Wang Hsien-Chi, Kao Yung-Ching, Chang Tien-Jye, Wong Min-Liang
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung 402, Taiwan.
Biochem Biophys Res Commun. 2005 Aug 26;334(2):631-7. doi: 10.1016/j.bbrc.2005.06.139.
Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation. Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine (or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work, we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase (enzyme catalyzing the conversion of arginine into ornithine and urea) treatment whereas this inhibition can be reversed by exogenous arginine, suggesting that arginine is essential for PRV proliferation. Western blotting was conducted to study the effect of arginine depletion on the levels of structural proteins of PRV in virus-infected cells. Four PRV structural proteins (gB, gE, UL47, and UL48) were chosen for examination, and results revealed that the levels of viral proteins were obviously reduced in long time arginase treatment. However, the overall protein synthesis machinery was apparently not influenced by arginase treatment either in mock or PRV-infected cells. Analyzing with native gel, we found that arginase treatment affected the mobility of PRV structural proteins, suggesting the conformational change of viral proteins by arginine depletion. Heat shock proteins, acting as molecular chaperons, participate in protein folding and translocation. Our results demonstrated that long time arginase treatment could reduce the expression of cellular heat shock proteins 70 (hsc70 and hsp70), and transcriptional suppression of heat shock protein 70 gene promoter was one of the mechanisms involved in this reduced expression.
伪狂犬病病毒（PRV）是α疱疹病毒属的成员；它是一种具有双链DNA基因组的包膜病毒。多胺（如精胺和亚精胺）在动物细胞中普遍存在，并参与细胞增殖和分化。我们实验室先前的结果表明，PRV在外源精胺（或亚精胺）存在或细胞内多胺耗尽的情况下都能完成裂解感染。氨基酸精氨酸是多胺生物合成的前体。在这项工作中，我们研究了精氨酸在PRV感染中的作用。发现用精氨酸酶（催化精氨酸转化为鸟氨酸和尿素的酶）处理可抑制PRV的噬斑形成，而这种抑制作用可被外源精氨酸逆转，这表明精氨酸对PRV增殖至关重要。进行蛋白质免疫印迹法以研究精氨酸耗竭对病毒感染细胞中PRV结构蛋白水平的影响。选择了四种PRV结构蛋白（gB、gE、UL47和UL48）进行检测，结果显示长时间用精氨酸酶处理后病毒蛋白水平明显降低。然而，无论是在未感染病毒的细胞还是PRV感染的细胞中，精氨酸酶处理显然都不会影响整体蛋白质合成机制。通过天然凝胶分析，我们发现精氨酸酶处理会影响PRV结构蛋白的迁移率，这表明精氨酸耗竭会导致病毒蛋白构象改变。热休克蛋白作为分子伴侣，参与蛋白质折叠和转运。我们的结果表明，长时间用精氨酸酶处理会降低细胞热休克蛋白70（hsc70和hsp70）的表达，热休克蛋白70基因启动子的转录抑制是这种表达降低所涉及 的机制之一。
