Molecular Cloning, Expression and Analysis of Antheraea mylitta Cypovirus Genome Segments 8 and 11

The genome segments 8 (S8) and 11 (S11) of the 11 double-stranded segmented RNA genome of Antheraea mylitta cypovirus (AmCPV) were converted to cDNA, cloned and sequenced. S8 consisted of 1677 nucleotides with a long ORF of 527 amino acids and could encode a protein of approximately 60 kDa, termed p60. AmCPV segment 11 consisted of 390 nucleotides and did not show presence of any ORF. No sequence similarity of S8 or S11 was found by searching nucleic acid and protein sequence databases using BLAST. Secondary structure prediction showed that p60 contained 13 alpha -helices and 18 extended beta -sheets along the entire length of the protein. S8 ORF was expressed as His-tagged fusion proteins in E. coli, purified through Ni-NTA chromatography and polyclonal antibody was raised indicating that p60 is strongly immunogenic. Immunoblot analysis of polyhedra, infected and uninfected cells with anti-p60 antibody showed that p60 is a viral structural protein. Motif scan search showed some similarity of p60 with inosine 5'-monophosphate dehydrogenase (IMPDH) containing a pair of Cystathionine Beta Synthase (CBS) domains and it was hypothesized that p60 may be involved in nucleotide biosynthesis and viral RNA replication. Due to absence of any ORF in S11 its function remained unclear.