Charles Anu-Susan, Chouljenko Vladimir N, Jambunathan Nithya, Subramanian Ramesh, Mottram Peter, Kousoulas Konstantin G
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA
J Virol. 2014 Jul;88(13):7618-27. doi: 10.1128/JVI.00657-14. Epub 2014 Apr 23.
The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-to-alanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.
We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.
单纯疱疹病毒1型（HSV-1）的UL20基因编码一种222个氨基酸的非糖基化包膜蛋白，该蛋白与病毒糖蛋白K（gK）形成复合物，在病毒粒子包膜形成、释放以及病毒诱导的细胞融合过程中发挥作用。为了研究UL20蛋白（UL20p）羧基末端在细胞质中病毒粒子包膜形成中的作用，构建并鉴定了一系列突变病毒。与野生型病毒相比，从UL20p羧基末端缺失六个氨基酸导致感染性病毒产量下降约1个对数级。令人惊讶的是，在氨基酸位置210处将苯丙氨酸替换为丙氨酸导致功能获得型表型，感染性病毒产量比野生型病毒增加了多达1个对数级。相反，将两个靠近膜的苯丙氨酸替换为丙氨酸导致感染性病毒粒子产量和细胞质中病毒粒子包膜形成受到严重抑制。对UL20p膜拓扑结构的预测表明，这两个氨基酸变化导致UL20p羧基末端从细胞内空间缩回。共聚焦显微镜显示，所构建的UL20突变均未影响UL20p向反式高尔基体网络膜的细胞内运输。此外，邻近连接分析表明，UL20突变均未影响UL20p的共定位以及与最近发现与gK/UL20蛋白复合物相互作用的UL37蛋白的潜在相互作用。总的来说，这些研究表明，UL20p羧基末端的苯丙氨酸残基参与细胞质中病毒粒子包膜形成和感染性病毒产量的调节。
我们之前已经表明，UL20/gK蛋白复合物在细胞质中病毒粒子包膜形成中起关键作用，并且它与UL37衣壳蛋白相互作用以促进细胞质中病毒粒子包膜形成。在本研究中，我们研究了UL20p羧基末端苯丙氨酸残基的作用，因为已知芳香族和疏水氨基酸通过其芳香结构的堆积参与蛋白质-蛋白质相互作用。对携带苯丙氨酸（Phe）到丙氨酸（Ala）突变的突变病毒的表征表明，两个靠近膜的Phe残基对于正确的UL20p膜拓扑结构以及有效的病毒粒子包膜形成和感染性病毒产量至关重要。令人惊讶的是，位于UL20p羧基末端大约中间位置的Phe到Ala的变化显著增强了细胞质包膜形成和感染性病毒粒子的总体产量。这项工作表明，UL20p羧基末端的Phe残基参与细胞质中病毒粒子包膜形成和感染性病毒产量的调节。
