Infectious salmon anemia virus (ISAV) genomic segment 3 encodes the viral nucleoprotein (NP), an RNA-binding protein with two monopartite nuclear localization signals (NLS)

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Infectious salmon anemia virus (ISAV) is an orthomyxovirus causing serious disease in Atlantic salmon(Salmo salar L.). This study presents the characterization of the ISAV 50-kDa glycoprotein encoded by segment 5, here termed the viral membrane fusion protein (F). This is the first description of a separate orthomyxovirus F protein, and to our knowledge, the first pH-dependent separate viral F protein described. The ISAV F protein is synthesized as a precursor protein, F 0 , that is proteolytically cleaved to F 1 and F 2 , which are held together by disulfide bridges. The cleaved protein is in a metastable, fusion-activated state that can be triggered by low pH, high temperature, or a high concentration of urea. Cell-cell fusion can be initiated by treatment with trypsin and low pH of ISAV-infected cells and of transfected cells expressing F, although the coexpression of ISAV HE significantly improves fusion. Fusion is initiated at pH 5.4 to 5.6, and the fusion process is coincident with the trimerization of the F protein, or most likely a stabilization of the trimer, suggesting that it represents the formation of the fusogenic structure. Exposure to trypsin and a low pH prior to infection inactivated the virus, demonstrating the nonreversibility of this conformational change. Sequence analyses identified a potential coiled coil and a fusion peptide. Size estimates of F 1 and F 2 and the localization of the putative fusion peptide and theoretical trypsin cleavage sites suggest that the proteolytic cleavage site is after residue K 276 in the protein sequence. Infectious salmon anemia virus (ISAV) is an enveloped virusbelonging to the family Orthomyxoviridae and the genus Isavirus, and it causes serious disease in Atlantic salmon (Salmo salar L.) (51,58,68,71,80). The ISAV genome is composed of eight negative-sense, single-stranded RNA segments, and while nucleotide sequences of all segments have been published (9,43,44,56,66,67,74,75), much remains to be elucidated with respect to protein identification and characterization. A total of 12 proteins have been detected by immunoprecipitation of lysates from radiolabeled infected cells (40), while four major structural proteins have been recognized in purified ISAV particles, including the matrix (M1; 22 to 24 kDa) (7, 24), the nucleoprotein (NP; 66 to 71 kDa) (3, 24), and two membrane glycoproteins (24). The receptor-binding and receptor-destroying activities are associated with the 42-kDa glycoprotein encoded by segment 6, termed the hemagglutininesterase (HE) (24,25,34,42,43,66), while the activities of the second glycoprotein, glycoprotein 50 (gp50), encoded by segment 5 (9), have not been described previously.ISAV pursues a nuclear replication strategy similar to that of the influenza viruses (3, 26) which is initiated by receptor binding and internalization into cellular endosomes, where the viral and cellular membranes fuse in response to low pH (21). The addition of trypsin to the culture medium during ISAV replication has been demonstrated to have a benefici...

Section: Resultssupporting

confidence: 74%

Section: Infectious Salmon Anemia Virus (Isav) Is An Enveloped Virusmentioning

confidence: 99%

Section: Infectious Salmon Anemia Virus (Isav) Is An Enveloped Virusmentioning

confidence: 99%

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Characterization of the Infectious Salmon Anemia Virus Fusion Protein

Aspehaug

Mikalsen

Snow

et al. 2005

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119

“…The function of most proteins encoded by the segments of ISAV has been assigned according to their similarities to the proteins encoded by the influenza A virus. Thus, polymerase, which in influenza A virus synthesizes both mRNA and vRNA, is constituted by three putative proteins coded by segment 1 (polymerase basic 2, PB2 [72]), segment 2 (polymerase basic 1, PB1 [41]), and segment 4 (polymerase acid, PA [3,64,72]). Segment 3 codes for nucleoprotein, NP, which participates in vRNA transport to the nucleus (3).…”

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confidence: 99%

Bioinformatic Analysis of the Genome of Infectious Salmon Anemia Virus Associated with Outbreaks with High Mortality in Chile

Cottet

Martı́n

Tello

et al. 2010

The infectious salmon anemia virus (ISAV), an orthomyxovirus, is the major cause of outbreaks of high mortality rates in salmon in Chile. It has been proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase and fusion glycoproteins. However, based on current information, the contribution of other viral genes cannot be ruled out. To study this, we isolated and determined the complete coding sequence of two high-prevalence Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and ISAV901_09. These isolates were compared to 15 Norwegian isolates that exhibit differences in their virulence. For this purpose, we performed bioinformatic analyses of (i) functional domains, (ii) specific mutations, (iii) Bayesian phylogenetics, and (iv) structural comparisons between ISAV and influenza virus glycoproteins by using molecular modeling. Phylogenetic analysis shows two genogroups for each protein, one of them containing the Chilean isolates. The gene sequence of the polymerase complex and nucleoprotein indicated that they are closely related to homologues from highly pathogenic Norwegian viruses. Notably, seven of the eight mutations that are present only in the Chilean isolates are on the polymerase complex and nucleoprotein. Structural modeling of hemagglutinin-esterase shows patches of variable residues on its surface. Fusion protein modeling shows that insertions are flexible regions that could affect proteolytic processing, increasing either the accessibility or the number of recognition sites for specific proteases. We found antigenic drift processes related to insertion into the isolated segment 5 of the ISAV752_09. Our results confirm the European origin of Chilean isolates to be the result of reassortments from Norwegian ancestors.

“…The functions of most proteins encoded by the segments of ISAV have been assigned according to their similarities to the proteins encoded by influenza A virus, suggesting that ISAV uses its own polymerase to copy and transcribe its genome. Thus, ISAV polymerase synthesizes both mRNA and viral RNA (vRNA) and seems to be constituted by three subunits: polymerase basic 2 (PB2), encoded by segment 1 (43); polymerase basic 1 (PB1), encoded by segment 2 (24); and polymerase acid (PA), encoded by segment 4 (3,38,43). Moreover, as with influenza virus, RNA replication and transcription occur in the cell nucleus, while mRNA and vRNA are transported to the cytoplasm for translation and packaging, respectively (18).…”

mentioning

confidence: 99%

Inhibitory Effect of a Nucleotide Analog on Infectious Salmon Anemia Virus Infection

Rivas‐Aravena

Vallejos‐Vidal²,

Martı́n

et al. 2011

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-␤-D-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC 50 ) of 0.02 g/ml and an IC 90 of 0.4 g/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo.