Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture

Abstract

Salmonid alphavirus (SAV) is the cause of pancreas disease and sleeping disease in farmed salmonid fish in Europe. The spread of these diseases has been difficult to control with biosecurity and current vaccination strategies, and increased understanding of the viral pathogenesis could be beneficial for the development of novel vaccine strategies. N-glycosylation of viral envelope proteins may be crucial for viral virulence and a possible target for its purposed attenuation. In this study, we mutated the N-glycosylation consensus motifs of the E1 and E2 glycoproteins of a SAV3 infectious clone using site-directed mutagenesis. Mutation of the glycosylation motif in E1 gave a complete inactivation of the virus as no viral replication could be detected in cell culture and infectious particles could not be rescued. In contrast, infectious virus particles could be recovered from the SAV3 E2 mutants (E2319Q, E2319A), but not if they were accompanied by lack of N-glycosylation in E1. Compared to the non-mutated infectious clone, the SAV3-E2319Q and SAV3-E2319A recombinant viruses produced less cytopathic effects in cell culture and lower amounts of infectious viral particles. In conclusion, the substitution in the N-linked glycosylation site in E2 attenuated SAV3 in cell culture. The findings could be useful for immunization strategies using live attenuated vaccines and testing in fish will be desirable to study the clone’s properties in vivo.

Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of Atlantic salmon

“…Protein secondary structure predictions were performed using PSIPRED 4.0 (Jones, 1999). We have previously predicted the N‐glycosylation site in SAV3 E2 using the NetNGlyc 1.0 server (available at http://www.cbs.dtu.dk/services/NetNGlyc/) supplemented by data from structure homology modelling of the protein using iTasser (Aksnes et al., 2020).…”

“…Additional mutations were introduced in the capsid by substituting two lysines (K) with alanines (A) in a predicted bipartite nuclear localization signal (NLS) at positions 79 and 81. In the E2 protein, an asparagine (N) was substituted with alanine (A) at position 319 in a predicted N‐linked glycosylation site as previously described (Aksnes et al., 2020). Also, a XbaI site originally introduced in the junction region of prSAV3 as a tag to distinguish between the infectious clone from that of wild‐type SAV3 (Karlsen, Villoing, et al., 2010) was replaced with the wild‐type sequence prior to the main challenge experiment in the present study.…”

“…Following the incubation period, three serial passages of supernatant were performed (P2‐P4). To quantify viral RNA from the P1‐P4 supernatants, RT‐qPCRs were performed as previously described (Aksnes et al., 2020). Viral RNA from infected cells (P4) was also Sanger‐sequenced following PCR to verify that the mutations were present.…”

“…Viral RNA from infected cells (P4) was also Sanger‐sequenced following PCR to verify that the mutations were present. Propagation and quantification (TCID 50 ) of isolates were performed with CHH‐1 cells using standard techniques as previously described (Aksnes et al., 2020).…”

“…Studies have shown that alterations of glycosylation motifs, and thus prevention of glycan attachment, may alter the infectivity of alphaviruses (Knight et al., 2009; Nelson et al., 2016). Previously, we have shown that a mutation in the predicted N‐linked glycosylation site in E2 attenuated SAV3 replication properties in cell culture, causing less cytopathogenic effects and production of infectious virus, while similar mutations in glycosylation site in E1 inactivated the virus (Aksnes et al., 2020). In addition, the capsid has been shown to be associated with inhibition of cellular proliferation, assumed to be important for the cytopathic effect caused by SAV (Karlsen, Yousaf, et al., 2010).…”