Enterovirus manipulation of autophagic trafficking pathways

肠道病毒 D-68 (EV-D68) 会导致儿童严重呼吸窘迫，并可能与急性弛缓性脊髓炎和吉兰-巴利综合征有关。过去十年中，EV-D68 的爆发频率和规模均有所增加。尽管这种小核糖核酸病毒在 50 多年前就被发现了，但人们对其细胞内生命周期的细节知之甚少。在这里，我们提供的数据显示 EV-D68 诱导自噬体形成以促进其自身复制。自噬体是细胞中发现的双膜囊泡，是自噬的所有标志，自噬是细胞用来降解和回收受损蛋白质和细胞器的降解途径。自噬也是对饥饿或感染等压力的反应。 EV-D68 感染与其他小核糖核酸病毒感染一样，会诱导宿主细胞自噬。对于其他病毒，自噬途径涉及病毒颗粒成熟为其感染形式以及病毒在包膜中的释放。我们发现 EV-D68 以几种新的方式调节自噬以促进其自身复制。 EV-D68 诱导 SQSTM1/p62 自噬货物受体裂解，这可能导致货物装载到自噬体中受到抑制。 SNARE 蛋白 SNAP29 是介导酸性自噬体和溶酶体之间融合的复合物的一部分，在感染过程中被病毒 3C 蛋白酶切割。我们假设 SNAP29 裂解促进含有病毒的自噬体来源的囊泡重定向，防止它们与溶酶体融合并促进与质膜融合和病毒释放。我们研究了同一家族中的另一个 SNARE SNAP47，它与内体 SNARE VAMP7 相关，发现它是正常自噬和 EV-D68 复制所必需的。由于酸化自噬体需要内体，并且我们已经证明酸化促进相关病毒脊髓灰质炎病毒的衣壳成熟，因此我们预测SNAP47可能促进病毒颗粒成熟为感染性病毒。这些数据表明，该病毒至少通过两种机制破坏自噬降解：阻止货物装载到自噬体中，并抑制自噬体递送至溶酶体。为了了解 EV-D68 抑制和重定向自噬途径的机制，在第一个目标中，我们将定义 SNAP29 在病毒生命周期的多个点上的作用。在 Aim II 中，我们研究了 SNAP47 在自噬和 EV-D68 复制中的具体作用。在最终目标中，我们将分析这些重定向膜与质膜融合并从细胞中释放的机制，包括 SNARE 蛋白 STX17 和 SNAP23。我们假设自噬降解的失调是病毒在宿主内从一个细胞传播到另一个细胞的重要组成部分，我们预计了解这一途径将为开发一类治疗方法指明道路，这些治疗方法可以限制肺部感染的持续传播，或者更重要的是，限制病毒传播到包括中枢神经系统在内的其他器官。

Enterovirus D-68 (EV-D68) causes severe respiratory distress in children and may be associated with acute flaccid myelitis and Guillain-Barré syndrome. Outbreaks of EV-D68 have increased in frequency and size over the last decade. Despite being discovered over 50 years ago, little is known about the specifics of the intracellular life cycle of this picornavirus. Here we present data showing that EV-D68 induces autophagosome formation to promote its own replication. Autophagosomes are double-membraned vesicles found in cells and all the hallmark of autophagy, a pathway of degradation which is used by cells to degrade and recycle damaged proteins and organelles. Autophagy is also a response to stresses such as starvation or infection. EV-D68 infection, like infection with other picornaviruses, induces autophagy in host cells. For other viruses, the autophagy pathway is involved in maturation of the virus particle into its infectious form and release of virus in enveloped packets. We have found that EV-D68 dysregulates autophagy in several novel ways to promote its own replication. EV-D68 induces cleavage of the SQSTM1/p62 autophagic cargo receptor, which likely results in inhibition of cargo loading into autophagosomes. The SNARE protein SNAP29, part of a complex that mediates fusion between acidic autophagosomes and lysosomes, is cleaved by the viral 3C protease during infection. We hypothesize that SNAP29 cleavage promotes redirection of virus-containing autophagosome- derived vesicles, preventing them from fusing with lysosomes and promoting fusion with the plasma membrane and release of virus. We have investigated another SNARE in the same family, SNAP47, which associates with the endosomal SNARE VAMP7, and found that it is required for normal autophagy and EV-D68 replication. Since endosomes are required for acidifying autophagosomes, and we have shown that acidification promotes capsid maturation of the related virus poliovirus, we predict SNAP47 may promote virion maturation into infectious virus. These data indicate that the virus disrupts autophagic degradation by at least two mechanisms: blocking cargo loading into autophagosomes, and inhibiting autophagosome delivery to lysosomes. To understand the mechanisms of EV-D68 inhibiting and redirecting the autophagy pathway, in the first Aim we will define the roles of SNAP29 at multiple points of the virus life cycle. In Aim II, we investigate the specific role of SNAP47 in autophagy and EV-D68 replication. In the final Aim, we will analyze mechanisms by which these redirected membranes fuse with the plasma membrane and are released from cells, including SNARE proteins STX17 and SNAP23. We hypothesize that dysregulation of autophagic degradation is an important part of virus dissemination from cell to cell within a host, and we anticipate that understanding this pathway will point the way to a class of therapeutics which could limit continued spread of infections within the lung, or importantly, spread of the virus to other organs including the central nervous system.