Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9

Abstract

Genome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced. In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa. Secondary structure prediction showed the presence of nine α-helices in the central and terminal domains with localized similarity to RNA-binding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases. Nucleotide sequences were 99·6% identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases. The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E. coli and polyclonal antibody was raised against the purified protein. Immunoblot as well as immunofluorescence analysis with anti-NSP38 antibody showed that the protein was not present in polyhedra or uninfected cells but was present in AmCPV-infected host midgut cells. NSP38 was expressed in insect cells as soluble protein via a baculovirus expression vector and shown to possess the ability to bind poly(rI)–(rC) agarose, which was competitively removed by AmCPV viral RNA. These results indicate that NSP38 is expressed in virus-infected cells as a non-structural protein. By binding to viral RNA, it may play a role in the regulation of genomic RNA function and packaging.