Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylittacytoplasmic polyhedrosis virus

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.

Interactions among Dendrolimus punctatus cypovirus proteins and identification of the genomic segment encoding its A-spike

“…Moreover, our results revealed that VP2 interacted with VP1 (Fig. S1c), which is consistent with the report that VP2 maintains the stability of the capsid shell (Chakrabarti et al , 2010).…”

“…The RNA-dependent RNA polymerase (RdRp), which is a main component of transcriptase enzyme complexes, attaches to the inner surface of the capsid shell at the icosahedral fivefold vertices (Xia et al , 2003; Zhang et al , 1999). VP2 is associated with the CSPs as an inner capsid to maintain the stability of the capsid shell (Chakrabarti et al , 2010). VP4 binds to viral RNA and helps in replication and transcription through ATP binding and hydrolysis (Chavali et al , 2008), but its localization on the particle remains unknown.…”

Genomic and Biological Characterization of a New Cypovirus Isolated from Dendrolimus punctatus

“…The deduced amino acid sequence encoded by S1 shows greatest homology to P2 of Heliothis armigera cypovirus 5 (HaCPV-5), encoded by its genomic segment 2 (GenBank accession number: YP_001883322). It is also similar (26%) to the minor capsid protein of AmCPV-4, encoded by its genomic segment 1 [19]. The minor capsid protein of AmCPV-4 associates with the major capsid protein to maintain its stability [19].…”

“…It is also similar (26%) to the minor capsid protein of AmCPV-4, encoded by its genomic segment 1 [19]. The minor capsid protein of AmCPV-4 associates with the major capsid protein to maintain its stability [19].…”

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles

“…Abundant evidence exists that the CSP monomers of BmCPV could assemble themselves into virus-like particles (VLPs) [10]. In addition to BmCPV, many other viruses have similar abilities, such as the VP1 protein of norovirus, the VP1 and VP3 proteins of Antheraea mylitta CPV, and the VP2 and VP3 proteins of rotaviruses [11][12][13]. VLPs have received increasing interest for their biotechnological applications.…”