Markus H. Beck

A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway

Pathogens often suppress the melanization response of host insects, but the underlying molecular mechanisms are largely unknown. Here we report that Microplitis demolitor bracovirus (MdBV) carried by the wasp M. demolitor produces a protein, Egf1.0, which inhibits the phenoloxidase (PO) cascade. Egf1.0 belongs to a larger gene family that shares a cysteine-rich motif with similarities to the trypsin inhibitor-like (TIL) domains of small serine proteinase inhibitors (smapins). Gain-of-function and RNAi experiments indicated that the Egf genes are the only MdBV-encoded factors responsible for disabling the insect melanization response. Known smapins bind target proteinases in a substrate-like fashion and are cleaved at a single reactive site bond. The P1–P1′ position for Egf1.0 has the sequence Arg-Phe, which suggested that its target proteinase is a prophenoloxidase-activating proteinase (PAP). Wild-type Egf1.0 inhibited PAP-3 from Manduca sexta, whereas Egf1.0R51A, whose reactive-site arginine was replaced with an alanine, had no PAP-3 inhibitory activity. Other experiments using wild-type and mutant constructs indicated that Egf1.0 blocks activation of the PO cascade via PAP inhibition. Overall, our results identify a novel inhibitor of the PO cascade and indicate that suppression of the host melanization response is functionally important for both the virus and its associated wasp.

Glc1.8 from Microplitis demolitor Bracovirus Induces a Loss of Adhesion and Phagocytosis in Insect High Five and S2 Cells

ABSTRACT Polydnaviridae is a unique family of DNA viruses that are symbiotically associated with parasitoid wasps. Upon oviposition, wasps inject these viruses into their hosts, where they cause several physiological alterations, including suppression of the cellular immune response. Here we report that expression of the glc1.8 gene from Microplitis demolitor bracovirus (MdBV) causes a loss of adhesion by two hemocyte-like cell lines, namely, High Five cells from the lepidopteran Trichoplusia ni and S2 cells from the dipteran Drosophila melanogaster. The expression of recombinant Glc1.8 also greatly reduced the ability of these cells to phagocytize foreign targets. Glc1.8 is characterized by a signal peptide at its N terminus, an extracellular domain comprised of five nearly perfect tandem repeats of 78 amino acids, and a C-terminal hydrophobic domain that encodes a putative membrane anchor sequence. The expression of a Glc1.8 mutant lacking the anchor sequence resulted in a secreted protein that had no effect on adhesion or phagocytosis. In contrast, sequential deletion of the repeats in the extracellular domain resulted in a progressive reduction in immunosuppressive activity. Since each repeat and its associated glycosylation sites are nearly identical, these results suggested that adhesion-blocking activity depends more on the overall number of repeats in the extracellular domain than on the specific determinants within each repeat. While it severely compromised adhesion and phagocytic functions, Glc1.8 did not cause cell death. Collectively, these results indicate that Glc1.8 is a major pathogenic determinant of MdBV that is involved in suppression of the insect cellular immune response.

RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells.

The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes.

The viral protein EGF1.0 is a dual activity inhibitor of prophenoloxidase activating proteinases 1 and 3 from Manduca sexta

Some pathogens are capable of suppressing the melanization response of host insects, but the virulence factors responsible are largely unknown. The insect pathogen Microplitis demolitor bracovirus encodes the Egf family of small serine proteinase inhibitors. One family member, Egf1.0, was recently shown to suppress melanization of hemolymph in Manduca sexta in part by inhibiting the enzymatic activity of prophenoloxidase activating proteinase 3 (PAP3). However, other experiments suggested this viral protein suppresses melanization by more than one mechanism. Here we report that Egf1.0 inhibited the amidolytic activity of PAP1 and dose-dependently blocked processing of pro-PAP1 and pro-PAP3. Consistent with its PAP inhibitory activity, Egf1.0 also prevented processing of pro-phenoloxidase, serine proteinase homolog (SPH) 1, and SPH2. Isolation of Egf1.0-protein complexes from plasma indicated that Egf1.0 binds PAPs through its C-terminal repeat domain. Egf1.0 also potentially interacts with SPH2 and two other proteins, ferritin and gloverin, not previously associated with the phenoloxidase cascade. Overall, our results indicate that Egf1.0 is a dual activity PAP inhibitor that strongly suppresses the insect melanization response.

Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.

Egf1.5 is a second phenoloxidase cascade inhibitor encoded by Microplitis demolitor bracovirus

The three-member Egf gene family from the polydnavirus Microplitis demolitor bracovirus (MdBV) encodes novel proteins distinguished by a shared cysteine-rich motif. Prior studies determined that one family member, Egf1.0, inhibits melanization of hemolymph from the moth Manduca sexta by disabling phenoloxidase activating proteinases (PAPs). Here we characterized a second family member, Egf1.5, which shares an identical cysteine-rich motif with Egf1.0, but possesses an extended C-terminal repeat domain. Similar to Egf1.0, Egf1.5 inhibited processing and the amidolytic activity of PAP1 and PAP3 from M. sexta. Egf1.5 also bound PAP1, PAP3 and serine proteinase homolog 2 (SPH2). Comparative studies indicated that Egf1.5 and Egf1.0 similarly inhibited melanization of plasma from two lepidopterans (Pseudoplusia includens and Helicoverpa zea) that are permissive hosts for M. demolitor and MdBV, and two lepidopterans (M. sexta and Bombyx mori) that are nonpermissive hosts. Expression studies showed that transcript abundance of egf1.5 and egf1.0 was also similar in MdBV-infected P. includens and H. zea. Taken together, our results indicate that Egf1.5 and Egf1.0 are functionally similar paralogs.

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

ABSTRACT Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.