Characterization of a glycoprotein entry complex from a novel orthomyxovirus

一种新的正粘病毒感染性鲑鱼贫血病毒(ISAV)的糖蛋白进入复合体的特征会导致鲑鱼严重贫血，死亡率接近100%。在过去的30年里，ISAV在加拿大、美国和智利的养殖三文鱼中引发了重大疾病暴发。2012年，纽芬兰确认爆发疫情，导致45万条养殖三文鱼被销毁。该病毒已造成重大经济损失，并对加拿大三文鱼养殖业的生存能力构成威胁。目前，ISAV的生命周期定义不明确，疫苗只能提供部分保护。需要更多的研究来了解这种病毒如何进入其鲑鱼宿主的基本生物学。ISAV属于正粘病毒科ISAV属，由8个单链RNA片段组成，编码至少10种蛋白质。虽然ISAV与流感病毒关系密切，但其进入病毒的机制是独特的。ISAV病毒粒子有两个表面糖蛋白(GP)：42 kDa的血凝素-酯酶(HE)，负责附着、趋向性和受体的破坏/释放；50 kDa的融合(F)GP，参与宿主和病毒脂双层的合并。在之前的NSERC发现资助期间，我们确定了ISAV HE的晶体结构(Cook等人。2017年PNAS)和核聚变后ISAV F(Cook等人2015 JBC)。这项工作提供了ISAV条目中涉及的各个全科医生的初始快照。导致病毒融合和进入的初始附着后的分子事件仍然没有明确的定义。例如，我们不知道ISAV HE和F如何相互作用来协调病毒融合，也不知道病毒GP的寡聚化如何影响进入。我们有初步数据表明，在融合的pH值下，ISAV HE经历了从三聚体到单体的转变。ISAV HE单体被假设为触发融合所需的ISAV F的融合前构象的变化。这是一种完全独特的分子机制，因为还没有观察到病毒GP进入复合体经历这样的事件。使用创新的生物物理和结构方法相结合，我们能够进一步描述ISAV的进入机制。我们的具体目标是：1.阐明ISAV HE和F融合中间产物的分子作用AIM 2.通过冷冻电子断层扫描确定ISAV HE和F的相互作用。对病毒GP的结构和功能的研究是必要的，因为关于ISAV HE和F相互作用的结构信息将为ISAV进入和致病的基本生物学过程提供新的知识，从而为基于结构的新型、经济可行的疫苗的开发提供蓝图，用于加拿大养鱼场。我们在全科医生方面的大量初步数据、独特的工具集和广泛的专业知识使我们能够在这一重要和及时的举措上取得快速进展。

Characterization of a glycoprotein entry complex from a novel orthomyxovirus Infectious salmon anemia virus (ISAV) causes severe anemia in salmon and has mortality rates that approach 100%. Over the last 30 years, ISAV has caused major disease outbreaks in farmed salmon in Canada, the US, and Chile. In 2012, a confirmed outbreak in Newfoundland prompted the destruction of 450,000 farmed salmon. The virus has caused major economic losses and is a threat to the viability of Canadian salmon farming. Currently, the ISAV lifecycle is poorly defined and vaccines offer only partial protection. Additional studies are needed to understand the basic biology of how this virus enters its salmon host. ISAV belongs to the Isavirus genus in the family orthomyxoviridae and consists of eight single-stranded RNA segments that encode for at least 10 proteins. Although ISAV is closely related to influenza viruses, its mechanism of viral entry is unique. ISAV virions have two surface glycoproteins (GPs): a 42-kDa hemagglutinin-esterase (HE) that is responsible for attachment, tropism, and receptor destruction/release and a 50-kDa fusion (F) GP that is involved in the merger of the host and viral lipid bilayers. During the previous NSERC Discovery Grant funding period, we determined the crystal structures of ISAV HE (Cook et al. 2017 PNAS) and the postfusion ISAV F (Cook et al. 2015 JBC). This work provides initial snapshots of the individual GPs involved in ISAV entry. The molecular events after initial attachment that lead to fusion and entry of the virus remain poorly defined. For example, we do not know how ISAV HE and F interact to coordinate viral fusion nor do we understand how oligomerization of the viral GPs influences entry. We have preliminary data that suggest at the pH of fusion, ISAV HE undergoes a transition from a trimer to a monomer. The ISAV HE monomer is hypothesized to trigger changes to the prefusion conformation of ISAV F necessary for fusion. This is an entirely unique molecular mechanism, as no viral GP entry complex has been observed to undergo such an event. Using a combination of innovative biophysical and structural approaches, we are in position to further delineate the ISAV entry mechanism. Our specific aims are to: AIM 1. Elucidate the molecular roles of ISAV HE and F fusion intermediates AIM 2. Determine the interplay of ISAV HE and F by cryo-electron tomography Structural and functional studies of viral GPs warrant a significant effort as structural information on the interplay of ISAV HE and F will provide new knowledge on the fundamental biological process of ISAV entry and pathogenesis, leading to a blueprint for the structure-based development of novel, economically feasible vaccines to be used in Canadian fish farms. Our substantial preliminary data, unique tool set, and extensive expertise in GPs position us to make rapid progress on this important and timely initiative.