Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral Envelope Glycoprotein B and Regulates Its Expression on the Cell Surface
Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral Envelope Glycoprotein B and Regulates Its Expression on the Cell Surface
复制标题
DOI：
10.1128/jvi.01451-08
复制
发表时间：
2009-01-01
影响因子：
5.4
通讯作者：
Kawaguchi, Yasushi
中科院分区：
文献类型：
--
作者：
Kato, Akihisa;Arii, Jun;Kawaguchi, Yasushi
Us3是由单纯疱疹病毒1号（HSV-1）编码的丝氨酸-苏氨酸蛋白激酶。正如本文所报道的，我们试图在hsv -1感染的细胞中鉴定先前未报道的Us3生理底物。我们的结果如下。(i)生物信息学分析预测了病毒包膜糖蛋白B （gB）在密码子557 ~ 562 （RRVSAR）和密码子884 ~ 889 （RRNTNY）两个假定的Us3磷酸化位点。（ii）在体外激酶实验中，位于gB结构域887位的苏氨酸残基（Thr-887）被Us3特异性磷酸化，而位于560位的丝氨酸残基没有被Us3特异性磷酸化。（iii）利用抗体特异性检测感染野生型HSV-1的Vero细胞中gB Thr-887的磷酸化，该抗体识别-3和-2位置的精氨酸磷酸化的丝氨酸或苏氨酸残基。（iv）感染细胞中gB Thr-887的磷酸化依赖于Us3的激酶活性。(v)用丙氨酸替代Thr-887显著上调了感染细胞中gB的细胞表面表达，而用天冬氨酸替代（有时模仿组成磷酸化）则恢复了野生型表型。用重组HSV-1编码催化失活Us3的细胞感染后，细胞表面gB表达上调。这些结果支持了Us3磷酸化gB并下调hsv -1感染细胞中gB的细胞表面表达的假设。
Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the -3 and -2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.