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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 AmCPV S1 encoded proteins using anti-p141 polyclonal antibody. (A) SDS-8% PAGE and (B) Western Blot. Lane M, Prestained protein molecular weight marker (Fermentas); lane 1, purified insect cell expressed recombinant p141 protein; lane 2, uninfected midgut of A. mylitta; lane 3, infected midgut of A. mylitta; lane 4, purified polyhedra and lane 5, purified virion particle. Arrow indicates the position of immunoreactive protein.
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Figure 3: Immunoblot analysis of AmCPV S1 encoded proteins using anti-p141 polyclonal antibody. (A) SDS-8% PAGE and (B) Western Blot. Lane M, Prestained protein molecular weight marker (Fermentas); lane 1, purified insect cell expressed recombinant p141 protein; lane 2, uninfected midgut of A. mylitta; lane 3, infected midgut of A. mylitta; lane 4, purified polyhedra and lane 5, purified virion particle. Arrow indicates the position of immunoreactive protein.

Mentions: Sf9 cells infected with S1 and S3 recombinant baculovirus expressed these proteins in soluble form as 141 and 137 kDa, respectively [Fig 3A and 4A (lane 1)]. This was confirmed by immunoblot analysis (Fig. 3B and 4B, lane 1). Expression of predicted same size proteins both in bacteria and insect cells indicate that although a number of glycosylation sites are present in both these genes but they are not used for post translational modification. In E. coli M15 cells the expressed proteins might not fold properly into correct conformation and thus the incorrectly folded protein may have aggregated to produce insoluble inclusion bodies but in insect (Sf9) cells via baculovirus expression system due to proper folding soluble proteins are produced.


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 AmCPV S1 encoded proteins using anti-p141 polyclonal antibody. (A) SDS-8% PAGE and (B) Western Blot. Lane M, Prestained protein molecular weight marker (Fermentas); lane 1, purified insect cell expressed recombinant p141 protein; lane 2, uninfected midgut of A. mylitta; lane 3, infected midgut of A. mylitta; lane 4, purified polyhedra and lane 5, purified virion particle. 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 3: Immunoblot analysis of AmCPV S1 encoded proteins using anti-p141 polyclonal antibody. (A) SDS-8% PAGE and (B) Western Blot. Lane M, Prestained protein molecular weight marker (Fermentas); lane 1, purified insect cell expressed recombinant p141 protein; lane 2, uninfected midgut of A. mylitta; lane 3, infected midgut of A. mylitta; lane 4, purified polyhedra and lane 5, purified virion particle. Arrow indicates the position of immunoreactive protein.
Mentions: Sf9 cells infected with S1 and S3 recombinant baculovirus expressed these proteins in soluble form as 141 and 137 kDa, respectively [Fig 3A and 4A (lane 1)]. This was confirmed by immunoblot analysis (Fig. 3B and 4B, lane 1). Expression of predicted same size proteins both in bacteria and insect cells indicate that although a number of glycosylation sites are present in both these genes but they are not used for post translational modification. In E. coli M15 cells the expressed proteins might not fold properly into correct conformation and thus the incorrectly folded protein may have aggregated to produce insoluble inclusion bodies but in insect (Sf9) cells via baculovirus expression system due to proper folding soluble proteins are produced.

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