Qualitative and quantitative reporters of <i>in vivo</i> viral replication.

<p><b>A.</b> Adult, anesthetized flies expressing SinR-GFP using eye-specific driver GMR-GAL4. Virtually no GFP expression is detectable. <b>B.</b> Quantification of viral replication <i>in vivo</i>, using Luciferase-expressing, replication-competent replicon SinR-Luc. Mutations in <i>imd</i> resulted in significantly higher activity. In contrast, knock-down of Akt and Pi3K using UAS-RNAi constructs resulted in a significant decrease. (Luminometer counts in relative units per fly, per µL of homogenate). <b>C.</b> Strong GFP expression can be observed in the entire eye, when RNAi is inhibited using homozygous <i>Dcr2</i> mutants. <b>D.</b> Quantification of RNAi effects on viral replication <i>in vivo</i>, using SinR-Luc in combination with different ways of inhibiting RNAi (UAS-B2 co-expression, homozygous <i>Drc2</i> mutants). Inhibition of RNAi greatly increased SinR-Luc activity. Note that UAS-Luciferase control levels are unaffected by suppression of RNAi. <b>E.</b> SinR-GFP[GVD] with a point mutated RNA-dependent RNA Polymerase never results in GFP expression as detected by <i>in vivo</i> fluorescence. <b>F.</b> Rescue of GFP expression from SinR-GFP[GVD] in <i>trans</i>, using GMR-GAL4, through co-expression of non-fluorescent replicon SinR-Luc, providing an active replicase.</p

Quantification of Sindbis replicon expression in different tissues.

<p><b>A,C.</b> Examples of SinR-GFP expression in different tissues. Labeled are adult neurons (<i>NSyb</i>-GAL4; A) or adult fat body (<i>r4</i>-GAL4; C). For both tissues, RNAi was inhibited using UAS-B2. <b>B.</b> Quantification of SinR-Luc Luciferase activity in neurons. Significantly higher levels of Luciferase activity were obtained in homozygous <i>Dcr2</i> mutants, comparable to those obtained with UAS-Luciferase controls. Inactivation of RNAi using UAS-B2 had weaker yet comparable effects while <i>Drc2</i> heterozygotes show little to no effect. <b>D.</b> Similar effects were obtained in other tissues, like the adult fat body). All luminometer counts in relative units per fly, per uL of homogenate.</p

Trans-activation of defective helper replicons.

<p><b>A.</b> Expression of replication-defective replicon DH-TOM in the adult eye, using GMR-GAL4 in <i>Dcr2</i> homozygotes. Low levels were visible as ‘pseudopupil’, in the center of the eye, most likely due low-level ribosomal read-through (despite numerous nonsense ATG's). <b>B.</b> Strong expression of DH-TOM activated in <i>trans</i> from a 2^nd replicon (SinR-GFP), contributing an intact RdRP. However, red fluorescence is sparse, and co-expression of GFP and Tomato is rare. <b>C.</b> The point-mutated replicon SinR-GFP[GVD] always failed to trans-activate defective replicon DH-TOM. <b>D.</b> Third instar larval eye discs dissected from flies co-expressing UAS-B2, SinR-GFP, and DH-TOM reveal a low level of myr:Tomato trans-activation (C′). <b>E.</b> Luciferase activity (in relative units per fly, per µL of homogenate) of defective DH-Luc in different genetic backgrounds. Significant levels of trans-activation by an intact replicon (SinR-GFP) were observed when RNAi was inactivated (+UAS-B2, or <i>Dcr2</i> homozygotes). However, the absolute levels were very low when compared to SinR-Luc activity in the same backgrounds. <b>F.</b> Co-expression of replicons with deleted replicase ORF (DH-Tom), or a point-mutation (SinR-GFP[GVD]) never trans-activated Luciferase activity of DH-Luc.</p

A <i>Drosophila</i> Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

<div><p>Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile <i>Drosophila</i> toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication <i>in vivo</i>. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our <i>in vivo</i> system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development.</p></div

A toolkit of transgenic Sindbis replicons.

<p><b>A.</b> Schematic of Sindbis genome, a bicistronic single-stranded RNA with positive polarity: the 5′ end contains a ‘packaging signal’ (PS) for incorporation into the particle. An ‘internal Promoter’ (iP) can be found (on the ‘antigenome’; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112092#pone.0112092.s001" target="_blank">Figure S1A</a>) in between the viral ORF's. Abbreviations: nsp  =  ‘non-structural proteins’; sp  =  ‘structural proteins’. <b>B.</b> Four transgenic fly strains containing different Sindbis replicons (SinR) stably inserted into the genome. Each transgenic replicon is harboring different reporter genes, or mutations. Abbreviations: UAS  =  GAL4 ‘GAL4 Upstream activating sequence’; TATA  =  hsp70 TATA box; RBZ: Hepatitis Delta Ribozyme; GFP  =  membrane tagged mCD8:eGFP fusion protein; TOM  =  myristoylated Tomato; Luc  =  firefly luciferase; nsp[GVD]  =  point-mutated RNA-dependent RNA Polymerase. <b>C.</b> Four replication-incompetent replicons (SinR), all lacking ORF1 due to deletions in the Sindbis genomic DNA sequence, and harboring different sequences in ORF2. Note that DH-EB harbors a smaller deletion, thus retaining a ‘packaging signal’.</p

ORF2 does not induce exclusion and Model.

<p><b>A.</b> Quantitative test whether defective helper transgenes excluded replicon expression, using SinR-Luc. In homozygous <i>Dcr2</i> mutants, expression levels of SinR-Luc were unaffected by co-expression of either DH-TOM, DH-BB or DH-EB defective replicons, all harboring deletions of ORF1. Co-expression of foreign glycoproteins (UAS-G[VSV]) also had no significant effect on Luciferase expression. <b>B.</b> Model summarizing factors regulating <i>in vivo</i> replicon expression. While replicon expression is inhibited by cellular pathways (RNAi, NMD, innate immunity), a strong preference of the viral RdRP for the internal promoter on the ‘subgenomic RNA’ originating from the same transcript exists. As a result, trans-activation is weak, even from transgenes with a deleted RdRP.</p

Genetic Dissection of Photoreceptor Subtype Specification by the <i>Drosophila melanogaster</i> Zinc Finger Proteins Elbow and No ocelli

<div><p>The <i>elbow/no ocelli</i> (<i>elb</i>/<i>noc</i>) complex of <i>Drosophila melanogaster</i> encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the ‘dorsal rim area’ (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease.</p></div

Elb and Noc are crucial for Homothorax function in DRA specification.

<p><b>A.</b>–<b>C.</b> Homothorax fails to ectopically induce the DRA fate in absence of Elb and Noc. (Ommatidial schematic to the left summarizes the only ommatidial subtype found inside the dashed white boxes; pink dot: nuclear Sens expression in R8). <b>A.</b> Exclusion of Rh6 (green) from the DRA (labeled with Hth, purple), in wild type flies (dashed white box). <b>B.</b> Complete loss of Rh6 upon over-expression of Hth (purple), under LGMR-GAL4 control. <b>C.</b> Co-expression of Rh6 (green) and Hth (purple) in <i>elb,noc</i> double mutant flies ectopically expressing Hth (green) in all photoreceptors. <b>D.</b> Expression of <i>elb</i>-GAL4 (visualized using UAS-lacZ:NLS) is never expanded into R1-6 by over-expression of Hth (green). Instead, strong expression of βGal (red) is observed in all inner photoreceptors R7 and R8 (co-labeled with Spalt, green). <b>E</b>–<b>G.</b> Homothorax fails to repress Senseless in absence of <i>elb</i> and <i>noc</i>. Whole mounted pupal retina expressing Hth in all photoreceptors (LGMR-<i>hth</i>; red) with homozygous clones lacking both <i>elb</i> and <i>noc</i> marked by absence of <i>arm</i>-<i>lacZ</i> (blue, E). The vast majority of strong Sens expression (green) is observed in R8 cells inside homozygous clones (F), as well as in close vicinity to mutant clones (G).</p

Expression of <i>elbow</i> (<i>elb</i>) and <i>No ocelli</i> (<i>noc</i>) in photoreceptors.

<p><b>A.</b> Schematic of the <i>elb</i>/<i>noc</i> locus with GAL4 enhancer traps shown as red triangles. <b>B.</b> Schematic summarizing Rhodopsin expression in the three main ommatididal subtypes of <i>Drosophila</i>. L  =  lamina; M  =  medulla. <b>C.</b> Domain structure of Elb/Noc proteins: like most members of the NET family of transcriptional repressors they contain a single C2H2 zinc finger domain at the C-terminus (light blue), as well as a conserved SPLALLA motif of unknown function at the N-terminus (purple). In between the two lies a Groucho-binding motif (yellow). <b>D.</b> GFP fluorescence of <i>elb</i> > UAS-eGFP observed under water immersion. Fluorescence localizes to the central photoreceptors of each ommatidium (R7 or R8), as well as non-neuronal cells (green blur). <b>E.</b> Cryostat cross section through an adult eye expressing βGalactosidase (<i>elb</i> > <i>lacZ</i>). Strong expression was observed in R7 and R8 of the Dorsal Rim Area (red dashed box). Additional expression exists in the brain, in many R8 cells, as well as few R7 cells throughout the retina. <b>F.</b> Elbow expression (<i>elb</i> > <i>lacZ</i>:NLS; red) co-localizes with DRA marker Homothorax (Anti-Hth; green; white arrows). <b>G.</b> Expression of <i>elb</i>-GAL4 driving UAS-<i>lacZ</i>:NLS in the adult retina (visualized on a Cryostat cross section): expression is restricted to R7 and R8 nuclei, as well as non-neuronal cone cell nuclei above the R1-6 level. <b>H.</b> Adult expression of <i>noc</i>-GAL4 is virtually indistinguishable from <i>elb</i>. Expression is strongest in R7 and R8 in the DRA (red dashed box), as well as subsets of R7 and R8 cells outside the DRA. <b>I. </b><i>No ocelli</i> expression (<i>noc</i> > lacZ:NLS; red) also co-localizes with DRA marker Homothorax (Anti-Hth; green; white arrows).</p

VP16- and <i>en</i>[R]-fusions of Noc specifically affect R8 rhodopsin expression.

<p><b>A.</b> Schematic of VP16:<i>noc</i> fusion cDNA generated in UAS-constructs for over-expression (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>B</b>,<b>C.</b> Mild effect of VP16:<i>noc</i> over-expression on DRA development: Rh3 expression (red) in the DRA is weak (arrows), yet detectable (B), and Hth expression (green) is normal (C, arrows). <b>D,E.</b> VP16:<i>noc</i> has a strong activating effect on Rh5 expression (blue), resulting in a high ratio of pR8 cells with Rh5. Since Rh6 expression appears normal (green), many R8 cells now co-express the two R8 rhodopsins (white arrows). <b>F.</b> Schematic of Engrailed fusion cDNAs generated for Noc (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004210#s4" target="_blank">Materials and Methods</a>). <b>G.</b> Ectopic over-expression of <i>en[R]:noc</i> has no effect on R7 rhodopsin expression (Rh3 in red; Rh4 in cyan) and DRA specification (arrows). <b>H.</b> Expression of Rh5 (blue) is lost upon over-expression of <i>en[R]:noc</i>, and Rh6 (green) is the only R8 rhodopsin remaining.</p