Lim Byung-Kwan, Yun Soo-hyeon, Ju Eun-Seon, Gil Chae-Ok, Kim Duk-Kyung, Jeon Eun-Seok
Department of Medicine, Sungkyunkwan University School of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Seoul, Korea.
Biosci Biotechnol Biochem. 2012;76(6):1173-6. doi: 10.1271/bbb.120045. Epub 2012 Jun 7.
We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.
我们构建了一种具有心脏嗜性且能复制的嵌合柯萨奇病毒B3（CVB3）以表达乙醇脱氢酶（ADH）。尽管通过蛋白质免疫印迹分析发现了外源性表达的ADH，但其酶功能受到抑制。为了确定抑制ADH酶功能的因素，我们在CVB3 VP0衣壳蛋白的第二个氨基酸（MGAQEF···）处引入了一个定点突变，有效地将甘氨酸变为丙氨酸。已知该甘氨酸在感染细胞中病毒衣壳蛋白成熟过程中是一个肉豆蔻酰化位点。与未修饰的病毒相比，在突变的rCVB3-ADH（G2A）病毒中很容易检测到ADH的表达和酶功能。虽然ADH的表达需要CVB3 VP0肉豆蔻酰化位点发生突变才能正常发挥功能，但另一种表达绿色荧光蛋白的嵌合病毒（rCVB3-GFP（G或A））的作用则与肉豆蔻酰化位点无关。实际上，在荧光显微镜下，感染的HeLa细胞显示出绿色荧光蛋白。这些结果表明，VP0衣壳蛋白中的肉豆蔻酰化位点抑制了具有酶活性的ADH的表达，但不影响绿色荧光蛋白的表达。VP0肉豆蔻酰化对于嵌合CVB3病毒的复制是可有可无的。
