Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

在诱导裂解之前，裸病毒可以通过在细胞外囊泡 (EV) 中释放来逃离宿主细胞。这些纳米级 EV 将分泌的病毒颗粒隐藏在宿主衍生的膜中，从而改变了影响感染效率和抗病毒免疫的病毒-宿主相互作用。目前，关于调节这种病毒释放形式的病毒和宿主因素知之甚少。在这里，我们评估了脑心肌炎病毒 (EMCV) 前导蛋白的作用，这是一种破坏宿主抗病毒反应的“病毒安全蛋白”。使用高分辨率流式细胞术在单粒子水平上对野生型病毒或领导缺陷突变体感染后的 EV 释放进行了表征。Leader 的失活消除了感染期间的 EV 诱导，并大大减少了 EV 封闭的病毒释放。我们证明，Leader 通过刺激自噬的分泌臂来促进 EV 内病毒粒子的释放。EMCV 领导者这一新发现的角色增加了这种蛋白质影响病毒-宿主相互作用的多种机制。此外，这些数据提供了第一个证据，证明非结构病毒蛋白在通过包装在 EV 中非裂解释放小核糖核酸病毒中的关键作用。