VMCSs promote the morphogenesis and functioning of viral replication organelles.

<p>(A) The formation of invagination-type replication organelles is driven by vMCSs through direct protein–protein interactions of a viral replication protein (e.g., TBSV p33) with cellular ORPs and the ER residential VAPs. The vMCS, which is likely stabilized by co-opted actin filaments, facilitates the enrichment of sterols in the peroxisomal membrane. (B) An electron microscopic (EM) image of a vMCS and multiple vesicle-like structures (spherules), each of which harbors a viral replicase complex (VRC), which replicates the viral genome, is shown from a TBSV-infected plant cell [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005912#ppat.1005912.ref013" target="_blank">13</a>]. We propose that the vMCS forms transiently between two subdomains of apposing membranes, locally enriching sterols, then sliding onto new neighboring subdomains of the same organelles, thus acting as a molecular assembly line. (C) vMCSs between protrusion-type replication organelles and the ER depend on the recruitment of PI4Ks by VPs. At the replication organelle membranes, PI4Ks produce PI4P lipids to recruit OSBP. Then, OSBP mediates the accumulation of cholesterol in replication organelle membranes in a counter-exchange with PI4P, which is hydrolyzed after delivery at the ER by Sac1 phosphatase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005912#ppat.1005912.ref010" target="_blank">10</a>]. OSBP is naturally anchored to the ER by VAPs, which for some viruses are known to interact with VPs (see text).</p

Phenotypic characterization of the SL-A stem mutants.

<p>Phenotypic characterization of the SL-A stem mutants.</p

Phenotypic characterization of the SL-A loop mutants.

<p>Phenotypic characterization of the SL-A loop mutants.</p

Mutations affecting the mengovirus SL-A stem structure.

<p>(A). Mutations introduced by site-directed mutagenesis. (B) Luciferase activity produced by wild-type and the four SL-A loop mutants. Mengovirus OriL SL-A mutations were introduced into the mengovirus luciferase replicon pRzpM-Luz. At various time points after infection luciferase was measured.</p

BDCA1⁺ mDCs more efficiently phagocytose murine Min6 cells compared to BDCA3⁺ mDCs.

<p><b>A</b>) BDCA1⁺ mDCs or <b>B</b>) BDCA3⁺ mDCs were co-cultured overnight (o/n) with PKH67-labeled mock- or CVB-infected Min6 cells (M6/M and M6/CVB, respectively) stained for the CD11c or BDCA3 and analyzed by flow cytometry on viable, single cells (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.s001" target="_blank">S1 Fig</a>). Percentages in upper right corner represent the percentage of DCs that has engulfed Min6 material. This was calculated as follows: percentage PKH⁺ DCs/total DCs (i.e. total CD11c positive cells or total BDCA3 positive cells) <b>C</b>) DCs were analyzed as in A) and B), shown is average + SEM for >3 donors. * p<0.05, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

Mutations affecting the mengovirus SL-A loop structure.

<p>(A). Mutations introduced by site-directed mutagenesis. (B) Plaque assay of the wild-type mengovirus (up) and the ΔG43-A46 mutant (down). Plaque assay were performed in L929 cells. Cells were stained with crystal violet. (C) Luciferase activity produced by wild-type and the four SL-A loop mutants. Mengovirus OriL SL-A mutations were introduced into the mengovirus luciferase replicon pRzpM-Luz. At various time points after infection luciferase was measured.</p

Iminoproton assignment in the apical part of SL-A of the cardiovirus oriL.

<p>(A) Secondary structure of the RNA element used for NMR studies. (B) 1D iminoproton spectrum (600 MHz) recorded in H₂O/10% D₂O. Iminoproton assignments are indicated.</p

Conservation of cardiovirus OriL SL-A element.

<p>(A) Multiple alignment of SL-A of the cardiovirus OriL. The upper part shows the alignment of the EMCV subgroup of the cardiovirus genus. The lower part shows the alignment of the theiloviruses, including the human Theiler's like virus. The apical part of the stem-loop is shaded and includes the EMCV SL-A octaloop and the Theilovirus SL-A tetraloop with two closing base-pairs. (B) Secondary structures of OriL SL-A elements from cardioviruses and parechovirus 5. Structures were calculated using MFold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024818#pone.0024818-Zuker1" target="_blank">[14]</a>.</p

Type I and type III IFN responses are induced in mDC subsets upon encounter of CVB-infected cells.

<p><b>A</b>) DCs were co-cultured with mock- or CVB-infected Min6 cells (M6/M or M6/CVB), infected with CVB3 (MOI 50), stimulated with poly I:C, or left unstimulated (medium; Med) and after o/n incubation supernatant was harvested and analyzed for production of IFN-α2 (left panel) or IFN-λ1 (right panel). Shown are data from 4 (IFN-α2) and 3 (IFN-λ1) different donors. <b>B</b>) DCs were stimulated as in A) and after 6 hours mRNA expression was analyzed by qPCR. Shown are data from 4 donors (B, corresponding symbols represent the same donor). * p<0.05, ** p<0.01, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

EV, but not CVB infection impairs TLR-induced responses in BDCA1+ mDCs.

<p>A) Freshly isolated BDCA1⁺ mDCs were infected as indicated (MOI 50) and after 16 h all cells were stimulated with poly I:C (20 µg/ml). After an additional 24 h expression of cell surface markers was determined using flowcytometry. Data is shown as mean fluorescent intensity minus isotype control. B) Supernatant taken from mDCs infected and stimulated as in A) was analyzed for production of IL6 and TNF-α. Shown are averages of 2 experiments using different donors. Statistical significance determined by Students T-test, * p<0.05. **p<0.01.</p