Abstract
BackgroundAntheraea 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.ResultsIn 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 ~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 ~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.ConclusionOur 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.
Highlights
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
We have reported that the genome segments 6, 7, 8 of AmCPV encode viral structural proteins [24,25,26], segment 2 encodes viral RNA dependent RNA polymerase [27], segment 9 encodes a nonstructural protein, NSP38, having RNA binding property [28], segment codes for polyhedrin [29] and segment does not code for any protein [26]
segments 1 (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 ~141 kDa (p141)
Summary
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. BmCPV, the type Cypovirus, has a single layer capsid made up of 120 copies of the major capsid protein, VP1, which is decorated with 12 turrets on its icosahedral vertices [9,10] These hollow turrets are involved in post-transcriptional processing of viral mRNA and provide a channel through which newly synthesized 5′ capped viral RNA are released from the capsid into the cytoplasm of infected cells [10,11]. After translation of this mRNA into capsid, polymerase and other proteins, they assembled into viral procapsid within which one copy of each genome segments plus polarity RNA are packaged and replicated to form dsRNA. Besides BmCPV, capsids of other CPVs have not been studied well all the genome segments of DpCPV, LdCPV and TnCPV have been cloned and sequenced [6,19,20,21]
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