Production of Virus-Derived Ping-Pong-Dependent piRNA-like Small RNAs in the Mosquito Soma

The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication

Synthesis and antiproliferative activity of 3-aryl-2-[1H(2H)-benzotriazol-1(2)-yl]acrylonitriles variously substituted:Part 4

A new series of variously substituted 3-aryl-2-[1H(2H)-benzotriazol-1(2)-yl]acrylonitriles was synthesized and tested for antiproliferative and antitubercular activity as part of our continuing research program in the antimicrobial and antitumor fields. The most cytotoxic derivatives (5a,g,i,j,l and 7b) (CC50 < 3.0 microM against MT-4 cells) were evaluated against a panel of human cell lines derived from hematological and solid tumors, using 6-mercaptopurine (6-MP) and etoposide as reference drugs. In particular, E-2-(5,6-dimethyl-1H-benzotriazol-1-yl)-3-(3-nitrophenyl)acrylonitrile (5g) resulted more potent than 6-MP on all cell lines, even if 2-14-fold less potent than etoposide. In the antitubercular screening, the derivatives 5i,j and 7e showed moderate activity against some resistant strains of Mycobacterium tested

1,5-benzodiazepines XIV. Synthesis of new substituted 9H-bis-[1,2,4]triazolo[4,3-a:3’,4’-d][1,5]benzodiazepines and relate compounds endowed with in vitro cytotoxic properties

A series of 11 new 9H-bis-[1,2,4]triazolo[4,3-a:3',4'-d] [1,5]benzodiazepine derivatives 8e-o was synthesized. Ten of these compounds (8e-m,o), along with four analogues (8a-d) (previously synthesized by us) were tested in vitro in order to evaluate their cytotoxic and anti-HIV-1 properties. In this connection other six original compounds, i.e., five 9-substituted compounds prepared starting from the 6,12-diphenylderivative 8c (compounds 10, 11, 12, 13a,b) and the bis-triazolone derivative 14, were synthesized and tested for the same purpose. While none of the 20 compounds tested exhibited any appreciable anti-HIV-1 activity, some of them exhibited interesting cytotoxic properties, the best results being shown by compounds 8c,d,k and 11 (CC(50) range=3-12 microM). Therefore, these four compounds were further evaluated for their antiproliferative activity against a panel of human tumor cell lines; actually, compounds 8d, 8k and 11 showed antiproliferative properties against either or both leukemia- and lymphoma-derived cell lines in the low micromolar range

B2-mediated suppression of piRNA-like viral small RNAs in <i>dcr-2</i> null mutant cells.

<p>Size distribution and nucleotide analysis of virus-derived small RNAs in <i>dcr-2^FS−1</i> cells infected with CHIKV-B2 (FHV) (<b>A</b>) or CHIKV-B2 (C44A) (<b>B</b>). Single representative TruSeq libraries are shown (replicate #2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002470#ppat.1002470.s004" target="_blank">Table S1</a>). CHIKV (+) strands per virus-derived small RNA in 1 ug of total RNA (calculated from normalized 25–29 nt reads identified in replicate TruSeq libraries) (<b>C</b>). Error bars indicate the standard deviation among three biological replicates. Modulation of alphavirus infection by an antiviral piwi-like RNA pathway in <i>dcr-2^FS−1</i> (C6/36) cells (<b>D</b>). Time course of cytopathology in <i>dcr-2^FS−1</i> (C6/36) cells infected with recombinant CHIK viruses (20X magnification).</p

Production of piRNA-like viral small RNAs in the mosquito soma.

<p>Size distribution, density plots, and nucleotide analysis of virus-derived small RNAs in <i>A. aegypti</i> (<b>A</b>), <i>A. albopictus</i> (<b>B</b>) and head and thorax of <i>A. albopictus</i> (<b>C</b>) infected with CHIKV. Example weblogos are shown for the predominant size classes. Arrows denote approximate start of 26S mRNA.</p

Model for RNA-based immune pathways modulating alphavirus pathogenesis in the mosquito soma.

<p>Following entry and uncoating, the genomic (+) strand RNA of an alphavirus serves both as mRNA and as a template for the synthesis of complementary (−) strand RNA. The viral (−) strands then serve as templates for the synthesis of new genomic-length (+) strand RNAs, as well as for shorter subgenomic (+) strand RNAs (26S mRNA) that encode the virus' structural genes. Alphaviruses are thought to synthesize (−) strand RNAs for a limited duration of time early in the infection, establishing an upper limit on the number of dsRNA RIs present in the cell. However, production of the (+) single-stranded genomic (49S) and subgenomic (26S) RNAs continues much longer, ultimately becoming the predominant virus-specific RNAs present in the cell. In this model antiviral siRNA and piRNA-like viral small RNA biogenesis pathways compete for a limited number of precursor dsRNA RIs in the infected cell. While recognition of dsRNA activates both pathways, secondary piRNA-like viral small RNAs are preferentially generated from viral mRNAs. Efficient processing of dsRNA RIs by Dcr-2 may restrict the amount of precursor substrate available to enter the piRNA-like viral small RNA biogenesis pathway. The B2 protein binds both siRNA duplexes and long dsRNAs preventing the protein components of antiviral pathways access to dsRNAs, but inhibition is not absolute. Elevated levels of viral replication may increase amplification of secondary piRNA-like viral small RNAs from 49S and 26S mRNA substrates.</p

Identification of <i>dcr-2</i> null mutant mosquito cell lines.

<p>Size distribution and nucleotide analysis of virus-derived small RNAs in u4.4 cells (<b>A</b>), <i>dcr-2^FS−1</i> (C6/36) cells (<b>B</b>) and <i>dcr-2^{del 33}</i> (C7-10) cells (<b>C</b>) infected with CHIKV. Schematics indicating Dcr-2 domains (<b>D</b>). The <i>A. albopictus</i> Dcr-2 contains a DExH/D protein family domain (DEAD) and helicase conserved C-terminal domain (H); a domain of unknown function (DUF); a PAZ domain; and tandem RNase III domains. Asterisks indicate locations of deletions in <i>dcr-2</i> sequences.</p