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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.

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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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New gene expression model for segment 7 of ISAV of North American genotype. There are 3 ORFs consisting of the primary transcript (7-ORF1) 300 amino acids long, and 7-ORF1/2 and 7ORF1/3 each with an intron removed from 7-ORF1 and consisting of 159 and 81 amino acids, respectively.
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Figure 7: New gene expression model for segment 7 of ISAV of North American genotype. There are 3 ORFs consisting of the primary transcript (7-ORF1) 300 amino acids long, and 7-ORF1/2 and 7ORF1/3 each with an intron removed from 7-ORF1 and consisting of 159 and 81 amino acids, respectively.

Mentions: The rabbit antiserum against the GST-7 ORF1 fusion protein was also used to directly monitor the synthesis of the 7-ORF1 protein in ISAV-infected cells in cell culture. For this, TO cell monolayers were infected with ISAV isolate RPC/NB 980-049-1 at a multiplicity of infection of 10 and individual flasks were removed daily from 1–5 days and were radiolabeled for 20 hr with 100 μCi of [35S]methionine. The cell monolayers were then harvested using RIPA buffer and were then subjected to immunoprecipitation with the rabbit antiserum against the GST-7 ORF1 fusion protein. Four individual protein bands of 68, 32, 18, and 10.6 kDa were detected in ISAV-infected cell lysates but not in uninfected cell controls (Fig. 6). The 68-kDa band corresponds to the ISAV NP protein [7]; cross-reactivity with the antiserum against GST-7 ORF1 fusion protein was surprising since there is no amino acid sequence homology between NP and 7-ORF1 proteins. It is possible that the two proteins have a similar conformation epitope. We have noted a similar cross-reactivity with a monoclonal antibody against the ISAV NP protein [25]. The 32-kDa protein was immunoprecipitated from lysates of the ISAV-infected TO cells throughout the sampling period (Fig. 6, lanes 2–6); the protein is comparable in size to that immunoprecipitated from ISAV-infected TO cell lysates using antiserum against purified ISAV (Fig. 2B, lanes 2–4). Thus the molecular mass of 35 kDa for the viral protein in the purified virus (Fig. 3C, lane 1) is larger than the segment 7 ORF1 in the infected cells and the in vitro transcription-translation product, and we consider this additional evidence for the post-translational modifications of the mature segment 7 ORF 1 protein. The 18-kDa protein was seen only at 1 and 2 days (Fig. 6, lanes 2–3) whereas the 10.6 kDa protein was present only at day 2 (Fig. 6, lane 3) of sampling. We speculate that these two proteins are also encoded by ISAV segment 7 and the cross-reactivity with the GST-7 ORF1 antiserum is because they share the N-terminal 22 amino acids of the 7-ORF1 protein. Previously, only the segment 7 ORF2 product was suggested to have this sequence [10], and would correspond to the 18-kDa protein in our study. We now show for the first time that a third protein of 10.6 kDa may have similar sequence. This spliced protein would consist of the first 22 amino acids of the 7-ORF1 product and a 257 nucleotide intron spliced out so that the translation continues in the +3 reading frame for a total of 81 amino acids (aa), and a predicted molecular weight of 9.5 kDa. This is a new putative ISAV protein never before reported, as a result of which we have also proposed a new gene expression model of ISAV segment 7 (Fig. 7), distinct from the coding strategy of segment 7 of influenza A virus. Analysis of 10 ISAV isolates for which we have full-length sequences of the 7-ORF1 gene, ISAV isolates RPC/NB 98-049-1, RPC/NB 02-1179-4, RPC/NB 98-0280-2, RPC/NB 02-0775-14, 7833-1, RPC/NB 01-0593-1, RPC/NB 01-0973-3, RPC/NB 04-085-1, 485/9/97, and 390/98 revealed that the segment 7-ORF1/3 polypeptide of 81 aa is present in all ISAV isolates of the North American genotype, whereas it terminates after 62 or 32 aa in ISAV isolates of the European genotype (Table 4).


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)

New gene expression model for segment 7 of ISAV of North American genotype. There are 3 ORFs consisting of the primary transcript (7-ORF1) 300 amino acids long, and 7-ORF1/2 and 7ORF1/3 each with an intron removed from 7-ORF1 and consisting of 159 and 81 amino acids, respectively.
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Related In: Results  -  Collection

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Figure 7: New gene expression model for segment 7 of ISAV of North American genotype. There are 3 ORFs consisting of the primary transcript (7-ORF1) 300 amino acids long, and 7-ORF1/2 and 7ORF1/3 each with an intron removed from 7-ORF1 and consisting of 159 and 81 amino acids, respectively.
Mentions: The rabbit antiserum against the GST-7 ORF1 fusion protein was also used to directly monitor the synthesis of the 7-ORF1 protein in ISAV-infected cells in cell culture. For this, TO cell monolayers were infected with ISAV isolate RPC/NB 980-049-1 at a multiplicity of infection of 10 and individual flasks were removed daily from 1–5 days and were radiolabeled for 20 hr with 100 μCi of [35S]methionine. The cell monolayers were then harvested using RIPA buffer and were then subjected to immunoprecipitation with the rabbit antiserum against the GST-7 ORF1 fusion protein. Four individual protein bands of 68, 32, 18, and 10.6 kDa were detected in ISAV-infected cell lysates but not in uninfected cell controls (Fig. 6). The 68-kDa band corresponds to the ISAV NP protein [7]; cross-reactivity with the antiserum against GST-7 ORF1 fusion protein was surprising since there is no amino acid sequence homology between NP and 7-ORF1 proteins. It is possible that the two proteins have a similar conformation epitope. We have noted a similar cross-reactivity with a monoclonal antibody against the ISAV NP protein [25]. The 32-kDa protein was immunoprecipitated from lysates of the ISAV-infected TO cells throughout the sampling period (Fig. 6, lanes 2–6); the protein is comparable in size to that immunoprecipitated from ISAV-infected TO cell lysates using antiserum against purified ISAV (Fig. 2B, lanes 2–4). Thus the molecular mass of 35 kDa for the viral protein in the purified virus (Fig. 3C, lane 1) is larger than the segment 7 ORF1 in the infected cells and the in vitro transcription-translation product, and we consider this additional evidence for the post-translational modifications of the mature segment 7 ORF 1 protein. The 18-kDa protein was seen only at 1 and 2 days (Fig. 6, lanes 2–3) whereas the 10.6 kDa protein was present only at day 2 (Fig. 6, lane 3) of sampling. We speculate that these two proteins are also encoded by ISAV segment 7 and the cross-reactivity with the GST-7 ORF1 antiserum is because they share the N-terminal 22 amino acids of the 7-ORF1 protein. Previously, only the segment 7 ORF2 product was suggested to have this sequence [10], and would correspond to the 18-kDa protein in our study. We now show for the first time that a third protein of 10.6 kDa may have similar sequence. This spliced protein would consist of the first 22 amino acids of the 7-ORF1 product and a 257 nucleotide intron spliced out so that the translation continues in the +3 reading frame for a total of 81 amino acids (aa), and a predicted molecular weight of 9.5 kDa. This is a new putative ISAV protein never before reported, as a result of which we have also proposed a new gene expression model of ISAV segment 7 (Fig. 7), distinct from the coding strategy of segment 7 of influenza A virus. Analysis of 10 ISAV isolates for which we have full-length sequences of the 7-ORF1 gene, ISAV isolates RPC/NB 98-049-1, RPC/NB 02-1179-4, RPC/NB 98-0280-2, RPC/NB 02-0775-14, 7833-1, RPC/NB 01-0593-1, RPC/NB 01-0973-3, RPC/NB 04-085-1, 485/9/97, and 390/98 revealed that the segment 7-ORF1/3 polypeptide of 81 aa is present in all ISAV isolates of the North American genotype, whereas it terminates after 62 or 32 aa in ISAV isolates of the European genotype (Table 4).

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