Nachrichten aus der Brüder-Gemeine, 1828, no. 01

The Moravian Church's monthly journal of its worldwide activities, including in Labrador, published from 1819-94

Mutualistic Polydnaviruses Share Essential Replication Gene Functions with Pathogenic Ancestors

<div><p>Viruses are usually thought to form parasitic associations with hosts, but all members of the family <i>Polydnaviridae</i> are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus <i>Bracovirus</i> (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from <i>Microplitis demolitor</i> bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (<i>lef-4, lef-9</i>), capsid assembly (<i>vp39, vlf-1</i>), and envelope formation (<i>p74, pif-1</i>) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for <i>vp39, vlf-1, p74</i>, and <i>pif-1</i> as structural components of MdBV virions. Additional experiments suggested that <i>vlf-1</i> together with the nudivirus-like gene <i>int-1</i> also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation.</p></div

Knockdown of <i>vlf-1</i> and <i>int-1</i> disables excision of genomic segment B from the <i>M.</i><i>demolitor</i> genome.

<p>(A) Alignment of MdBV <i>vlf-1</i> and <i>integrase</i> family members to family members from select nudiviruses (<i>Paenaeus monodon</i> nudivirus (PmNV), <i>Heliothis zea</i> nudivirus (HzNV-1), <i>Gryllus bimaculatus</i> nudivirus (GbNV), <i>Oryctes rhinoceros</i> nudivirus (OrNV)), the baculovirus <i>Autographa californica</i> multinnucleopolyhedrosis virus (AcMNPV), and <i>Chelonus inanitus</i> bracovirus (CiBV). The alignment shows only the portion of each where the predicted conserved tyrosine (Y) residue required for recombinase activity by other tyrosine recombinase family members is located (box). Select other conserved residues are highlighted with other background colors. (B) Schematic illustrating the qPCR assays to quantify copy number of MdBV genomic segment B (6307 bp) in <i>M. demolitor</i> ovaries <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348-Burke1" target="_blank">[7]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348-Webb1" target="_blank">[14]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348-Beck2" target="_blank">[30]</a>. Episomal segment B is specifically amplified using the primers MdSegBLL and MdSegBRR, which can only generate a product if segment B is circularized. Copy number of the rejoined empty locus in <i>M. demolitor</i> is specifically amplified using the primers MdSegBLR2 and MdSegBRL, which can only generate a product if segment B has been excised and the wasp flanking domains are rejoined. (C) Copy number of the rejoined empty locus from excision of segment B in the ovaries of newly emerged <i>M. demolitor</i> adult females pretreated with ds-<i>eGFP</i>, ds-<i>int-1</i> or ds-<i>vlf-1</i>. The ovaries from individual, newly emerged adults wasps were dissected, followed by DNA isolation and qPCR analysis as outlined in (B). Error bars, N values, and statistical significance are indicated as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>.</p

Knockdown of <i>vp39</i> and <i>vlf-1</i> increases DNAse sensitivity and reduces infectivity.

<p><i>M. demolitor</i> larvae were injected with ds-<i>eGFP</i>, ds-<i>vp39</i> or ds-<i>vlf-1</i> as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>. (A) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged <i>M. demolitor</i> adult females pretreated with ds-<i>eGFP</i> or ds-<i>vp39</i>. (B) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged <i>M. demolitor</i> adult females pretreated with ds-<i>eGFP</i> or ds-<i>vlf-1</i>. (C) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-<i>eGFP</i> or ds-<i>vp39</i>. (D) Copy number of MdBV episomal genomic segment B in CiE1 cells infected with MdBV from wasps pretreated with ds-<i>eGFP</i> or ds-<i>vlf-1</i>. (E) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-<i>eGFP</i> or ds-<i>vp39</i>. (F) Normalized fraction of CiE1 cells expressing the gene product GLC1.8 on their surface after infection with MdBV from wasps pretreated with ds-<i>eGFP</i> or ds-<i>vlf-1</i>. Error bars, N values, and statistical significance are indicated as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>.</p

BV conserved gene products detected in MdBV virions.

<p>The predicted conserved gene set for BVs (gray boxes) is shown to the right relative to the predicted core genes for nudiviruses (black boxes) and baculoviruses (hatched boxes) as previously detailed <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348-Burke1" target="_blank">[7]</a>. Black circles in hatched boxes represent baculovirus core gene products that are detected in mature occlusion-derived baculovirus virions <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348-Hou1" target="_blank">[55]</a>. Two independently prepared protein samples for MdBV virions were analyzed and are indicated as replicate 1 and 2 in the figure. Each gray box for the two replicates with a number or asterisk (*) indicates that peptides matching this conserved gene product were detected. The number in each gray box indicates the total number of unique peptides that matched the product. Gray boxes with asterisks indicate that the MdBV proviral genome contains two or more homologs of nudivirus/baculovirus genes and peptides matching multiple homologs were identified (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat.1003348.s001" target="_blank">Table S1</a>). Gray boxes with no number or asterisk indicate that no peptides were identified that matched the product.</p

Knockdown of <i>lef-4</i> and <i>lef-9</i> reduces expression of MdBV structural genes.

<p><i>M. demolitor</i> larvae were injected with ds-<i>eGFP</i>, ds-<i>lef-4</i> or ds-<i>lef-9</i> as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated followed by measurement of copy number per ng of total RNA of: (A) <i>vp39</i>, (B) <i>p74</i>, (C) <i>ef1α</i>, (D) <i>dnapolδ</i>. (E) Copy number of DNase-protected MdBV episomal genomic segment B in the ovaries of newly emerged <i>M. demolitor</i> adult females. The ovaries from individual, newly emerged adults wasps were dissected and treated with DNAse. DNA was then isolated and total copy number of episomal genomic segment B determined. Error bars, N values, and statistical significance are indicated as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>.</p

Electron microscopy analysis shows defects in MdBV morphogenesis after <i>vlf-1</i> and <i>vp39</i> knockdown.

<p><i>M. demolitor</i> larvae were injected with ds-<i>eGFP</i>, ds-<i>vp39</i> or ds-<i>vlf-1</i> as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003348#ppat-1003348-g002" target="_blank">Figure 2</a>. (A) Image of calyx fluid in the lumen of the reproductive tract of a newly emerged adult female pretreated with ds-<i>eGFP</i>. Each virion consists of one electron dense nucleocapsid (arrowhead) surrounded by a single elongate envelope (arrow). (B) Image of calyx fluid from a wasp pretreated with ds-<i>vlf-1</i>. Note the lower density of virions present relative to (A). (C) Image of calyx fluid from a wasp pretreated with ds-<i>vp39</i>. Scale bars in A-C equal 500 nm. (D) Graph showing that virion density in calyx fluid from wasps treated with ds-<i>vlf-1</i> was significantly lower than in wasps treated with ds-<i>eGFP</i> or ds-<i>vp39</i>. (E) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-<i>eGFP</i>. The left side of the image shows a large array of assembled MdBV virions. Virions in the process of assembly are visible in the lower right of the image. The insert in the upper right shows virion assembly at higher magnification. Note that assembled virions (*), <i>de novo</i> forming envelopes, empty capsids (arrowhead) and electron dense nucleocapsids in the process of being surrounded by envelopes (arrow). (F) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-<i>vlf-1</i>. Unlike normal calyx cells, few virions are assembled into arrays (*). Most of these virions have envelopes that are rounded. Numerous electron dense nucleocapsids without envelopes (arrows) and rounded envelopes without electron dense nucleocapsids are visible. The insert in the upper right shows these defects at higher magnification. It also shows that some rounded envelopes contain empty capsids (arrow). (G) Image showing a portion of a calyx cell nucleus from a wasp pretreated with ds-<i>vp39</i>. Arrays of assembled virions are visible but they are much smaller than the arrays observed in calyx cell nuclei from control wasps. The insert in the upper right shows virions with elongate envelope and electron dense nucleocapsid similar to virions from control wasps. Scale bar for images and inset images E–G equal 500 nm.</p

RNAi knockdown of six MdBV putative replication genes.

<p><i>M. demolitor</i> larvae were injected with double-stranded RNA (dsRNA) specific for (A) <i>lef-4</i>, (B) <i>lef-9</i>, (C) <i>vp39</i>, (D) <i>vlf-1</i>, (E) <i>p74</i>, (F) <i>pif-1</i>, (G) <i>int-1</i>. Control larvae were injected with ds-<i>eGFP</i>. The ovaries from individual, newly emerged adults wasps were then dissected and total RNA isolated. The bars in each graph compare copy number of each target gene per ng of total RNA in wasps treated with dsRNA specific for the target gene versus ds-<i>eGFP</i> (control). Error bars represent one standard error from the mean. The number of individuals examined for each treatment is indicated by the N value at the bottom of each bar. Statistical significance is indicated by asterisks: *, <i>p</i><0.05; **, <i>p</i><0.001; ***, <i>p</i><0.0001; N. S., not significant. (H) Knockdown of <i>lef-9</i> depletes LEF-9 protein. <i>M. demolitor</i> larvae were injected with ds-<i>lef-9</i> as in (B). Total protein from the ovaries of a newly emerged adult wasp was loaded into lanes of an SDS-PAGE gel followed by immunoblot analysis using an MdBV anti-LEF-9 antibody (1∶5000). The left lanes show results from 5 individual <i>M. demolitor</i> females treated with ds-<i>lef-9</i> (knockdown), while the lanes on the right show results from 4 females treated with ds-<i>eGFP</i> (control). The predicted size of MdBV LEF-9 is 74.4 kDa.</p

Bondholder Coercion: The Problem of Constrained Choice in Debt Tender Offers and Recapitalizations

<p>The effect MATE1, MATE2 and PMAT inhibitors on the [³H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with MATE1 inhibitor famotidine (1 μM), MATE2 inhibitor nifekalant (3 μM) or PMAT inhibitor lopinavir (2 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n =.4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.</p