Goldstein J N, Weller S K
Department of Microbiology, University of Connecticut Health Center, Farmington 06030-3205, USA.
Virology. 1998 May 10;244(2):442-57. doi: 10.1006/viro.1998.9129.
The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear localization, suggesting that they have retained an intact global conformation. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determined whether expression of mutant proteins from amplicon plasmids could complement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so. Loss of exonuclease activity therefore correlates with loss of in vivo function.
单纯疱疹病毒1型（HSV-1）的UL12基因编码一种碱性pH依赖的脱氧核糖核酸酶，称为碱性核酸酶。重组UL12基因敲除突变体AN-1的生长严重受损，对该突变体的分析表明，UL12在处理复杂的DNA复制中间体中起作用（R. 马丁内斯、R. T. 萨里斯基、P. C. 韦伯和S. K. 韦勒，（1996年）《病毒学杂志》70卷，2075 - 2085页）。从细胞核向细胞质输出的有功能的衣壳的产生可能需要这一处理步骤。在本报告中，我们探讨了AN-1生长表型是否由于UL12催化活性丧失的问题。我们在UL12的一个高度保守区域（基序II）构建了两个点突变，并从杆状病毒表达系统中纯化了野生型和突变型酶。两种突变蛋白都是稳定的、可溶的，并且能够正确地定位于细胞核，这表明它们保留了完整的整体构象。然而，两种突变蛋白都不表现出核酸外切酶活性。为了研究这些突变在体内的作用，我们确定了来自扩增子质粒的突变蛋白的表达是否能够互补AN-1。虽然野生型质粒能够互补空突变体的生长，但两种UL12突变体都不能。因此，核酸外切酶活性的丧失与体内功能的丧失相关。
