Characterization of a glycoprotein entry complex from a novel orthomyxovirus

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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.