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Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus.

Chakrabarti M, Ghorai S, Mani SK, Ghosh AK - Virol. J. (2010)

Bottom Line: Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability.Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India.

ABSTRACT

Background: Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.

Results: In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of approximately 141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of approximately 137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV), Lymantria dispar CPV (LdCPV), and Dendrolimus punctatus CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.

Conclusion: Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

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Immunoblot analysis of recombinant VLPs using anti-p137 and anti-p141 antibodies. (A) SDS-8% PAGE, (B) western blot with anti-p137 antibody and (C) western blot with anti-p141 antibody. Lane M, Prestained molecular weight marker (GE Healthcare Bio-Science); lane 1, VLPs from cells infected with AmCPVS1 recombinant baculovirus, lane 2, VLPs from cells infected with AmCPVS1 and AmCPV S3 recombinant baculovirus; lane 3, purified p137 protein; lane 4, purified p141 protein; lane 5, purified native virion particles. Arrow indicates the position of immunoreactive protein.
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Figure 6: Immunoblot analysis of recombinant VLPs using anti-p137 and anti-p141 antibodies. (A) SDS-8% PAGE, (B) western blot with anti-p137 antibody and (C) western blot with anti-p141 antibody. Lane M, Prestained molecular weight marker (GE Healthcare Bio-Science); lane 1, VLPs from cells infected with AmCPVS1 recombinant baculovirus, lane 2, VLPs from cells infected with AmCPVS1 and AmCPV S3 recombinant baculovirus; lane 3, purified p137 protein; lane 4, purified p141 protein; lane 5, purified native virion particles. Arrow indicates the position of immunoreactive protein.

Mentions: To further confirm the protein content of these VLPs obtained from recombinant baculovirus infected Sf9 cells, immunoblot analysis was performed using anti-p137 and anti-p141 antibodies. Immunoblot study using anti-p137 antibody (Fig. 6B) showed a single major immunoreactive band at 137 kDa in purified VLPs from cells infected with AmCPV S3 baculovirus recombinants (lane 1), purified VLPs from cells co-infected with both AmCPV S1 and S3 baculovirus recombinants (lane 2), purified p137 protein (lane 3) and native virion particles (lane 5). Similar immunoblot study, using anti-p141 antibody showed a single major immunoreactive band at 141 kDa in purified VLPs obtained from cells expressing both AmCPV S1 and S3 (Fig. 6C, lane 2), purified recombinant p141 protein (lane 4) and native virion particles (lane 5). Since in SDS-PAGE, after Coomassie blue staining two bands (137 kDa and 141 kDa) were observed in purified VLPs from cells co-infected with both AmCPV S1 and S3 baculovirus recombinants (lane 2), and also in purified native virion particles (lane 5) and reacted with both anti-p141 and p137 antibodies, these results indicate that p137 is involved in the formation capsid outer shell and p141 is associated in the inner side of capsid (VLPs). Three dimensional structure of BmCPV has shown presence of spike molecules and transcription enzyme complexes along the icosahedral five fold axis both inside and outside of the core particles [10,16]. Similar studies are required to understand the association of AmCPV S1 and S3 encoded proteins in the viral capsid.


Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus.

Chakrabarti M, Ghorai S, Mani SK, Ghosh AK - Virol. J. (2010)

Immunoblot analysis of recombinant VLPs using anti-p137 and anti-p141 antibodies. (A) SDS-8% PAGE, (B) western blot with anti-p137 antibody and (C) western blot with anti-p141 antibody. Lane M, Prestained molecular weight marker (GE Healthcare Bio-Science); lane 1, VLPs from cells infected with AmCPVS1 recombinant baculovirus, lane 2, VLPs from cells infected with AmCPVS1 and AmCPV S3 recombinant baculovirus; lane 3, purified p137 protein; lane 4, purified p141 protein; lane 5, purified native virion particles. Arrow indicates the position of immunoreactive protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927528&req=5

Figure 6: Immunoblot analysis of recombinant VLPs using anti-p137 and anti-p141 antibodies. (A) SDS-8% PAGE, (B) western blot with anti-p137 antibody and (C) western blot with anti-p141 antibody. Lane M, Prestained molecular weight marker (GE Healthcare Bio-Science); lane 1, VLPs from cells infected with AmCPVS1 recombinant baculovirus, lane 2, VLPs from cells infected with AmCPVS1 and AmCPV S3 recombinant baculovirus; lane 3, purified p137 protein; lane 4, purified p141 protein; lane 5, purified native virion particles. Arrow indicates the position of immunoreactive protein.
Mentions: To further confirm the protein content of these VLPs obtained from recombinant baculovirus infected Sf9 cells, immunoblot analysis was performed using anti-p137 and anti-p141 antibodies. Immunoblot study using anti-p137 antibody (Fig. 6B) showed a single major immunoreactive band at 137 kDa in purified VLPs from cells infected with AmCPV S3 baculovirus recombinants (lane 1), purified VLPs from cells co-infected with both AmCPV S1 and S3 baculovirus recombinants (lane 2), purified p137 protein (lane 3) and native virion particles (lane 5). Similar immunoblot study, using anti-p141 antibody showed a single major immunoreactive band at 141 kDa in purified VLPs obtained from cells expressing both AmCPV S1 and S3 (Fig. 6C, lane 2), purified recombinant p141 protein (lane 4) and native virion particles (lane 5). Since in SDS-PAGE, after Coomassie blue staining two bands (137 kDa and 141 kDa) were observed in purified VLPs from cells co-infected with both AmCPV S1 and S3 baculovirus recombinants (lane 2), and also in purified native virion particles (lane 5) and reacted with both anti-p141 and p137 antibodies, these results indicate that p137 is involved in the formation capsid outer shell and p141 is associated in the inner side of capsid (VLPs). Three dimensional structure of BmCPV has shown presence of spike molecules and transcription enzyme complexes along the icosahedral five fold axis both inside and outside of the core particles [10,16]. Similar studies are required to understand the association of AmCPV S1 and S3 encoded proteins in the viral capsid.

Bottom Line: Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability.Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India.

ABSTRACT

Background: Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.

Results: In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of approximately 141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of approximately 137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV), Lymantria dispar CPV (LdCPV), and Dendrolimus punctatus CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.

Conclusion: Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

Show MeSH
Related in: MedlinePlus