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Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus.

Kibenge FS, Xu H, Kibenge MJ, Qian B, Joseph T - Virol. J. (2007)

Bottom Line: Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge.The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals.The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada. kibenge@upei.ca

ABSTRACT

Background: Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV.

Results: In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the same antiserum revealed the protein(s) to be localized in the cytoplasm. Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge.

Conclusion: Collectively, our observations suggest that the product of ISAV segment 7 primary transcript (7-ORF1) is a structural protein. The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals. The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.

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Autoradiograph of SDS-PAGE of expressed proteins labeled with [35S]methionine in a coupled in vitro TNT System with the different constructs in pCR®II-TOPO® plasmid and in TO cells infected with ISAV as in Fig. 1, immunoprecipitated with rabbit antiserum to purified ISAV. The prestained protein standards (Invitrogen Life Sciences) are drawn in on the left while corresponding molecular masses of immunoprecipitated ISAV proteins are indicated on the right. (A) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ORFs in ISAV segments 3–6, respectively. (B) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ISAV segment 7 ORFs 1 and 2 and segment 8 ORFs 1 and 2, respectively.
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Figure 2: Autoradiograph of SDS-PAGE of expressed proteins labeled with [35S]methionine in a coupled in vitro TNT System with the different constructs in pCR®II-TOPO® plasmid and in TO cells infected with ISAV as in Fig. 1, immunoprecipitated with rabbit antiserum to purified ISAV. The prestained protein standards (Invitrogen Life Sciences) are drawn in on the left while corresponding molecular masses of immunoprecipitated ISAV proteins are indicated on the right. (A) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ORFs in ISAV segments 3–6, respectively. (B) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ISAV segment 7 ORFs 1 and 2 and segment 8 ORFs 1 and 2, respectively.

Mentions: To eliminate the background bands in ISAV-infected cell preparations and in vitro translation products and identify all the structural viral proteins, we carried out immunoprecipitation analysis with rabbit antiserum against purified whole ISAV. Analysis of the immunoprecipitates revealed up to six major protein bands in ISAV-infected TO cells consisting of the 77 kDa, 44.5 kDa, 37 kDa, 30.5 kDa, 23.5 kDa, and 16.5 kDa proteins, with the most intense bands being of the 77 kDa and 23.5 kDa proteins. The 77 kDa, 44.5 kDa, 37 kDa, and 23.5 kDa proteins co-migrated with the gene products of segments 3, 5, 6, and 8 ORF2, respectively (Figs 2A and 2B), which in purified ISAV correspond to NP, F, HE, and M proteins, respectively. The 30.5 and 16.5 kDa proteins in immunoprecipitates of ISAV-infected TO cells corresponded to segment 7 ORFs 1 and 2 products, of 32.5 and 16.0 kDa respectively, which were also immunoprecipitated with whole ISAV antiserum (Fig. 2B). Indeed in gels ran with different sets of protein molecular weight standards, the corresponding immunoprecipitates of ISAV-infected TO cells were 32 and 18 kDa, respectively, indicating that one or both segment 7 products are components of ISAV virions. We previously reported a 29–30 kDa protein which was immunoprecipitated in different infected TO cell lysates for 5 different ISAV strains using the same antiserum against purified ISAV as in the present study [15]. But others reported the segment 7 ORF 1 encoded protein not to react with ISAV antiserum, and it was concluded that it was either non-structural or a minor structural protein [12]. However, the fact that both segment 7 ORFs 1 and 2 products could be immunoprecipitated with antiserum against purified ISAV in the present study indicates that they are components of ISAV virions. We conclude from these experiments that (a) the four major polypeptides of 71 kDa, 53 kDa, 43 kDa, and 24 kDa, observed in gel electrophoresis of purified ISAV [16,17] correspond to products of segments 3, 5, 6, and 8-ORF2, respectively, and (b) only the products of segments 5, 6, and 7-ORF1 are glycosylated when expressed in presence of canine microsomal membranes, suggesting that the 7-ORF1 product could also be a membrane protein. The segments 5 and 6 products correspond to the two previously reported surface glycoproteins of ISAV, the F protein [9] and the HE protein [7], respectively.


Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus.

Kibenge FS, Xu H, Kibenge MJ, Qian B, Joseph T - Virol. J. (2007)

Autoradiograph of SDS-PAGE of expressed proteins labeled with [35S]methionine in a coupled in vitro TNT System with the different constructs in pCR®II-TOPO® plasmid and in TO cells infected with ISAV as in Fig. 1, immunoprecipitated with rabbit antiserum to purified ISAV. The prestained protein standards (Invitrogen Life Sciences) are drawn in on the left while corresponding molecular masses of immunoprecipitated ISAV proteins are indicated on the right. (A) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ORFs in ISAV segments 3–6, respectively. (B) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ISAV segment 7 ORFs 1 and 2 and segment 8 ORFs 1 and 2, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851003&req=5

Figure 2: Autoradiograph of SDS-PAGE of expressed proteins labeled with [35S]methionine in a coupled in vitro TNT System with the different constructs in pCR®II-TOPO® plasmid and in TO cells infected with ISAV as in Fig. 1, immunoprecipitated with rabbit antiserum to purified ISAV. The prestained protein standards (Invitrogen Life Sciences) are drawn in on the left while corresponding molecular masses of immunoprecipitated ISAV proteins are indicated on the right. (A) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ORFs in ISAV segments 3–6, respectively. (B) Lanes 1 to 8 contain radiolabeled proteins synthesized in uninfected TO cells, ISAV-infected TO cells at 24, 48 and 96 hr post-infection, and in TNT System with constructs of ISAV segment 7 ORFs 1 and 2 and segment 8 ORFs 1 and 2, respectively.
Mentions: To eliminate the background bands in ISAV-infected cell preparations and in vitro translation products and identify all the structural viral proteins, we carried out immunoprecipitation analysis with rabbit antiserum against purified whole ISAV. Analysis of the immunoprecipitates revealed up to six major protein bands in ISAV-infected TO cells consisting of the 77 kDa, 44.5 kDa, 37 kDa, 30.5 kDa, 23.5 kDa, and 16.5 kDa proteins, with the most intense bands being of the 77 kDa and 23.5 kDa proteins. The 77 kDa, 44.5 kDa, 37 kDa, and 23.5 kDa proteins co-migrated with the gene products of segments 3, 5, 6, and 8 ORF2, respectively (Figs 2A and 2B), which in purified ISAV correspond to NP, F, HE, and M proteins, respectively. The 30.5 and 16.5 kDa proteins in immunoprecipitates of ISAV-infected TO cells corresponded to segment 7 ORFs 1 and 2 products, of 32.5 and 16.0 kDa respectively, which were also immunoprecipitated with whole ISAV antiserum (Fig. 2B). Indeed in gels ran with different sets of protein molecular weight standards, the corresponding immunoprecipitates of ISAV-infected TO cells were 32 and 18 kDa, respectively, indicating that one or both segment 7 products are components of ISAV virions. We previously reported a 29–30 kDa protein which was immunoprecipitated in different infected TO cell lysates for 5 different ISAV strains using the same antiserum against purified ISAV as in the present study [15]. But others reported the segment 7 ORF 1 encoded protein not to react with ISAV antiserum, and it was concluded that it was either non-structural or a minor structural protein [12]. However, the fact that both segment 7 ORFs 1 and 2 products could be immunoprecipitated with antiserum against purified ISAV in the present study indicates that they are components of ISAV virions. We conclude from these experiments that (a) the four major polypeptides of 71 kDa, 53 kDa, 43 kDa, and 24 kDa, observed in gel electrophoresis of purified ISAV [16,17] correspond to products of segments 3, 5, 6, and 8-ORF2, respectively, and (b) only the products of segments 5, 6, and 7-ORF1 are glycosylated when expressed in presence of canine microsomal membranes, suggesting that the 7-ORF1 product could also be a membrane protein. The segments 5 and 6 products correspond to the two previously reported surface glycoproteins of ISAV, the F protein [9] and the HE protein [7], respectively.

Bottom Line: Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge.The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals.The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada. kibenge@upei.ca

ABSTRACT

Background: Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV.

Results: In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the same antiserum revealed the protein(s) to be localized in the cytoplasm. Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge.

Conclusion: Collectively, our observations suggest that the product of ISAV segment 7 primary transcript (7-ORF1) is a structural protein. The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals. The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.

Show MeSH
Related in: MedlinePlus