Jambunathan Nithya, Charles Anu-Susan, Subramanian Ramesh, Saied Ahmad A, Naderi Misagh, Rider Paul, Brylinski Michal, Chouljenko Vladimir N, 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.
Department of Biological Sciences, College of Basic Science, Louisiana State University, Baton Rouge, Louisiana, USA.
J Virol. 2015 Dec 9;90(5):2230-9. doi: 10.1128/JVI.02468-15.
We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses.
Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted β-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.
我们之前已经表明，缺乏完整糖蛋白K（gK）表达或表达有38个氨基酸缺失的gK（gKΔ31 - 68突变）的单纯疱疹病毒1型（HSV - 1）在小鼠眼部感染后无法感染神经节神经元。我们构建了一个gK预测三维结构的新模型，发现gKΔ31 - 68突变跨越gK氨基末端一个明确的β - 折叠结构，该结构在甲型疱疹病毒中保守。使用一种新型病毒进入邻近连接分析（VEPLA），在体外培养的大鼠神经节细胞培养中测试了HSV - 1（McKrae）gKΔ31 - 68病毒进入神经节神经元轴突的能力。在该分析中，通过gD与其同源受体nectin - 1在受感染神经元表面的共定位来检测细胞表面结合的病毒粒子。通过病毒粒子被膜蛋白UL37的共定位来检测已进入细胞质的衣壳，病毒粒子衣壳加载到微管上进行逆行转运至细胞核需要动力蛋白。HSV - 1（McKrae）gKΔ31 - 68与野生型HSV - 1（McKrae）一样有效地附着于细胞培养中Vero细胞和神经节轴突的细胞表面。然而，与野生型病毒不同，突变病毒未能进入神经节神经元的轴质。这项工作表明，gK的氨基末端是进入神经元轴突的关键决定因素，并且可能对其他甲型疱疹病毒发挥类似的保守功能。
与β和γ疱疹病毒不同，甲型疱疹病毒具有感染神经元并在其中建立潜伏状态的独特能力。糖蛋白K（gK）和膜蛋白UL20在所有甲型疱疹病毒中保守。我们在此表明，在甲型疱疹病毒中保守的一个预测β - 折叠结构域在HSV - 1进入神经元轴突中起作用，表明它可能对其他疱疹病毒发挥类似功能。这些结果与我们之前的观察结果一致，即该gK结构域的缺失会阻止病毒在小鼠眼部感染后成功感染神经节神经元。
