Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.

当感染发生时，多数RNA病毒会重构其衣壳（capsid），以将基因组释放至宿主细胞胞质内完成复制。该过程通常由受体结合及/或内体的酸性环境所触发。在肠道病毒属（Enterovirus）——该属包含150余种可引发普通感冒的人鼻病毒（human rhinovirus, HRV）血清型——中，已有令人信服的证据表明，病毒RNA会通过蛋白壳上形成的通道以单链形式逸出。我们通过特异性寡核苷酸杂交结合荧光相关光谱检测，明确了56℃孵育病毒粒子（virions）时，HRV2的RNA末端随时间的释放动态。研究发现，补骨脂素（psoralen）紫外交联（UV crosslinking）可阻断RNA的完全释放，借此得以鉴定出衣壳内残留的RNA序列。我们还解析了脱衣壳中间体（uncoating intermediates）的结构：此类中间体中部分RNA发生浓缩，形成指向二十面体二重对称轴（假定的RNA出口位点）的杆状结构。综上，与教科书及综述中常见的模型相悖，我们的研究结果证实RNA的释放起始于3'端。这提示基因组的包装同样以有序方式进行，使得3'-聚腺苷酸（poly-(A)）尾在病毒粒子转化为亚病毒颗粒的过程中，定位于孔道形成的邻近位置。这种定向的基因组释放机制，可能广泛存在于众多二十面体无包膜单链RNA病毒中。