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（HSV） 1和2是常见的α-疱疹病毒，具有终身潜伏期。虽然大多数粘膜组织的HSV感染是相对良性的，但在脑炎和角膜HSV感染的罕见病例中，会产生炎症病理，即疱疹间质角膜炎（HSK）。HSK经常涉及反复感染，通常持续数年，由病毒引起，病毒在感觉神经节重新激活，然后传播到角膜，产生疤痕，最终导致失明。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性原因。粘膜和眼睛的复发性HSV感染源于神经元中潜伏病毒的再激活，随后是神经元轴突的顺行运输，病毒颗粒搭乘运动蛋白将病毒从神经元细胞体运送到轴突尖端的过程。我们的研究将研究顺行运输的两个阶段。第一阶段包括病毒颗粒在细胞质中的组装，随后是这些病毒颗粒的分期或分类，以便运输到神经元轴突。第二阶段是病毒颗粒沿着轴突内的微管通过运动蛋白马达进行运输。我们对这一过程第一阶段的研究将集中在两种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。我们最近证明，HSV gE-/US9-双突变体不能组装包膜病毒颗粒，相反，病毒衣壳积聚在细胞质膜上。也有证据表明，gE/gI和US9参与了随后的过程，包括将包膜病毒体分选为轴突。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如何在神经元中起作用的新范例。Aim 1的研究将验证两个假设：i) gE/gI和US9通过在病毒包膜位点的细胞质膜上收集其他病毒组装蛋白来促进病毒组装；ii)在包膜之后，gE/gI和US9转运序列促进病毒颗粒分选到轴突。gE/gI-和us9介导的组装和分选所涉及的分子机制将使用新的高分辨率成像技术，结合一组病毒突变体和生化分析进行研究。在目标2中，我们将研究顺行运输的第二阶段，解决两个基本重要的问题：i)在轴突中运输HSV颗粒的众多运动蛋白中的哪一个，ii)哪些病毒蛋白系在运动蛋白上？为了解决这些问题，我们将利用最近的进展，利用杆状病毒转导神经元来传递荧光货物分子和运动蛋白，“分裂运动蛋白”和mirna来沉默运动蛋白，使我们能够确定哪些运动蛋白对HSV顺行运输具有重要的功能。基于我们最近对运输HSV的某些驱动蛋白的鉴定，我们现在有机会鉴定捆绑在这些驱动蛋白马达上的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.