Characterization of a glycoprotein entry complex from a novel orthomyxovirus

传染性鲑鱼贫血病毒（ISAV）引起鲑鱼严重贫血，死亡率接近100%。在过去的30年里，ISAV在加拿大、美国和智利的养殖鲑鱼中引起了重大疾病爆发。2012年，在纽芬兰确诊的疫情导致45万条养殖鲑鱼被销毁。该病毒造成了重大的经济损失，并威胁到加拿大鲑鱼养殖的生存能力。目前，ISAV的生命周期定义不明确，疫苗只能提供部分保护。需要进一步的研究来了解这种病毒如何进入鲑鱼宿主的基本生物学原理。ISAV属于正黏液病毒科的is病毒属，由8个单链RNA片段组成，编码至少10种蛋白质。虽然ISAV与流感病毒密切相关，但其病毒进入的机制是独特的。ISAV病毒粒子有两种表面糖蛋白（GPs）：一种是42 kda的血凝素酯酶（HE），负责附着、趋向性和受体破坏/释放；另一种是50 kda的融合糖蛋白(F)，参与宿主和病毒脂质双分子层的合并。在之前的NSERC发现资助期间，我们确定了ISAV HE （Cook et al. 2017 PNAS）和融合后ISAV F （Cook et al. 2015 JBC）的晶体结构。这项工作提供了ISAV条目中涉及的单个gp的初始快照。最初附着后导致融合和病毒进入的分子事件仍然不清楚。例如，我们不知道ISAV HE和F如何相互作用以协调病毒融合，也不了解病毒gp的寡聚化如何影响进入。我们有初步的数据表明，在融合的pH值下，ISAV HE经历了从三聚体到单体的转变。ISAV HE单体被认为可以触发ISAV F预融合构象的变化，这是融合所必需的。这是一个完全独特的分子机制，因为没有观察到病毒GP进入复合物经历这样的事件。通过结合创新的生物物理和结构方法，我们能够进一步描述ISAV的进入机制。我们的具体目标是：阐明ISAV HE和F融合中间体AIM 2的分子作用。病毒gp的结构和功能研究需要付出巨大的努力，因为关于ISAV HE和F相互作用的结构信息将为ISAV进入和发病的基本生物学过程提供新的知识，为基于结构的新型、经济上可行的疫苗的开发提供蓝图，用于加拿大的养鱼场。我们拥有丰富的初步数据、独特的工具集和广泛的全科医生专业知识，使我们能够在这一重要而及时的倡议上取得迅速进展。

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.