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.

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A common strategy in the cellular response to stress signals is to shut down protein synthesis [1]. The cell contains several different latent translation initiation factor 2 alpha (eIF2a)-kinases that phosphorylate the a-subunit of the translation initiation factor eIF2 upon certain stimuli, such as viral infection, amino acid starvation, heme-depletion, and accumulation of misfolded proteins in the endoplasmic reticulum, leading to inhibition of protein synthesis [2]. Under normal conditions, eIF2 associates with GTP and Met-tRNA i to form a complex with the 40S ribosomal subunit [3]. As initiation proceeds, eIF2 responds to alignment of the initiator tRNA with the initiation codon by initiating hydrolysis of GTP and releasing Met-tRNA i into the partial P-site. Recycling of eIF2-GDP to the active GTP-bound form requires the GTP exchange factor, The translation initiation factor 2 alpha (eIF2a)-kinase, dsRNA-activated protein kinase (PKR), constitutes one of the major antiviral proteins activated by viral infection of vertebrates. PKR is activated by viral double-stranded RNA and subsequently phosphorylates the a-subunit of translation initiation factor eIF2. This results in overall down regulation of protein synthesis in the cell and inhibition of viral replication. Fish appear to have a PKR-like protein that has Z-DNA binding domains instead of dsRNA binding domains in the regulatory domain, and has thus been termed Z-DNA binding protein kinase (PKZ). We present the cloning of the Atlantic salmon PKZ cDNA and show its upregulation by interferon in Atlantic salmon TO cells and poly inosinic poly cytodylic acid in head kidney. We also demonstrate that recombinant Atlantic salmon PKZ, expressed in Escherichia coli, phosphorylates eIF2a in vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2a. PKZ activity, as measured by phosphorylation of eIF2a, was increased after addition of Z-DNA, but not by dsRNA. In addition, we show that wild-type Atlantic salmon PKZ, but not the kinase defective variant K217R, has a direct inhibitory effect on protein synthesis after transient expression in Chinook salmon embryo cells. Overall, the results support a role for PKZ, like PKR, in host defense against virus infection.Abbreviations As, Atlantic salmon; As-IFN, Atlantic salmon interferon; b-gal, b-galactosidase; CHSE, Chinook salmon embryonic; dsRBD, dsRNA binding domain; EGFP, enhanced green fluorescence protein; eIF2a, translation initiation factor 2 alpha; EST, expressed sequence tag; Hu, human; IFN, interferon; PERK, protein kinase-like endoplasmic reticulum eIF2a kinase; PKR, dsRNA-activated protein kinase; PKZ, Z-DNA binding protein kinase; poly dG:dC, poly deoxyguanosine poly deoxycytidine acid; poly I:C, poly inosinic poly cytodylic acid; SG, stress granule; ZBP1, Z-DNA binding protein 1.

Bacterial resistance is compromising the use of β-lactam antibiotics including carbapenems. The main resistance mechanism against β-lactams is hydrolysis of the β-lactam ring mediated by serine- or metallo-β-lactamases (MBLs). Although several inhibitors of MBLs have been reported, none has been developed into a clinically useful inhibitor. Mercaptocarboxylic acids are among the most prominent scaffolds reported as MBL inhibitors. In this study, the carboxylate group of mercaptocarboxylic acids was replaced with bioisosteric groups like phosphonate esters, phosphonic acids and NH-tetrazoles. The influence of the replacement on the bioactivity and inhibitor binding was evaluated. A series of bioisosteres of previously reported inhibitors was synthesized and evaluated against the MBLs VIM-2, NDM-1 and GIM-1. The most active inhibitors combined a mercapto group and a phosphonate ester or acid, with two/three carbon chains connecting a phenyl group. Surprisingly, also compounds containing thioacetate groups instead of thiols showed low IC values. High-resolution crystal structures of three inhibitors in complex with VIM-2 revealed hydrophobic interactions for the diethyl groups in the phosphonate ester (inhibitor 2b), the mercapto bridging the two active site zinc ions, and tight stacking of the benzene ring to the inhibitor between Phe62, Tyr67, Arg228 and His263. The inhibitors show reduced enzyme activity in Escherichia coli cells harboring MBL. The obtained results will be useful for further structural guided design of MBL inhibitors.

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 m2 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-b signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins l1 and s2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins m1, s1 and s3 suggested differences regarding cellular interactions between the reovirus genera. However, for s1, amino acid residues central for MRV binding to sialic acids, and cleavage-and myristoylation sites in m1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV s3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding s3 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.