Abstract
In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.
Highlights
Infectious Salmon Anaemia Virus (ISAV) is an orthomyxovirus which causes disease in farmed Atlantic salmon (Salmo salar L.)
The first step involved assessing the expression level of each surface glycoprotein by immunofluorescence to verify that mutations introduced did not negatively affect glycoprotein folding and transport through the secretory pathway to the cell surface
All 7 mutant F proteins and the highly polymorphic region (HPR)-deleted HE mutant were expressed at levels similar to those of their respective wild type (WT) homologues (Table 3)
Summary
Infectious Salmon Anaemia Virus (ISAV) is an orthomyxovirus which causes disease in farmed Atlantic salmon (Salmo salar L.). The virus is enveloped with a genome consisting of 8 single-stranded RNA segments in negative orientation. Segments 5 and 6 encode two surface glycoproteins: the Fusion (F) protein and Haemagglutinin-esterase (HE), respectively. In the HE, the haemagglutinin function allows ISAV to attach to 4-O-Acetylated sialic acid receptors on the surface of host cells [7,8], while the acetyl esterase function destroys the sialic acid bonds promoting virus release [7,8]. The F protein is responsible for virus and host cell membrane fusion that allows the delivery of the viral genetic material into the cell [9]. There is mounting evidence that both the ISAV HE and F proteins act in a cooperative manner to trigger fusion during the early stage of infection [9,10]
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