Identification and Characterization of Viral Structural Proteins of Infectious Salmon Anemia Virus

Abstract

Infectious salmon anemia virus (ISAV) is an unclassifiedOrthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42-and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-3 H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.

First field evidence of the evolution from a non-virulent HPR0 to a virulent HPR-deleted infectious salmon anaemia virus

“…Atlantic salmon kidney (ASK) cells [64] were cultured and used for virus propagation as outlined previously [14,65]. For primary virus isolation, cells in 24-well tissue culture plates (Nunc) were inoculated and inspected every 2 days for development of CPE.…”

Comparative aspects of infectious salmon anemia virus, an orthomyxovirus of fish, to influenza viruses

“…ISAV has a morphology resembling that of orthomyxoviruses [12][13][14]. Like the FluA and FluB the ISAV genome consists of eight single-stranded RNA segments of negative polarity [15][16][17].…”

“…receptor binding), receptor release and fusion activities. However, these activities are associated with two surface proteins called the hemaglutinin-esterase (HE) and the fusion (F) glycoproteins [14].…”

Completion of the full-length genome sequence of the infectious salmon anemia virus, an aquatic orthomyxovirus-like, and characterization of mAbs

“…ISAV is an enveloped virus composed of four major structural proteins: the nucleoprotein (66-72 kDa), the matrix protein (22-25 kDa) and two envelope glycoproteins that are the haemagglutinin-esterase glycoprotein (42 kDa) and the fusion protein (47-50 kDa) (Clouthier et al, 2002;Falk et al, 2004). Sequence characterization of ISAV genome segments is almost fully achieved, except for several segment end UTRs (Sandvik et al, 2000).…”

“…The ISAV genome encodes at least 10 proteins. ISAV genome segments 1-6 encode one protein each and by analogy to other orthomyxoviruses and additional characterizations, the ISAV proteins are as follows: PB2, component of RNA polymerase (Snow et al, 2003); PB1, RNA-dependent RNA polymerase core motif (Krossoy et al, 1999); NP, nucleoprotein (Aspehaug et al, 2004;Ritchie et al, 2001;); PA; component of RNA polymerase (Ritchie et al, 2001); F, fusion protein (Aspehaug et al, 2005;Clouthier et al, 2002), and HE, haemagglutininesterase glycoprotein containing both receptor-binding and receptor-destroying enzyme domains (Hellebo et al, 2004; Kristiansen et al, 2002;Krossoy et al, 2001;Rimstad et al, 2001). Similar to influenza viruses, the two smallest ISAV The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are HQ259671-HQ259678.…”