这些结果证实UL9基因产物对病毒生长和DNA复制至关重要。此外，由于在感染hr94的细胞中未检测到UL9蛋白合成，这种病毒为进一步的结构-功能分析提供了有用的遗传背景，因为不会表达潜在干扰性的无功能UL9蛋白。我们在强诱导性HSV-1 ICP6启动子的控制下表达了UL9开放阅读框，并获得了含有不同拷贝数ICP6::UL9构建体的Vero细胞系。该构建体拷贝数超过约120的细胞无法支持野生型病毒高效形成噬斑。该构建体拷贝数低的细胞系能够弥补hr27、hr156和hr94。
