Copyright
IIT Kharagpur
Abstract
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is the most widespread pathogen of Indian non-mulberry silkworm Antheraea mylitta. The genome segments 1 (S1), 3 (S3), 8 (S8) and 11 (S11) were converted to cDNA, cloned and sequenced. S1 (3852 nucleotides), S3 (3784 nucleotides) and S8 (1677 nucleotides) showed ORFs of 1245, 1210 and 528 amino acids residues, respectively, and could encode proteins of approximate molecular weight of 141 kDa (p141), 137 kDa (p137) and 60 kDa (p60), respectively. AmCPV segment 11 consisted of 390 nucleotides and did not show presence of any ORF. S1, S3 and S8 ORFs were expressed as His- tagged fusion proteins in E. coli as insoluble proteins, purified through Ni-NTA chromatography and polyclonal antibodies were raised indicating that p141, p137 and p60 were strongly immunogenic. Immunoblot analysis of polyhedra, infected, uninfected midgut cells of Antheraea mylitta and purified native virion particles with the anti-p141, anti-p137 and anti-p60 antibodies showed that p141, p137 and p60 were structural proteins. All these proteins were expressed in insect cells (Sf 9) via baculovirus expression system. TEM analysis of virion like particles isolated from Sf 9 cells infected by recombinant baculoviruses of AmCPV S3 showed self assembly of p137 proteins to form single-shelled virion like particles. Sf9 cells co-infected with recombinant baculoviruses expressing p141 and p137 also showed the formation of virus like particles in vitro. Immunoblot analysis of purified virus like particles from S3 baculovirus infected, and S1 and S3 baculovirus co-infected Sf 9 cells using anti-p137 and anti-p141 antibodies confirms the presence of only p137, and p141 and p137 together, respectively in those particles. Immunogold staining of the particles using purified anti-p137 antibodies confirmed that the p137 protein was exposed to the outer surface of the virus like particles, whereas p141 was present as inner capsid for maintaining stability of virus like particles. Electrophoretic mobility shift assay and experiment using poly (I:C)-sepharose showed binding and interaction of AmCPV S8 encoded p60 with viral RNA and optimal binding occurs at ~200 mM NaCl concentration at 30 °C but denaturation of p60 with urea abolished the binding, indicating p60 binds with viral RNA and may participates in viral RNA replication, and/or it’s packaging.
Characterization of S1 and S3 encoded capsid proteins, and S8 encoded RNA binding protein will help to understand the interaction of viral capsid-host cell receptor during viral entry and its genome replication/packaging during formation of virion particles. In vitro production of viral capsid will also help to produce monoclonal antibody against it for early detection of viral infection in infected larvae.