Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Abstract

Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.Rhabdoviruses cause highly damaging diseases in fish reared by the worldwide salmon culture industry. Among fish rhabdoviruses (novirhabdoviruses), the viral hemorrhagic septicemia virus (VHSV), which originated in Europe but has recently been found in America (26, 41), affects not only salmonids but also cod, turbot (38), sea bass, eels, flatfish, and shrimp (8,24,27). Despite many efforts, including successful DNA vaccines at the laboratory level, a commercial vaccine against VHSV is not yet available (1,18,25,30).The whole genome of VHSV has been sequenced (23), and the disulfide structure of its protein G (pG) has been elucidated (12). Reverse genetic methods have been developed for the VHSV-related rhabdovirus infectious hematopoietic necrosis virus (5-7), which could allow the design of new vaccines. The study of rhabdovirus fusion in the VHSV model could be important in the design of alternative vaccines to fight these diseases.The pG mutants of vesicular stomatitis virus (VSV), a wellstudied mammalian rhabdovirus, with an altered extent of fusion and/or optimal fusion pH in vitro, had mutations located either in the fusion peptide or in carboxy-terminal regions affecting the low-pH conformational changes required for fusion (39,40,43). Alignment of the pG sequence of VSV with those of 14 o...

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

“…Indeed, these loops are much less hydrophobic than the amino-terminal fusion peptides of class I proteins (even when the three fusion domains of G are grouped together in the post-fusion conformation). That these loops are indeed an essential part of the membrane interacting motif is consistent with previous mutagenesis work performed on rhabdoviruses [59,60] (Table 1) and has since been confirmed for both VSV G [61] (Table 1) and Herpesviruses gB [62,63].…”

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

“…Because the highest sequence variability on the protein G of VHSV was localized in amino acid 245e300 [2,4], new frg15 or frg16-like recombinants which will avoid the 245e300 hypervariable region, might be required to increase the scope of VHSV isolates which could be detected by those frgs. On the other hand, most of the variability found in frg11 was only restricted to 2 short amino acid stretches [29] and therefore frg11 might detect more isolates than frg15 or frg16. An extensive work with more VHSV isolates and/or with the corresponding different sequence variations in frgs, will have to be performed to demonstrate their ability to detect Abs in trout infected with any VHSV isolate.…”

“…3, data not differentially labelled). Because frg11 was the smallest G frg located at a region with the lowest amino acid variability [2,4,29] and showed the lowest background values, it was chosen to carry out the rest of the analysis with this frg. Fig.…”

Interferon response following infection with genetically similar isolates of viral haemorrhagic septicaemia virus (VHSV) exhibiting contrasting virulence in rainbow trout

“…Using a model devised by Walker & Kongsuwan [47], Rocha et al [48] proposed hypothetical fusion peptides are positioned between residues 142 and 159. While the G gene from DK-F1 and DK-M.Rhabdo isolates are reasonably conserved, there are still 10 amino-acid (1.97%) substitutions between isolates at residues 7, 15, 46, 217, 270, 288, 373, 388, 446 and 506, critically revealing no substitutions within the proposed fusogenic region proposed by Rocha et al [48]. Thus, in comparative terms the absence of mutations within this domain between isolates seemingly suggests the fusogenic potential of the marine isolate is not being impaired, therefore the inability of the marine isolate to successfully achieve cellular entry and replication remains to be determined.…”