Okoye Mercy E, Sexton Gerry L, Huang Eugene, McCaffery J Michael, Desai Prashant
Molecular Virology Laboratories, Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, The Johns Hopkins University, Baltimore, MD 21231, USA.
J Virol. 2006 Jan;80(2):929-40. doi: 10.1128/JVI.80.2.929-940.2006.
The triplex of herpesvirus capsids is a unique structural element. In herpes simplex virus type 1 (HSV-1), one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent VP5 molecules. The interaction between VP19C and VP23 was inferred by yeast cryoelectron microscopy studies and subsequently confirmed by the two-hybrid assay. In order to define the functional domains of VP19C and VP23, a Tn7-based transposon was used to randomly insert 15 bp into the coding regions of these two proteins. The mutants were initially screened for interaction in the yeast two-hybrid assay to identify the domains important for triplex formation. Using genetic complementation assays in HSV-1-infected cells, the domains of each protein required for virus replication were similarly uncovered. The same mutations that abolish interaction between these two proteins in the yeast two-hybrid assay similarly failed to complement the growth of the VP23- and VP19C-null mutant viruses in the genetic complementation assay. Some of these mutants were transferred into recombinant baculoviruses to analyze the effect of the mutations on herpesvirus capsid assembly in insect cells. The mutations that abolished the interaction in the yeast two-hybrid assay also abolished capsid assembly in insect cells. The outcome of these experiments showed that insertions in at least four regions and especially the amino terminus of VP23 abolished function, whereas the amino terminus of VP19C can tolerate transposon insertions. A novel finding of these studies was the ability to assemble herpesvirus capsids in insect cells using VP5 and VP19C that contained a histidine handle at their amino terminus.
疱疹病毒衣壳的三聚体是一种独特的结构元件。在单纯疱疹病毒1型（HSV-1）中，一个VP19C分子和两个VP23分子形成一个三叉结构，该结构通过与相邻的VP5分子相互作用来稳定衣壳外壳。VP19C和VP23之间的相互作用通过酵母冷冻电子显微镜研究推断得出，随后通过双杂交试验得到证实。为了确定VP19C和VP23的功能结构域，使用基于Tn7的转座子将15个碱基对随机插入这两种蛋白质的编码区域。最初在酵母双杂交试验中筛选突变体的相互作用，以鉴定对三聚体形成重要的结构域。在HSV-1感染的细胞中使用遗传互补试验，同样发现了病毒复制所需的每种蛋白质的结构域。在酵母双杂交试验中消除这两种蛋白质之间相互作用的相同突变，在遗传互补试验中同样无法补充VP23和VP19C缺失突变病毒的生长。将其中一些突变体转移到重组杆状病毒中，以分析这些突变对昆虫细胞中疱疹病毒衣壳组装的影响。在酵母双杂交试验中消除相互作用的突变也消除了昆虫细胞中的衣壳组装。这些实验的结果表明，至少在四个区域特别是VP23的氨基末端的插入会消除功能，而VP19C的氨基末端可以耐受转座子插入。这些研究的一个新发现是能够在昆虫细胞中使用在其氨基末端含有组氨酸标签的VP5和VP19C组装疱疹病毒衣壳。
