Arbovirus-Derived piRNAs Exhibit a Ping-Pong Signature in Mosquito Cells

The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed

GFAP-Driven GFP Expression in Activated Mouse Muller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background: Muller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Muller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.Methodology/Principal Findings: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Muller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Muller glial cells, several other inner retinal cell types were transduced. To obtain Muller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Muller glial cells aligning retinal blood vessels.Conclusions/Significance: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells

Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

Contains fulltext : 136405.pdf (publisher's version ) (Open Access)The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors

Posaconazole inhibits dengue virus replication by targeting oxysterol-binding protein

Dengue virus (DENV) is associated with an estimated 390 million infections per year, occurring across approximately 100 countries in tropical and sub-tropical regions. To date, there are no antiviral drugs or specific therapies to treat DENV infection. Posaconazole and itraconazole are potent antifungal drugs that inhibit ergosterol biosynthesis in fungal cells, but also target a number of human proteins. Here, we show that itraconazole and posaconazole have antiviral activity against DENV. Posaconazole inhibited replication of multiple serotypes of DENV and the related flavivirus Zika virus, and reduced viral RNA replication, but not translation of the viral genome. We used a combination of knockdown and drug sensitization assays to define the molecular target of posaconazole that mediates its antiviral activity. We found that knockdown of oxysterol-binding protein (OSBP) inhibited DENV replication. Moreover, knockdown of OSBP, but not other known targets of posaconazole, enhanced the inhibitory effect of posaconazole. Our findings imply OSBP as a potential target for the development of antiviral compounds against DENV

<i>Aedes aegypti</i> Aag2 cells produce transposon-derived piRNAs with a ping-pong signature.

<p><b>A.</b> Size distribution of the small RNA reads that match with 0 mismatches against an <i>Aedes aegypti</i> transposon dataset that contain full-length non-composite transposons sequences (TEfam: <a href="http://tefam.biochem.vt.edu/tefam/index.php" target="_blank">http://tefam.biochem.vt.edu/tefam/index.php</a>). <b>B.</b> Heat map for 25–29 nt small RNAs that mapped to individual retrotransposons with more than 1000 reads. Read count and log-transformed ratios of antisense/sense small RNAs are presented. <b>C</b>. Profile of 25–29 nt reads that mapped to the transposon Copia Ele56 (TF000691) allowing 0 mismatch during alignment. Transposon-derived piRNAs that mapped to the sense and antisense strand of the transposon sequence are shown in red and blue, respectively. <b>D.</b> Conservation and relative nucleotide frequency per position of 25–29 nt reads that mapped to the sense (top) and the antisense (bottom) strands of the entire transposon dataset. n indicates the number of reads used to generate each logo.</p

U4.4 cells produce vsiRNAs and vpiRNAs through a ping-pong mechanism upon (+) ssRNA arbovirus infection.

<p>Profile of 21 nt vsiRNAs (<b>A</b>) and 25–29 nt (<b>B</b>) SINV-GFP-derived small RNAs allowing 0 mismatch during alignment. Viral small RNAs that mapped to the sense and antisense strand of the SINV-GFP genome are shown in red and blue, respectively. <b>C.</b> Conservation and relative nucleotide frequency per position of 25–29 nt SINV-GFP-derived reads that mapped to the sense (top) and the antisense (bottom) strands of the SINV-GFP genome. The overall height of the nucleotide stack indicates the sequence conservation; the height of the nucleotides within each stack represents their relative frequency at that position. n indicates the number of reads used to generate each logo. <b>D.</b> Frequency map of the distance between 25–29 nt small RNAs that mapped to opposite strands of the SINV-GFP genome. The peak at position 9 on the sequence (the first nucleotide being position 0) indicates the position of maximal probability of finding the 5′ end of a complementary small RNA.</p

<i>Aedes albopictus</i> U4.4 cells are Dcr-2 competent and produce two populations of viral small RNAs.

<p><b>A.</b> Dicer assay in uninfected U4.4 cells. Lane 3 shows processing of a 113-bp dsRNA substrate into 21-nt siRNAs after incubation in a U4.4 cell extract. Synthetic siRNA (21-nt) and input dsRNA (113-nt) are used as size markers in lanes 1 and 2, respectively. <b>B.</b> RNAi reporter assay. Co-transfection of firefly luciferase specific dsRNA with reporter plasmids encoding firefly and <i>Renilla</i> luciferase into U4.4 cells results in silencing of the firefly luciferase reporter. GFP dsRNA was used as non-specific dsRNA control. <i>Renilla</i> luciferase activity was used as internal control to normalize the firefly luciferase activity. Error bars represent the standard deviations of three individual samples. <b>C.</b> Size distribution of the small RNA reads that match the genome of SINV-GFP with 0 mismatches.</p

VP1 suppressor activity is species-specific.

<p>(<b>A</b>) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from <i>D. melanogaster</i> Nora virus (DmelNV) and <i>D. immigrans</i> Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1^ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s002" target="_blank">Figure S2A</a>, lane 5, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256-vanMierlo2" target="_blank">[14]</a>). (<b>B</b>) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and <i>Renilla</i> luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (<b>C</b>) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1^ΔN284 or DimmNV VP1^ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's <i>post hoc</i> test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** <i>P</i><0.01; *** <i>P</i><0.001; ns, not significant.</p