在全球拥有80多个会员,Perbunyaviridae家族中的直齿轮状病毒属是包膜RNA病毒的大属,其中许多是人类和牲畜中新兴的病原体。正双股病毒(OBV)如何穿透并感染哺乳动物宿主细胞仍然缺乏表征。这里,我们研究了OBVGermiston(GERV)的进入机制。通过低温电子显微镜观察病毒颗粒,并大致呈球形,平均直径为98nm。用荧光染料标记病毒不会对其感染性产生不利影响,并且可以监测固定细胞和活细胞中的单个颗粒。使用这种方法,我们发现结合病毒的内吞内化是异步的,发生在30~40分钟内.病毒进入Rab5a阳性(Rab5a)早期内体,随后,含Rab7a但不含LAMP-1的晚期内体空泡。感染进入不需要蛋白水解切割,和内体酸化是足够和必要的病毒融合。酸激活的渗透在病毒内化开始后15至25分钟开始,并依赖于早期内体到晚期内体的成熟。病毒膜融合的最佳pH值略低于6.0,当钾流入被取消时,渗透受到阻碍。总的来说,我们的研究提供了GERV进入宿主细胞的实时可视化,并证明了晚期内体成熟在促进OBV穿透中的重要性.重要性正杆状病毒(OBVs),其中包括LaCrosse,Oropouche,和Schmallenberg病毒,对全球人类和家畜的威胁越来越大。理想情况下,预防OBV传播需要针对感染早期阶段的方法,即,病毒进入。然而,对OBV进入和感染宿主细胞的分子和细胞机制知之甚少。这里,我们开发了准确的,敏感的工具和检测方法来研究GERV的渗透过程。我们的数据强调了晚期内体成熟在GERV进入中的核心作用,提供OBV感染早期阶段的全面概述。我们的研究还带来了一个完整的创新方法工具箱,用于研究固定细胞和活细胞中OBV进入程序的每个步骤,从病毒结合和内吞到融合和渗透。本文获得的信息为旨在阻止OBV进入的抗病毒策略的开发奠定了基础。
With more than 80 members worldwide, the Orthobunyavirus genus in the
Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.