Characterization of a glycoprotein entry complex from a novel orthomyxovirus

新型正粘病毒糖蛋白进入复合物的表征

• 批准号: RGPIN-2019-05703
• 负责人: Lee, Jeffrey
• 金额: $ 3.64万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715754
• 关键词: Characterization; glycoprotein; entry; complex; novel

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.

一种新的正粘病毒糖蛋白进入复合物的鉴定 传染性鲑鱼贫血病毒（ISAV）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，美国和智利的养殖鲑鱼中引起了重大疾病爆发。2012年，纽芬兰爆发了一场确诊的疫情，导致45万条养殖鲑鱼被销毁。该病毒已造成重大经济损失，并对加拿大鲑鱼养殖的可行性构成威胁。目前，ISAV的生命周期定义不清，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其鲑鱼宿主的基本生物学。 ISAV属于正粘病毒科中的Isavirus属，并且由编码至少10种蛋白质的8个单链RNA区段组成。虽然ISAV与流感病毒密切相关，但其病毒进入机制是独特的。ISAV病毒粒子具有两种表面糖蛋白（GP）：负责附着、向性和受体破坏/释放的42-kDa血凝素酯酶（HE）和参与宿主和病毒脂质双层的合并的50-kDa融合（F）GP。 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进入所涉及的各个GP的初始快照。最初附着后导致病毒融合和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，ISAV HE经历了从三聚体到单体的转变。假设ISAV HE单体触发融合所需的ISAV F融合前构象的变化。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。 使用创新的生物物理和结构的方法相结合，我们能够进一步描绘ISAV进入机制。我们的具体目标是： AIM 1.阐明ISAV HE和F融合中间体的分子作用 AIM 2.通过冷冻电子断层扫描确定ISAV HE和F的相互作用 病毒GP的结构和功能研究保证了一个重大的努力，因为ISAV HE和F的相互作用的结构信息将提供新的知识，ISAV进入和发病机制的基本生物过程，导致一个蓝图的结构为基础的开发新的，经济上可行的疫苗将用于加拿大的养鱼场。我们大量的初步数据、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举措上取得快速进展。