A Polydnavirus ANK Protein Acts as Virulence Factor by Disrupting the Function of Prothoracic Gland Steroidogenic Cells

<div><p>Polydnaviruses are obligate symbionts integrated as proviruses in the genome of some ichneumonoid wasps that parasitize lepidopteran larvae. Polydnavirus free viral particles, which are injected into the host at oviposition, express virulence factors that impair immunity and development. To date, most studies have focused on the molecular mechanisms underpinning immunosuppression, whereas how viral genes disrupt the endocrine balance remains largely uninvestigated. Using <i>Drosophila</i> as a model system, the present report analyzes the function of a member of the <i>ankyrin</i> gene family of the bracovirus associated with <i>Toxoneuron nigriceps</i>, a larval parasitoid of the noctuid moth <i>Heliothis virescens</i>. We found that the <i>TnBVank1</i> expression in the <i>Drosophila</i> prothoracic gland blocks the larval-pupal molt. This phenotype can be rescued by feeding the larvae with 20-hydroxyecdysone. The localization of the <i>Tn</i>BVANK1 is restricted to the cytoplasm where it interacts with Hrs and Alix marked endosomes. Collectively, our data demonstrate that the <i>Tn</i>BVANK1 protein acts as a virulence factor that causes the disruption of ecdysone biosynthesis and developmental arrest by impairing the vesicular traffic of ecdysteroid precursors in the prothoracic gland steroidogenic cells.</p></div

<i>TnBVank1</i> expression causes the block of the transition from larval to pupal stage.

<p>(A) Light micrographs of <i>yw;h-Gal4</i> larva and pupa (control) and <i>h-Gal4>TnBVank1</i> larvae at different days AED. The scale bar is 500 µm. (B) Larval length of different genotypes, at 96 h AED. Five larvae of each genotype were analyzed and as control we measured larval length of <i>yw</i> and <i>h-Gal4</i> and <i>UASp-TnBVank1</i> stocks and <i>yw;h-Gal4</i>. Graph represents mean ± standard deviation (s.d.); there is no significant (NS) length difference between <i>h-Gal4>TnBVank1</i> (2680±83 µm) and <i>yw;h-Gal4</i> larvae (2580±82 µm). (C) Larval length of <i>h-Gal4>TnBVank1</i> increases during the extended larval life. Five <i>h-Gal4>TnBVank1</i> larvae were measured at different days AED; values are the mean ± s.d. of three independent experiments. The mean values of <i>h-Gal4>TnBVank1</i> larval length at four and eighteen days AED are shown above the bars.</p

<i>Tn</i>BVANK1 protein colocalizes with Hrs- and Alix-positive vesicles.

<p>Confocal images of PG of <i>yw;phm-Gal4</i> (A–C) and <i>TnBVank1</i> (D–I) larvae stained for Hrs (cyan), Alix (red) and <i>Tn</i>BVANK1 (green). In the control cells Alix (B) and Hrs (A) are widely distributed in the cytoplasm and their signals partially overlap (C). In <i>TnBVank1</i> cells (D,F) a number of vesicles marked by Hrs have different shape compared to those present in controls (A,C). These modified vesicles show a strong colocalization with <i>Tn</i>BVANK1 signal (E,F), demonstrating that <i>Tn</i>BVANK1 protein is associated with Hrs-marked vesicles. In <i>TnBVank1</i> cells (G) most of Alix-marked vesicles have different shape compared to those present in controls (B). Immunostaining with anti-Alix and anti-<i>Tn</i>BVANK1 shows a strong colocalization of <i>Tn</i>BVANK1 and Alix signals in the stroke-shaped vesicles (H). In these modified vesicles the Alix and the Hrs signals are both detected (I). PGs in all panels are at the same magnification and the reference scale bar is 5 µm indicated in A.</p

<i>TnBVank1</i> PG cells show lipids accumulation.

<p>(A) In the control <i>yw;phm-Gal4</i> there are few lipid droplets stained with Oil Red O, while in <i>TnBVank1</i> cells several lipid droplets are detected (B,C). (E,F) In <i>TnBVank1</i> there is also a sterol accumulation, shown by filipin staining, which is absent in control PG (D). 60 PGs were stained for each experiment. PGs in panels A,B,D,E are at the same magnification and the reference scale bar 50 µm is showed in A. Boxed regions are magnified in C,F and the reference scale bar is 5 µm indicated in C.</p

<i>TnBVank1</i> PG cells have an altered cytoskeleton.

<p>Phalloidin staining in control (A,B) and in <i>TnBVank1</i> (C,D) PG cells. F-actin shows an altered distribution, characterized by thick masses of filaments in <i>TnBVank1</i> PG cells. (E–H) α-tubulin-GFP fusion protein was expressed in <i>yw;phm-Gal4</i> and <i>TnBVank1</i> PG to investigate the microtubule network. Compared to control (E,F), in <i>TnBVank1</i> the microtubule cytoskeleton is strongly affected and forms bundles (G,H). (I–L) Immunostaining with anti-Dynein heavy chain shows that, compared to control (I,J), in <i>TnBVank1</i> PG cells the cortical localization of this protein is reduced and characterized by an evident dotted distribution (K,L). For each immunostaining we analyzed 60 PGs of five days AED larvae. PGs in panels A,C,E,G,I,K are at the same magnification and the reference scale bar is 25 µm and showed in A. Boxed regions are magnified in B,D,F,H,J,I and the reference scale bar 5 µm is indicated in B.</p

The expression of <i>TnBVank1</i> in prothoracic gland affects the E biosynthesis.

<p>(A) Ring gland includes the prothoracic gland (PG; yellow), the corpora allata (CA; orange) and the corpora cardiaca (CC; red). (B) The expression of the <i>TnBVank1</i> gene is driven in the different ring gland compartments, highlighted in green, by three <i>Gal4</i> drivers. <i>P0206-Gal4>TnBVank1</i>, expressed in PG and CA, causes the developmental arrest at the last larval stage; <i>aug21-Gal4>TnBVank1</i> (CA) does not induce any developmental defects; <i>phm-Gal4>TnBVank1</i> (PG) blocks the transition from larval to pupal stage. (C) Total 20E titer in five larvae of <i>UASp-TnBVank1</i> stock (white bars), <i>phm-Gal4/TM6B</i> (grey bars) and <i>phm-Gal4>TnBVank1</i> (black bars), at different time (hours AED). In the control stocks <i>UASp-TnBVank1</i> and <i>phm-Gal4/TM6B</i>, the 20E peak which induces the pupariation is present at 120 h AED. Instead, this peak is absent in <i>phm>TnBVank1</i> larvae at 120 h AED and during the extended larval life. Error bars represent s.d.; *** = p<0.0001 versus controls (<i>UASp-TnBVank1</i> and <i>phm-Gal4/TM6B</i>). The mean values of total 20E at 120 h AED of different genotype larvae are shown above the bars. (D) Feeding <i>TnBVank1</i> larvae with medium supplemented with 20E induces the pupariation (red), while <i>TnBVank1</i> larvae fed with medium containing ethanol (EtOH) do not reach the pupal stage (green). Values are the mean ± s.d. of three independent experiments. The <i>yw;phm-Gal4</i> larvae serve as background control (blue). Immunostaining with anti-Dib in <i>yw;phm-Gal4</i> (E) and <i>TnBVank1</i> (F) PG reveals that the expression of Dib is strongly reduced in all <i>TnBVank1</i> PGs analyzed. Panels E,F are at the same magnification and the reference scale bar is 25 µm indicated in E.</p

<i>Tn</i>BVANK1 disrupts the endocytic pathway in PG cells.

<p>60 PGs stained for Rab5 (A,B), Rab11 (C,D) and Rab7 (E,F) in <i>yw;phm-Gal4</i> and <i>TnBVank1</i> larvae at five days AED. The distribution of endosomes marked with Rab5 (A,B) and Rab11 (C,D) is not affected by <i>TnBVank1</i> expression, while a reduction in number was observed for late endosomes marked with Rab7 (E,F). All panels are at the same magnification and the reference scale bar is 5 µm showed in A.</p

<i>Tn</i>BVANK1 distribution in the PG cells and its effects on PG.

<p>The immunolocalization of <i>Tn</i>BVANK1 in PG cells (marked with mCD8::GFP, green), analyzed with anti-<i>Tn</i>BVANK1 (cyan), shows its presence in stroke-shaped particles (A–C), which are distributed only in cytoplasm (nucleus is stained with Propidium Iodide, red) (B,C). (D) At five days AED, the PG of <i>yw;phm-Gal4</i> larvae, marked with GFP, is significantly larger (+54%) than the <i>TnBVank1</i> PG (E). (F) The graph represents the mean ± s.d.; 50 PGs were analyzed; *** = p<0.0001. (G) Measurement of PG cell area shows no difference between <i>yw;phm-Gal4</i> and <i>TnBVank1</i>. 50 PGs were analyzed; NS: not significant. (H–J) Immunostaining with anti-Cleaved Caspase-3 (red) or TUNEL labeling (red) in PG cells marked with GFP. In the control <i>yw;phm-Gal4</i> no caspase or TUNEL signal is detected (H,K), while in <i>TnBVank1</i> PG few cells undergo apoptosis (I,J,L,M). PGs in panels A,D,E,H,I,K,L are at the same magnification and their scale bar is 25 µm and is showed in A. Scale bar in B and C is 5 µm and showed in B. Boxed regions are magnified in J and M and the reference scale bar is in B. (N) Larvae of <i>yw;tub-Gal80^ts/+;phm-Gal4/+</i> (<i>Gal80^ts-phm-Gal4</i>) and <i>UASp-TnBVank1;UASp-TnBVank1/tub-Gal80^ts;phm-Gal4/+</i> (<i>Gal80^ts-TnBVank1)</i> were raised at 21°C (cyan) for different time intervals, then shifted at 31°C (red) and dissected at 120 h AED. PG size from larvae incubated at 21°C until 96 h AED or until 72 h AED shows no significant (NS) differences from control larvae. PG size is strongly reduced in <i>Gal80^ts-TnBVank1</i> larvae incubated at 21°C until 48 h AED compared to PG from control larvae (*** = p<0.0001). Graph represents mean ± s.d.; 10 PGs were analyzed for each experiment.</p

Seasonal dynamics of bees in colonies with low and high levels of mite infestation.

<p>(<b>A</b>) Estimated bee numbers recorded in each hive in October, when a sudden decrease of bee population was observed in highly infested colonies. (<b>B</b>) Bee mortality over time. The error bars indicate the standard deviation; mean values significantly different are denoted with asterisks (*<i>P</i>≤0.05; **<i>P</i>≤0.01). Bee population in highly infested colonies reached minimum levels in October, because of a marked increase of bee mortality.</p

Effect of the down-regulation of the transcription factor <i>dorsal-1A</i> by RNAi on DWV replication in bees.

<p>(<b>A</b>) <i>Dorsal-1A</i> transcript level in bees fed for different times with a sucrose/protein solution, containing dsRNA of honeybee <i>dorsal-1A</i> (dsRNA Dorsal) or dsRNA of Green Fluorescent Protein (dsRNA GFP) as a control. (<b>B</b>) Deformed wing virus genome copies in bees treated as above. The error bars indicate the standard deviation. The significant rate (H = 7.00, df = 1: <i>P</i> = 0.008) of silencing of the target gene triggered a significant increase (H = 9.61, df = 1: <i>P</i> = 0.002) of viral replication.</p