Identification and characterization of viral structural proteins of infectious salmon anemia virus

Abstract

Infectious salmon anemia virus (ISAV) is an unclassified Orthomyxovirus 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.

FIG. 1.

(a) Autoradiograms of [³⁵S]methionine-labeled ISAV proteins resolved in SDS-12.5% polyacrylamide gels. Lanes: 1, whole virus purified by sucrose gradient centrifugation; 2, whole virus purified by affinity purification with immunomagnetic beads and an anti-HA MAb; 3, immunoprecipitation of cell lysates from virus-infected cell cultures with an anti-whole virus antibody (30- and 82-kDa proteins are indicated by asterisks); 4, control lysates of [³⁵S]methionine-labeled cells. (b) Detection of glycoproteins. Lanes: 1, Western blot of purified virus stained with biotinylated concanavalin A; 2, immunoprecipitation of cell lysates from virus-infected cells labeled with [³H]mannose; 3, control lysates of [³H]mannose-labeled cells; 4, [³⁵S]methionine-labeled ISAV proteins were immunoprecipitated, followed by deglycosylation; 5, nondeglycosylated control preparation; 6, control lysates of [³⁵S]methionine-labeled cells. (c) Detection of phosphoproteins. Lanes: 1, immunoprecipitation of cell lysates from virus-infected cells labeled with ³²P by using an anti-whole virus antibody; 2, control lysates of ³²P-labeled cells.

FIG. 2.

(a) Effect of trypsin treatment of immunoprecipitated [³⁵S]methionine-labeled ISAV HA. Following treatment, proteins were resolved in SDS-12.5% polyacrylamide gels. Lanes: 1, trypsin treatment of proteins immunoprecipitated with an anti-HA MAb; 2, nontreated control of proteins in lane 1; 3, nonreduced sample of trypsin-treated proteins in lane 1; 4, nontreated control of proteins in lane 3. (b) SDS-8 to 18% polyacrylamide gels of nonreduced, non-heat-treated samples of the ISAV HA protein immunoprecipitated from [³⁵S]methionine-labeled ISAV cell lysate, demonstrating that the HA protein exists at least as dimers and probably as trimers (lane 1). Lane 2 is included for reference and contains proteins immunoprecipitated with a polyclonal anti-whole virus antibody.

FIG. 3.

Dissociation of ISAV with NP-40. [³⁵S]methionine-labeled virus was purified by sucrose gradient centrifugation, treated with 1% NP-40, loaded onto a 20% sucrose cushion, and centrifuged. The supernatant (lanes 1) and pellet (lanes 2) were then examined by SDS-12.5% PAGE. (a) Autoradiograms of the two fractions. (b) Western blot analysis of the two fractions stained with a PAb to recombinant vp22. Lane 3 is included for reference and contains purified virus stained with a polyclonal anti-whole virus antibody.

FIG. 4.

Immunofluorescent staining of ISAV-infected ASK cells with PAbs to recombinant vp22 (green) (a and b) and whole virus (green) (c and d). All preparations were double stained with the anti-HA MAb (red). Neither vp22 nor HA was detected at 12 h p.i. (a and c), while virus protein was detected in the nucleus (c). At 24 h p.i., vp22 was detected in the nucleus (b), while HA was detected in the cytoplasm (b and d). The nuclear staining in panel d probably represents both the nucleoprotein and vp22.

FIG. 5.

Inhibition of virus elution by DFP and DCIC. Four HA units of virus was incubated with either 0.1 to 5 mM DFP or 12.5 to 100 μM DCIC, followed by addition of 0.75% rabbit RBC. Controls: 0, no inhibitor; C, no virus. (a) Incubation for 1 h, showing complete agglutination in all wells. (b) After 6 h of incubation, the agglutination had eluted in the control wells but not in wells containing esterase inhibitors.

FIG. 6.

Analysis of [1,3-3H]DFP-labeled ISAV protein. Purified ISAV was incubated with [1,3-³H]DFP for 1 h at room temperature, followed by an overnight incubation at 4°C. Viral proteins were analyzed by electrophoresis on a reducing SDS-12% polyacrylamide gel (a) or a nonreducing SDS-8% polyacrylamide gel (b). The gels were then cut into two pieces, and one part of each gel was blotted onto nitrocellulose and probed with ISAV-specific antiserum. The rest of the gel was incubated with Amplify (Amersham-Pharmacia), dried, and subjected to fluorography. Lanes: 1, Western blot; 2, [1,3-³H]DFP.

FIG. 7.

Alignment of amino acid sequences around the active-site residues. The HA sequences of ISAV Glesvaer/2/90 and ISAV Bay of Fundy/97 were aligned by using the CLUSTAL algorithm with the sequences around the active-site serine residue (A) and Asp-352/His-355 (B) of the HE proteins of influenza C virus, mouse hepatitis virus strain JHM (MHV JHM), bovine coronavirus (BCoV) strain Mebus, and Breda torovirus. Amino acid residues identical to the consensus sequence are in boldface, and gaps are indicated by hyphens. Amino acid residues are numbered according to the GenBank entries. Active-site residues are marked by asterisks.