Sequence analysis of the genome of piscine orthoreovirus (PRV) associated with heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar)

Abstract

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Figure 1. The PRV genome.

Gene segments are assigned according to mammalian reoviruses. Open reading frames (ORFs) and putative encoded proteins are indicated by regions in grey, with start and end positions indicated. Non-translated regions (UTR’s) at gene segment ends are shown in black. Gene segments L2, S1 and S2 are possibly polycistronic.

Figure 2. Expression and subcellular localization of the S1-encoded σ3 and p13 proteins in mammalian VERO and salmonid CHSE cells.

Immunofluorescent staining of σ3 or p13 (green colour), and staining with the trans-Golgi marker WGA (red colour). Transfected VERO cells (A) and CHSE cells (B) expressing both σ3 and p13 from the large S1 ORF (upper panels), and p13 expression from the S1 internal ORF (lower panels). Nuclei are stained with DAPI (blue colour). Yellow colour indicates colocalization of p13 and WGA. Non-transfected cells stained with WGA and anti-p13 serum was used as controls (CTRL).

Figure 3. PSIPRED secondary structure predictions of PRV σ2 (green) and the homologous proteins from the reovirus prototype strains MRV T3D (σ2, pink), ARV-138 (σA, blue) and GCRV-873 (VP6, brown).

Figure 4. Multiple sequence alignment of PRV σ1 with MRV T3D σ1.

Black lines represent putative nuclear export signals (NEP) in MRV and PRV, respectively, as predicted by NetNes 1.1. ▪ = L₁₄₉ in the MRV protein involved in a second predicted NES. ▴ = residues in the MRV protein involved in binding to sialic acid residues. The alignment has been manually adjusted.