Cairns Tina M, Milne Richard S B, Ponce-de-Leon Manuel, Tobin Deanna K, Cohen Gary H, Eisenberg Roselyn J
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Virol. 2003 Jun;77(12):6731-42. doi: 10.1128/jvi.77.12.6731-6742.2003.
In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.
在甲型疱疹病毒中，糖蛋白B（gB）、gD、gH和gL对于病毒进入至关重要。一种具有复制能力的gL缺失型伪狂犬病病毒（PrV）（B.G. Klupp和T.C. Mettenleiter，《病毒学杂志》73:3014 - 3022，1999年）被证明可表达一种gDgH融合蛋白，该蛋白在细胞 - 细胞融合和空病毒互补试验中可替代gD、gH和gL。为了在单纯疱疹病毒1型（HSV - 1）中研究这一现象，我们构建了四种gDgH嵌合体，将gD的前308个氨基酸与gH不同的N端截短体相连。这些嵌合体根据其截短的gH的第一个氨基酸命名：22、259、388和432。所有嵌合体均用针对构象表位的gD和gH抗体进行免疫沉淀。通常情况下，gH转运至细胞表面需要形成gH - gL复合物。嵌合体22包含与gD308融合的全长gH。与PrV gDgH不同，嵌合体22转运至转染的Vero细胞表面需要gL。有趣的是，尽管嵌合体259未能到达细胞表面，但嵌合体388和432表现出不依赖gL的转运。为了检测gD和gH结构域的功能，在细胞 - 细胞融合和空病毒互补试验中对每个嵌合体进行了测试。与PrV gDgH不同，HSV - 1的嵌合体在融合试验中均不能替代gL。只有嵌合体22能够替代gH进行融合，并且在互补试验中也能替代gH或gD。令人惊讶的是，尽管该嵌合体能够互补gD缺失型病毒并结合HSV进入受体（HveA和nectin - 1），但其在融合试验中作为gD替代物的表现非常差。嵌合体388和432含有与嵌合体22相同的gD部分，在融合试验中替代gD的效率为野生型水平的25%至50%。然而，这些嵌合体在gD缺失型病毒互补试验中的功能较差。结果突出了这样一个事实，即这两种功能试验测量的是两个相关但不同的过程。
