Herpes simplex virus egress from neurons into axons mediated by HSV membrane proteins gE/gI and US9 and axonal transport by kinesin motors.

单纯疱疹病毒1型和2型是常见的α疱疹病毒，可建立终生潜伏期。虽然大多数粘膜组织的HSV感染是相对良性的，但也有罕见的脑炎病例，角膜的HSV感染会产生称为疱疹间质角膜炎(HSK)的炎性病理。HSK经常涉及反复感染，通常是多年后，由病毒在感觉神经节重新激活产生，然后传播到角膜，产生疤痕，最终可能导致失明。在美国，每年有6万例HSK病例，HSV仍然是导致失明的主要传染病。黏膜和眼睛中反复出现的HSV感染源于神经元中潜伏病毒的重新激活，随后是神经元轴突的顺行运输，这是一个病毒颗粒搭乘运动蛋白马达的过程，运动蛋白马达将病毒从神经元细胞体运送到轴突尖端。我们的研究将研究顺行运输的两个阶段。第一阶段包括在细胞质中组装病毒颗粒，然后对这些病毒颗粒进行分期或分选，以便运输到神经元轴突中。第二个阶段涉及到由动蛋白马达沿着轴突内的微管运输病毒颗粒。我们对这一过程的第一阶段的研究将集中在两个HSV膜蛋白Ge/Gi和US9上，它们共同促进病毒颗粒的组装和颗粒向轴突的极化分选。我们最近证实，HSV Ge-/US9-Double突变体不能组装被包裹的病毒颗粒，而是聚集在细胞质膜上的病毒衣壳。也有证据表明GE/GI和US9参与了随后的过程，包括将被包裹的病毒粒子分类为轴突。这些观察结果代表了神经元中病毒组装和分类中神经元特异性缺陷的新例子，并代表了GE/GI和US9在神经元中如何发挥作用的新范式。AIM 1的研究将检验两个假设：i)GE/GI和US9通过在作为病毒被膜部位的细胞质膜上收集其他病毒组装蛋白来促进病毒组装；ii)在被膜后，GE/GI和US9运输序列促进病毒颗粒分选到轴突中。涉及GE/GI-和US9介导的组装和分选的分子机制将使用新的高分辨率成像技术结合一组病毒突变和生化分析来研究。在目标2中，我们将研究顺行运输的第二阶段，解决两个基本的重要问题：i)在轴突中哪些动蛋白马达运输HSV颗粒；ii)哪些病毒蛋白系在动蛋白上？为了解决这些问题，我们将利用利用杆状病毒转导神经元的能力的最新进展，利用杆状病毒传递荧光货物分子和动链蛋白、“分裂动链蛋白”和沉默动链蛋白的miRNAs，从而使我们能够确定哪些动链蛋白对HSV顺行运输具有重要功能。基于我们最近发现的运输HSV的某些驱动蛋白，我们现在有机会识别与这些驱动蛋白马达相连的HSV蛋白。

Herpes simplex virus (HSV) 1 and 2 are common α-herpesviruses that establish lifelong latency. While most HSV infections of mucosal tissues are relatively benign, there are rare cases of encephalitis and HSV infections of the cornea can produce inflammatory pathology known as herpes stromal keratitis (HSK). HSK frequently involves recurring infections, often over years, produced by virus that reactivates in sensory ganglia that then travels to the cornea, producing scarring which can eventually lead to blindness. In the U.S. there are 60,000 cases of HSK/annually and HSV remains the leading infectious cause of blindness. Recurrent HSV infections in mucosa and the eye stem from reactivation of latent virus in neurons followed by anterograde transport in neuronal axons, a process by which virus particles hitchhike on kinesin motors that ferry virus from neuron cell bodies to axon tips. Our research will study two stages of anterograde transport. The first stage involves assembly of virus particles in the cytoplasm, followed by staging or sorting of these virus particles for transport into neuronal axons. The second stage involves transport of virus particles by kinesin motors along microtubules within axons. Our studies of first stage of this process will focus on two HSV membrane proteins gE/gI and US9 that cooperate to promote the assembly of virus particles and the polarized sorting of particles into axons. We recently demonstrated that HSV gE-/US9- double mutants were unable to assemble enveloped virus particles and, instead, viral capsids accumulated on cytoplasm membranes. There was also evidence that gE/gI and US9 participate in a subsequent process, involving sorting of enveloped virions into axons. These observations represent a novel example of neuron-specific defects in virus assembly and sorting in neurons and represent a new paradigm for how gE/gI and US9 function in neurons. The research in Aim 1 will test two hypotheses: i) gE/gI and US9 promote virus assembly by collecting other viral assembly proteins on cytoplasmic membranes that are sites of virus envelopment and ii) following envelopment, gE/gI and US9 trafficking sequences promote sorting of virus particles into axons. The molecular mechanisms involved in gE/gI- and US9-mediated assembly and sorting will be investigated using novel high resolution imaging techniques coupled with a panel of viral mutants and biochemical assays. In Aim 2, we will study the second stage of anterograde transport addressing two fundamentally important questions: i) which of the many kinesin motors transport HSV particles in axons and ii) which viral proteins tether onto kinesins? To address these questions, we will take advantage of recent advances in our ability to transduce neurons using baculoviruses to deliver fluorescent cargo molecules and kinesins, “split kinesins” and miRNAs to silence kinesins allowing us to determine which kinesins are functionally important for HSV anterograde transport. Based on our recent identification of certain kinesins that transport HSV, we now have the opportunity to identify HSV proteins that tether onto these kinesin motors.