Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes

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 MdPDVinfected haemocyte cDNA library identified a 0n4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0n4. 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 egf1n5 and egf1n0. Sequencing of the genomic clone pMd-10 indicated that it contained the egf0n4 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 1n8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1n8. 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 egf0n4 and glc1n8 were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1n8 localized to the cell surface.

The Encapsidated Genome of Microplitis demolitor Bracovirus Integrates into the Host Pseudoplusia includens

“…Thus, members of the ptp gene family consist of one predicted gene located on genomic segment D (ptp-D1), five on segment H (ptp-H1, ptp-H2, ptp-H3, ptp-H4, and ptp-H5), four on segment J (ptp-J1, ptp-J2, ptp-J3, and ptp-J4), and three on segment N (ptp-N1, ptp-N2, and ptp-N3) (25,42). Members of the ank gene family are named similarly (ank-C1, ank-C2, ank-F4, ank-F5, ank-G3, ank-G4, ank-H4, ank-I1, ank-J4, ank-N1, ank-N4, and ank-N5), whereas all egf (egf0.4, egf1.0, and egf1.5) and glc (glc1.8 and glc3.2) genes reside on genomic segment O and are named by the size of their corresponding cDNAs (4,36,39,42).…”

“…After protein concentrations were determined by use of the Micro BCA protein assay kit (Pierce), samples were resolved on 4-to-20% gradient SDS-PAGE gels (Lonza), immunoblotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore), and blocked (20). The membrane was probed with a murine monoclonal antibody (55F2E7) specific for Glc1.8 and Glc3.2 (1:10,000 dilution) (2,39). The primary antibody was detected by using a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution) (Jackson Laboratory), followed by visualization using a chemiluminescent substrate (ECL Advance kit; GE Healthcare) (20).…”

“…MdBV disables encapsulation by preventing hemocytes called granulocytes and plasmatocytes from adhering to wasp eggs and causing some granulocytes to apoptose (33,34,39). The loss of adhesion is due primarily to the expression of the glc genes in infected hemocytes, which encode very similar cell surface glycoproteins (3,4,25), while apoptosis is associated with the expression of ptp-H2 (37).…”

Bracoviruses Contain a Large Multigene Family Coding for Protein Tyrosine Phosphatases

“…Polydnaviruses have some of the largest virus genomes (568 kb for CcBV) (17a), and gene families are common in both ichnoviruses (9,23,37,65) and bracoviruses (18,37,57,60). The organization of bracovirus genomes in large doublestranded DNA segments encapsidated in distinct nucleocapsids (1) and the fact that they are exclusively chromosomally transmitted (in the absence of virus replication within the host tissues) probably relieved constraints on viral genome size, allowing the formation of gene families.…”

Glc1.8 fromMicroplitis demolitorBracovirus Induces a Loss of Adhesion and Phagocytosis in Insect High Five and S2 Cells

“…Several MdBV genes are expressed in hemocytes and High Five cells, including glc1.8, which encodes a 514-amino-acid mucin-like protein that localizes to the surfaces of infected cells (30,40). Glc1.8 is characterized by a signal peptide at its N terminus, an extracellular domain comprised of five 78-amino-acid repeats arranged in a tandem array, and a C-terminal hydrophobic domain that encodes a putative anchor sequence (40).…”