MdBVe46 is an envelope protein that is required for virion formation by Microplitis demolitor bracovirus

Bracoviruses (BVs) are endogenized nudiviruses that braconid parasitoid wasps have coopted for functions in parasitizing hosts. Microplitis demolitor is a braconid wasp that produces Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of the moth Chrysodeixis includens. Some BV core genes are homologs of genes also present in baculoviruses while others are only known from nudiviruses or other BVs. In this study, we had two main goals. The first was to separate MdBV virions into envelope and nucleocapsid fractions before proteomic analysis to identify core gene products that were preferentially associated with one fraction or the other. Results indicated that nearly all MdBV baculovirus-like gene products that were detected by our proteomic analysis had similar distributions to homologs in the occlusion-derived form of baculoviruses. Several core gene products unknown from baculoviruses were also identified as envelope or nucleocapsid components. Our second goal was to functionally characterize a core gene unknown from baculoviruses that was originally named HzNVorf64-like. Immunoblotting assays supported our proteomic data that identified HzNVorf64-like as an envelope protein. We thus renamed HzNVorf64-like as MdBVe46, which we further hypothesized was important for infection of C. includens. Knockdown of MdBVe46 by RNA interference (RNAi) greatly reduced transcript and protein abundance. Knockdown of MdBVe46 also altered virion morphogenesis, near-fully inhibited infection of C. includens, and significantly reduced the proportion of hosts that were successfully parasitized by M. demolitor.

Microplitis demolitor Bracovirus Proviral Loci and Clustered Replication Genes Exhibit Distinct DNA Amplification Patterns during Replication

ABSTRACTPolydnaviruses are large, double-stranded DNA viruses that are beneficial symbionts of parasitoid wasps. Polydnaviruses in the genusBracovirus(BVs) persist in wasps as proviruses, and their genomes consist of two functional components referred to as proviral segments and nudivirus-like genes. Prior studies established that the DNA domains where proviral segments reside are amplified during replication and that segments within amplified loci are circularized before packaging into nucleocapsids. One DNA domain where nudivirus-like genes are located is also amplified but never packaged into virions. We recently sequenced the genome of the braconidMicroplitis demolitor, which carriesM. demolitorbracovirus (MdBV). Here, we took advantage of this resource to characterize the DNAs that are amplified during MdBV replication using a combination of Illumina and Pacific Biosciences sequencing approaches. The results showed that specific nucleotide sites identify the boundaries of amplification for proviral loci. Surprisingly, however, amplification of loci 3, 4, 6, and 8 produced head-to-tail concatemeric intermediates; loci 1, 2, and 5 produced head-to-head/tail-to-tail concatemers; and locus 7 yielded no identified concatemers. Sequence differences at amplification junctions correlated with the types of amplification intermediates the loci produced, while concatemer processing gave rise to the circularized DNAs that are packaged into nucleocapsids. The MdBV nudivirus-like gene cluster was also amplified, albeit more weakly than most proviral loci and with nondiscrete boundaries. Overall, the MdBV genome exhibited three patterns of DNA amplification during replication. Our data also suggest that PacBio sequencing could be useful in studying the replication intermediates produced by other DNA viruses.IMPORTANCEPolydnaviruses are of fundamental interest because they provide a novel example of viruses evolving into beneficial symbionts. All polydnaviruses are associated with insects called parasitoid wasps, which are of additional applied interest because many are biological control agents of pest insects. Polydnaviruses in the genusBracovirus(BVs) evolved ∼100 million years ago from an ancestor related to the baculovirus-nudivirus lineage but have also established many novelties due to their symbiotic lifestyle. These include the fact that BVs are transmitted only vertically as proviruses and produce replication-defective virions that package only a portion of the viral genome. Here, we studiedMicroplitis demolitorbracovirus (MdBV) and report that its genome exhibits three distinct patterns of DNA amplification during replication. We also identify several previously unknown features of BV genomes that correlate with these different amplification patterns.

PTP-H2 and PTP-H3 from Microplitis demolitor Bracovirus Localize to Focal Adhesions and Are Antiphagocytic in Insect Immune Cells

ABSTRACT Viruses in the family Polydnaviridae are symbiotically associated with parasitoid wasps. Wasps inject polydnaviruses (PDVs) when laying an egg into their insect host, and expression of viral gene products causes several physiological alterations, including immunosuppression, that allow the wasp's progeny to develop. As with other PDVs, most Microplitis demolitor bracovirus (MdBV) genes are related variants that form gene families. The largest MdBV gene family includes 13 members that encode predicted proteins related to protein tyrosine phosphatases (PTPs). Sequence analysis during the present study indicated that five PTP family members (PTP-H2, -H3, -N1, and -N2) have fully conserved catalytic domains, whereas other family members exhibited replacements, deletions, or rearrangements of amino acids considered essential for tyrosine phosphatase activity. Expression studies indicated that most MdBV PTP genes are expressed in virus-infected host insects, with transcript abundance usually being highest in hemocytes. MdBV-infected hemocytes also exhibited higher levels of tyrosine phosphatase activity than noninfected hemocytes. We produced expression constructs for four of the most abundantly expressed PTP family members and conducted functional studies with hemocyte-like Drosophila S2 cells. These experiments suggested that recombinant PTP-H2 and PTP-H3 are functional tyrosine phosphatases whereas PTP-H1 and PTP-J1 are not. PTP-H2 and -H3 localized to focal adhesions in S2 cells, and coexpression with another MdBV gene product, Glc1.8, resulted in complete inhibition of phagocytosis.