Enterovirus manipulation of autophagic trafficking pathways

肠病毒D-68 （EV-D68）在儿童中引起严重的呼吸窘迫，并可能与急性弛缓性脊髓炎和格林-巴-罗综合征有关。在过去十年中，EV-D68的暴发频率和规模都有所增加。尽管在50多年前被发现，但人们对这种小核糖核酸病毒的细胞内生命周期的细节知之甚少。在这里，我们提供的数据表明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，是细胞自噬的标志，是细胞降解和回收受损蛋白质和细胞器的一种降解途径。自噬也是对饥饿或感染等压力的反应。与其他小核糖核酸病毒感染一样，EV-D68感染可诱导宿主细胞自噬。对于其他病毒，自噬途径涉及病毒颗粒成熟到其感染形式，并在包膜包中释放病毒。我们发现EV-D68通过几种新的方式失调自噬以促进其自身复制。EV-D68诱导SQSTM1/p62自噬货物受体的切割，这可能导致货物装载到自噬体的抑制。SNARE蛋白SNAP29是介导酸性自噬体和溶酶体融合的复合体的一部分，在感染期间被病毒3C蛋白酶切割。我们假设SNAP29的裂解促进了含有病毒的自噬体衍生囊泡的重定向，阻止它们与溶酶体融合，促进与质膜的融合和病毒的释放。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核内体，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，我们预测SNAP47可能促进病毒粒子成熟为感染性病毒。这些数据表明，该病毒通过至少两种机制破坏自噬降解：阻断货物进入自噬体，抑制自噬体向溶酶体的递送。为了了解EV-D68抑制和重定向自噬途径的机制，在第一个Aim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。在最后的Aim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细胞间传播的重要组成部分，我们预计了解这一途径将为一类治疗方法指明道路，这些治疗方法可以限制感染在肺部的持续传播，或者重要的是，限制病毒向包括中枢神经系统在内的其他器官的传播。

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.