Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.
Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.
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DOI：
10.1371/journal.ppat.1006197
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发表时间：
2017-02
期刊：
影响因子：
6.7
通讯作者：
Rowlands DJ
中科院分区：
文献类型：
--
作者：
Groppelli E;Levy HC;Sun E;Strauss M;Nicol C;Gold S;Zhuang X;Tuthill TJ;Hogle JM;Rowlands DJ
小核糖核酸病毒是通过受体介导的内吞作用进入细胞的无包膜RNA病毒。由于它们缺乏包膜，小核糖核酸病毒面临着将其RNA基因组穿过内吞囊泡膜进入细胞质以引发感染的挑战。目前，基因组释放和跨膜易位的机制仍然知之甚少。在肠病毒属中，已经提出脊髓灰质炎病毒、鼻病毒2和鼻病毒16通过病毒诱导的孔在完整的内体膜上释放它们的基因组，而一项研究提出鼻病毒14在破坏内体膜后释放其RNA。对于亲缘关系较远的aphthovirus属（如口蹄疫病毒和马鼻炎A病毒），核内体的酸化导致病毒粒子分解成五聚体，并将病毒RNA释放到核内体的管腔中，但尚未阐明RNA如何穿过囊泡膜的细节。然而，最近的研究表明，病毒RNA是从完整的颗粒中释放出来的，解离成五聚体可能是一个后期事件。在本研究中，我们利用受体修饰脂质体模型研究了脊髓灰质炎病毒基因组易位对RNase A的敏感性，以及脊髓灰质炎病毒和马鼻炎A病毒感染对共内化RNase A的敏感性。结果表明，在脂质体模型中，脊髓灰质炎病毒基因组易位对RNase A不敏感，并且很少或根本没有释放到培养基中。我们还表明，脊髓灰质炎病毒和马鼻炎A病毒共内化RNase A并不会降低传染性。此外，我们表明，所有被内化到细胞中的脊髓灰质炎病毒基因组，而不仅仅是那些导致感染的脊髓灰质炎病毒基因组，都受到RNase a的保护。这些结果支持一个精细协调的、定向的病毒RNA传递模型，该模型涉及病毒蛋白和细胞膜。小核糖核酸病毒是一个重要的人类和动物病原体大家族，包括脊髓灰质炎病毒、人鼻病毒和口蹄疫病毒。小核糖核酸病毒通过劫持囊泡介导的细胞进入途径之一进入宿主细胞。然而，一旦病毒被内化，其传递病毒基因组穿过囊泡膜进入细胞质的机制仍不清楚，甚至存在争议。在这里，我们表明，对于脊髓灰质炎病毒（肠病毒属的一员），病毒RNA在感染期间直接从颗粒易位，穿过囊泡膜进入受体修饰的脂质体模型中的脂质体管腔或细胞质，而不暴露于脂质体周围的外部介质或进入囊泡的管腔。我们的研究结果表明，病毒颗粒和膜之间的相互作用导致了病毒基因组传递的特定机制，不仅指导而且保护RNA，使其作为一个完整的功能分子到达细胞质。此外，我们发现马鼻炎A病毒也是如此，这是一种aphthovirus属的成员，其基因组传递机制以前被认为与肠病毒使用的机制有很大不同，这表明整个小核糖核酸病毒家族的RNA传递机制可能是统一的。
Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes. Picornaviruses are a large family of important human and animal pathogens that include poliovirus, human rhinovirus and foot-and-mouth disease virus. Picornaviruses enter the host cell by hijacking one of the vesicle-mediated cellular entry routes. However, once the virus is internalized, the mechanism used to deliver the viral genome across the vesicle membrane and into the cytoplasm remains unclear and even controversial. Here we show that for poliovirus (a member of the enterovirus genus), viral RNA is translocated directly from the particle, across the vesicle membrane into the lumen of liposomes in a receptor-decorated liposome model, or cytoplasm during infection, without being exposed to external medium surrounding the liposomes or the lumen of the entry vesicle, respectively. Our results suggest that the interaction between the viral particle and the membrane results in a specific mechanism of viral genome delivery that not only directs but also protects the RNA so that it reaches the cytoplasm as an intact and functional molecule. Additionally, we show that this is also the case for equine rhinitis A virus, a member of the aphthovirus genus, whose genome delivery mechanism has previously been thought to differ significantly from the mechanism used by enteroviruses suggesting the possibility of a unified mechanism of RNA delivery for the entire picornavirus family.