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是建立终身潜伏期的常见α-疱疹病毒。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感染可产生称为疱疹性角膜基质炎（HSK）的炎性病理。HSK经常涉及反复感染，通常是多年，由病毒产生，在感觉神经节中重新激活，然后传播到角膜，产生疤痕，最终导致失明。在美国，每年有60，000例HSK病例，HSV仍然是导致失明的主要传染性原因。粘膜和眼睛中的复发性HSV感染源于潜伏病毒在神经元中的再活化，随后在神经元轴突中的顺行运输，这是病毒颗粒在驱动蛋白马达上搭便车的过程，驱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。第一阶段涉及病毒颗粒在细胞质中的组装，随后对这些病毒颗粒进行分期或分选以运输到神经元轴突中。第二阶段涉及驱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US 9，它们合作促进病毒颗粒的组装和颗粒到轴突中的极化分选。我们最近证明HSV gE-/US 9-双突变体不能组装包膜病毒颗粒，相反，病毒衣壳积累在细胞质膜上。还有证据表明，gE/gI和US 9参与了随后的过程，包括将包膜病毒粒子分选成轴突。这些观察结果代表了神经元中病毒组装和分选中神经元特异性缺陷的一个新例子，并代表了gE/gI和US 9在神经元中如何发挥作用的新范例。目的1中的研究将检验两个假设：i）gE/gI和US 9通过在作为病毒装配位点的细胞质膜上收集其他病毒装配蛋白来促进病毒装配，以及ii）在装配之后，gE/gI和US 9运输序列促进病毒颗粒分选到轴突中。参与gE/gI和US 9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体和生化检测进行研究。在目标2中，我们将研究顺行运输的第二阶段，解决两个根本性的重要问题：i）许多驱动蛋白马达中的哪一个在轴突中运输HSV颗粒，以及ii）哪些病毒蛋白质拴在驱动蛋白上？为了解决这些问题，我们将利用最近的进展，我们的能力，使用杆状病毒，以提供荧光货物分子和驱动蛋白，“分裂驱动蛋白”和miRNA沉默驱动蛋白，使我们能够确定哪些驱动蛋白是功能上重要的HSV顺行运输神经元。基于我们最近对某些运输HSV的驱动蛋白的鉴定，我们现在有机会鉴定连接到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.