Cargo, biogenesis and functions of extracellular vesicles released during HSV-1 infection

单纯疱疹病毒-1 （HSV-1）在机体入口处粘膜上皮细胞发生裂解性感染后，在感觉神经元中建立终身的、沉默的感染。由于免疫反应减弱或压力导致从良性唇疱疹到脑炎等严重疾病，病毒偶尔会被重新激活。1型单纯疱疹病毒会加剧阿尔茨海默氏症等神经退行性疾病，并促进HIV-1等其他病原体的感染。细胞外囊泡（Extracellular vesicles, EVs）是所有类型的细胞都可以释放的，因为它们是细胞间通讯的主要机制，并且它们可以影响受体细胞的功能。在1型单纯疱疹病毒感染期间，容易检测到两类ev。第一个种群是通过四聚氰胺生物发生途径产生的。这些是从HSV-1感染细胞中释放的最丰富的ev，它们富含CD63四蛋白，并携带宿主因子，如干扰素基因刺激因子（STING），这是细胞质中DNA的传感器。这些CD63+ ev可以激活未感染受体细胞的先天免疫反应并抑制随后的HSV-1感染。第二种ev是通过运输途径所需的内体分选复合体（ESCRT）产生的，因此我们将其命名为ESCRT+ ev。这些ev携带ESCRT成分以及选定的病毒被膜和包膜蛋白，它们似乎具有前病毒作用。除了这两个ev群体，早期的研究已经报道了HSV-1感染过程中产生的由被膜和包膜蛋白组成的无衣壳颗粒，但缺乏病毒基因组。这些粒子比HSV-1病毒粒子轻，被称为l粒子。l -颗粒与ESCRT+ ev相似，携带病毒蛋白，具有前病毒效应，但比ESCRT+ ev密度大，富含病毒蛋白。我们假设，在HSV- 1感染期间，不同的ev群体被释放，对感染有不同的影响。为了验证我们的假设，我们制定了三个目标。在目的1中，我们建议使用质谱方法将ESCRT+ ev和CD63+ ev的货物与HSV-1感染期间产生的l -颗粒进行比较。这种分析的结果将产生关于受感染细胞传递信息的重要信息。在Aim 2中，我们提出确定在HSV-1感染期间参与产生ESCRT+ EV、CD63+ EV和l -颗粒的EV生物发生途径。为此，我们将使用基因沉默方法来破坏选定的EV生物发生途径，或者我们将使用突变病毒来确定选定的病毒基因对EV生物发生的影响。在Aim 3中，我们建议确定CD63+ ev、ESCRT+ ev和l -颗粒在受体细胞中触发的反应类型，并确定感染的后果。这些研究将提供关于1型单纯疱疹病毒感染细胞释放的ev的生物发生、装载和功能的重要信息，这对于确定它们对1型单纯疱疹病毒发病机制的影响至关重要。

Following productive, lytic infection in mucosal epithelial cells located at the portal of entry in the body, herpes simplex virus-1 (HSV-1) establishes a lifelong, silent infection in sensory neurons. The virus is occasionally reactivated due to weakened immune response or stress causing diseases that range in severity from benign cold sores to encephalitis. HSV-1 contributes to exacerbation of neurodegenerative diseases such as Alzheimer's and facilitates infection by other pathogens such as HIV-1. Extracellular vesicles (EVs) are released by all types of cells, as they constitute a major mechanism of intercellular communication, and they can influence recipient cell functions. During HSV-1 infection two populations of EVs are readily detectable. The first population is produced through a tetraspanins biogenesis pathway. These are the most abundant EVs released from HSV-1 infected cells, which are enriched in CD63 tetraspanin and carry host factors such as the STimulator of INterferon Genes (STING), a sensor of DNA in the cytosol. These CD63+ EVs can activate innate immune responses in uninfected recipient cells and suppress a subsequent HSV-1 infection. The second population of EVs is produced through the endosomal sorting complex required for the transport (ESCRT) pathway, thus we named them ESCRT+ EVs. These EVs carry ESCRT components along with selected viral tegument and envelope protein and they appear to have a proviral role. In addition to these two population of EVs, earlier studies have reported the production of capsidless particles during HSV-1 infection composed of tegument and envelope proteins but lacking viral genome. These particles are lighter than HSV-1 virions and are known as L-particles. L-particles resemble the ESCRT+ EVs in that they carry viral proteins and have a proviral effect but are denser than ESCRT+ EVs and enriched in viral proteins. We hypothesize that distinct populations of EVs are released during HSV- 1 infection with different effects on the infection. To test our hypothesis, we have formulated three Aims. In Aim 1, we propose to compare the cargo of ESCRT+ EVs and CD63+ EVs with L-particles produced during HSV-1 infection using a mass spectrometry approach. The results of this analysis will generate important information about messages communicated by infected cells. In Aim 2, we propose to determine EV biogenesis pathways involved in the production of ESCRT+ EVs, CD63+ EVs and L-particles during HSV-1 infection. For this, we will use a gene silencing approach to compromise selected EV biogenesis pathways or we will use mutant viruses to determine the impact of selected viral genes on EV biogenesis. In Aim 3, we propose to determine the type of responses that CD63+ EVs, ESCRT+ EVs and L-particles trigger in recipient cells and determine the consequences to the infection. These studies will provide important information on the biogenesis, cargo, and functions of EVs released by HSV-1 infected cells, which are essential to determine their impact on HSV-1 pathogenesis.