ABSTRACT
The braconid wasp Microplitis demolitor carries M. demolitor polydnavirus (MdPDV) and parasitizes the larval stage of the moth Pseudoplusia includens. M. demolitor injects MdPDV into P. includens larvae when it lays an egg and the virus infects various cells including haemocytes. Two new MdPDV transcripts expressed in host haemocytes were characterized in this study. Screening of an MdPDV-infected haemocyte cDNA library identified a 0.4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0. 4. This protein contained a cysteine-rich epidermal growth factor (EGF)-like motif at its N terminus that was similar to the EGF-like domains in the previously identified MdPDV genes egf1.5 and egf1.0. Sequencing of the genomic clone pMd-10 indicated that it contained the egf0.4 gene, which consisted of two introns and three exons. This gene was located on MdPDV segment O and appeared to exist in multiple copies. A nucleic acid and expression screen identified a 1. 8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1.8. This protein consisted of a heavily glycosylated central core of six tandemly arranged repeats flanked by hydrophobic N- and C-terminal domains. Northern blotting and in situ hybridization studies indicated that both egf0.4 and glc1.8 were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1.8 localized to the cell surface.
Subject(s)
Hemocytes/metabolism , Hemocytes/virology , Moths/virology , Polydnaviridae/genetics , RNA, Viral/analysis , Wasps/virology , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Cell Membrane/chemistry , Cloning, Molecular , Epidermal Growth Factor/analysis , Epidermal Growth Factor/chemistry , Epidermal Growth Factor/genetics , Exons/genetics , Gene Expression Profiling , Gene Expression Regulation, Viral , Gene Library , Glycosylation , Hemocytes/parasitology , Immunohistochemistry , In Situ Hybridization , Introns/genetics , Molecular Sequence Data , Moths/parasitology , Multigene Family/genetics , Polydnaviridae/chemistry , Protein Structure, Tertiary , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/genetics , Sequence Alignment , Sequence Homology, Nucleic AcidABSTRACT
Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.
