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

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

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

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

Cooler Temperatures Destabilize RNA Interference and Increase Susceptibility of Disease Vector Mosquitoes to Viral Infection

<div><p>Background</p><p>The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.</p><p>Methodology/Principal Findings</p><p>We utilized transgenic “sensor” strains of <i>Aedes aegypti</i> to examine the role of temperature on RNA silencing. These “sensor” strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.</p><p>Conclusions/Significance</p><p> This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.</p></div

YFV infection of mosquitoes following rearing at 18°C or 28°C.

<p>Infectivity of YFV for <i>Ae. aegypti</i> (Lvp strain) and <i>Ae. albopictus</i> (Wise County) reared at 18°C or 28°C. Each bar represents the average of three biological replicates of 39–50 mosquitoes each; error bars indicate one standard deviation. Significance was assessed via a two-tailed Student's t-test.</p

Small RNA sequencing from 3×P3-sensor heads.

<p>Histogram displaying the length distribution of EGFP-derived small RNAs from 3×P3-sensor mosquito heads following rearing at 18°C or 28°C. Numbers above bars indicates fold increase from 28°C to 18°C. Sense (solid bar) and antisense (cross-hatched) siRNA totals are displayed in the same stack for each sample.</p

Low-temperature activation of RNAi sensor mosquitoes is reversible and can be induced in adults.

<p>Photographs taken at 7 or 14 days post emergence (7 d or 14 d) of typical individuals from <i>Ae. aegypti</i> RNAi sensor strain #2 following rearing at 18°C (<b>A</b>) or 28°C (<b>B</b>). Adult females were transferred to the indicated temperature at 1 day post-emergence; photographs are EGFP (top panel) or DsRED (bottom panel). Real-time qPCR of EGFP mRNA levels in 3×P3-sensor mosquito heads following rearing at 18°C (<b>C</b>) or 28°C (<b>D</b>), with newly emerged adults held at the alternate temperature for the indicated number of days. Error bars indicate one standard deviation corresponding to technical variation for a representative biological replicate. (<b>E</b>) Real-time qPCR of EGFP mRNA levels in transgenic RNAi sensor heads following rearing at 18°C or 28°C, with mosquitoes remaining at the same temperature as adults. Error bars indicate the standard deviation among three biological replicates; *** indicates significance at the p<0.001 level as determined by two-tailed Student's t-test.</p

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.−) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an <i>S</i>-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, ⁴⁷⁵<b>N</b>V<b><u>C</u></b>WA<b>K</b>⁴⁸⁰, which differs from papain’s (CGS²⁵<b><u>C</u></b>WAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in <i>k</i>_cat/<i>K</i>_m, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in <i>k</i>_cat/<i>K</i>_m. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased <i>K</i>_m 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly but not significantly increased <i>k</i>_cat and <i>K</i>_m. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity