Direct peptide profiling of AKH and AKHGK in control and <i>amon^C241Y</i> flies.

<p>(A) Model of the processing of the AKH prepropeptide (top) and profiling of the larval ring gland (left) and adult corpora cardiaca (right). AKH is processed by a concerted action of a signal peptidase (SP) and <i>amon</i>, likely followed by a two-step carboxypeptidase (CP) action that first removes the C-terminal R yielding the intermediate AKHGK. AKHG is than amidated to bioactive AKH (not shown). While AKH and AKHGK were detected in most preparations from control and rescued (continued heatshock once a day) flies, they were not detectable in <i>amon</i> larvae. (B) Original direct mass profiles from corpora cardiaca of adult control (above) and <i>amon</i> (below) flies. AKH only occurs as the characteristic [M+Na]⁺ and [M+K]⁺ adducts, whereas AKHGK also occurs as [M+H]⁺. In the control fly, both peptides show higher signal intensities as the stable isotope-labelled standard peptide (AKH*). In the <i>amon</i> fly, the signal intensity is clearly higher for AKH* than for the native peptides. As previously reported <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000967#pgen.1000967-Predel1" target="_blank">[41]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000967#pgen.1000967-Wegener1" target="_blank">[42]</a>, no other mass peaks occur in the range 990-1220 Da in direct mass spectrometric CC profiles. (C) Standard curve for adult corpora cardiaca obtained with a dilution series of AKH* added to the matrix salt, male OrR wild-type flies. The y axis shows the signal intensity ratio of native AKH/AKH*. Error bars are S.E.M. The relationship of AKH/AKH* is linear for AKH* concentrations of 50–500 nM. (D) Peptide quantification with the labeled AKH* standard at 400 nM. The concentrations of both AKH and AKHGK are significantly reduced in <i>amon</i> flies vs. controls five days after eclosion and final heatshock. *p<0.05, **p<0.01, Mann-Whitney.</p

Expression of <i>amon</i> in the AKH cells of an <i>amon^C241Y</i> mutant is sufficient to rescue hypoglycemia.

<p>The gray bar represents larvae in which <i>amon</i> expression has been restored in the AKH producing cells (<i>yw; uas-amon/hs-amon; Df(3R) Tl-X e/akh-gal4, amon^C241Y</i>) as compared to <i>amon</i> mutants (<i>yw; uas-amon/hs-amon; Df(3R) Tl-X e/amon^C241Y</i> white bar) and control siblings (<i>yw; uas-amon/hs-amon; Df(3R) Tl-X e</i> or <i>amon^C241Y/TM3 Sb Ser y+ e,</i> black bar). n = number of larvae assayed; larvae were pooled in groups of three. p<0.0015, one way ANOVA.</p

Ubiquitous expression of AKH rescues the hypoglycemic defect seen in <i>amon</i> knockdown larvae.

<p>The left black bar represents wild-type levels of combined glucose and trehalose (<i>yw; hs-akh/+; akh-gal4/+</i>). The left blue bar represents combined sugar levels of AKH ablated larvae (<i>yw; hs-akh/uas-reaper; akh-gal4/+</i>) while the left gray bar shows glucose and trehalose levels in which <i>amon</i> has been reduced in the AKH cells by RNAi (<i>yw; hs-akh/uas-amon-RNAi^28b; akh-gal4</i>). Bars denoted with a ‘+’ below the graph indicate combined glucose and trehalose levels following heatshock induced expression of <i>akh via</i> a <i>hs-akh</i> transgene. n = number of larvae assayed; larvae were pooled in groups of three. *p = 0.002, **p<0.0001, one-way ANOVA.</p

<i>amon</i> is required in the AKH producing cells for normal sugar regulation.

<p>(A) The black bar indicates <i>amon</i> transcript levels in control larvae, while the white bar indicates <i>amon</i> transcript levels when <i>amon-</i>RNAi is ubiquitously expressed. Primers specific to <i>amon</i> exon 10 were used to assess <i>amon</i> transcript levels by quantitative real time PCR. (B) Dorsal and ventral views of a control pupa (top). Middle panels represent <i>amon</i> mutants that are unable to complete metamorphosis, and die with defects in head eversion and abdominal differentiation. <i>amon</i> RNAi knockdown animals also die with phenotypes similar to <i>amon</i> mutants (bottom). (C) Combined glucose and trehalose levels of control larvae are shown in the black bar. The center blue bar shows hemolymph sugar levels in AKH ablated larvae, while the gray bar represents animals in which <i>amon</i> expression has been reduced in the AKH producing cells by RNAi. n = number of larvae assayed; larvae were pooled in groups of three. **p<0.0001, one-way ANOVA.</p

Calculated levels of AKH in the corpora cardiaca of individual flies.

<p>*Days post eclosion and last heatshock.</p><p>(mean ± sem in fmol, (n)).</p

Characterization of antibodies purified from immune and control mouse sera.

<p>Total immunoglobulin pool was affinity purified from immune or control sera collected at the peak time point of neutralizing activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">figure 3B</a>). The ELISA-binding (A) and NAb (B) activities were then measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">Figure 3</a> and showed that immunization elicited an immune response leading to the production of anti-XMRV immunoglobulins.</p

Detection of XMRV-specific antibody production in mouse sera.

<p>Time course of the production of (A) ELISA-binding antibodies and (B) NAb in Balb/C mice (10 animals in each group) immunized with pDP1-XMRV<i>envgag</i> (first arrow; P), Ad5-XMRV (second and third arrows; A) and XMRV VLP (fourth arrow; V). Determination of (C) endpoint dilution and (D) serum neutralizing titers at the peak time point indicated by asterisks in Panels A and B, respectively. The arrow indicates endpoint dilution. (E) The specificity of the serum neutralizing activity was determined by comparing XMRV and HIV-1 pseudoviruses and showed that the primary target for neutralization is the XMRV Env.</p

Expression of XMRV Env, Gag and VLP.

<p>(A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.</p

Characterization of XMRV pseudovirus and single-round neutralization assay.

<p>(A) Comparison of XMRV and control HIV-1 pseudoviruses in yield (p24 accumulation) and infectivity (IU/ml on TZM-bl cells). (B) Detection of antibody specificity to XMRV and HIV-1 pseudoviruses. Pseudoviruses were tested in the neutralization assay with mAb 83A25 that recognizes a shared epitope of MLV Env glycoprotein and with mAb b12 that recognizes HIV-1 Env glycoprotein. (C) Neutralization of the XMRV and HIV-1 pseudoviruses showing a broad range of sensitivity and specificity of the assay using polyclonal antibodies (anti Friend-MuLV).</p