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. 2019 Dec 2;13(12):e0007919.
doi: 10.1371/journal.pntd.0007919. eCollection 2019 Dec.

Polymorphism analyses and protein modelling inform on functional specialization of Piwi clade genes in the arboviral vector Aedes albopictus

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Polymorphism analyses and protein modelling inform on functional specialization of Piwi clade genes in the arboviral vector Aedes albopictus

Michele Marconcini et al. PLoS Negl Trop Dis. .

Abstract

Current knowledge of the piRNA pathway is based mainly on studies on Drosophila melanogaster where three proteins of the Piwi subclade of the Argonaute family interact with PIWI-interacting RNAs to silence transposable elements in gonadal tissues. In mosquito species that transmit epidemic arboviruses such as dengue and chikungunya viruses, Piwi clade genes underwent expansion, are also expressed in the soma and cross-talk with proteins of recognized antiviral function cannot be excluded for some Piwi proteins. These observations underscore the importance of expanding our knowledge of the piRNA pathway beyond the model organism D. melanogaster. Here we focus on the emerging arboviral vector Aedes albopictus and we couple traditional approaches of expression and adaptive evolution analyses with most current computational predictions of protein structure to study evolutionary divergence among Piwi clade proteins. Superposition of protein homology models indicate possible high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with several sites under positive selection. Analysis of the expression profiles of Piwi transcripts during mosquito development and following infection with dengue serotype 1 or chikungunya viruses showed a concerted elicitation of all Piwi transcripts during viral dissemination of dengue viruses while maintenance of infection relied on expression of primarily Piwi5. Opposite, establishment of persistent infection by chikungunya virus is accompanied by increased expression of all Piwi genes, particularly Piwi4 and, again, Piwi5. Overall these results are consistent with functional specialization and a general antiviral role for Piwi5. Experimental evidences of sites under positive selection in Piwi1/3, Piwi4 and Piwi6, that have complex expression profiles, provide useful knowledge to design tailored functional experiments.

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Conflict of interest statement

The authors have declared that no competing interests exist. Jacob E. Crawford, Bradley White and Teresa Lin are employed by a commercial company, Verily Life Sciences LLC. They have no competing interests.

Figures

Fig 1
Fig 1. Gene and transcript structure of Ae. albopictus Piwi5 and Piwi7.
A) Schematic representation of the DNA structure of Piwi5 and Piwi7 genes and their corresponding transcripts as obtained from cDNA amplification of single sugar-fed mosquito samples. Exons and introns are shown by blue boxes and black lines, respectively, with corresponding length in nucleotide below each. The positions of the predicted PAZ, MID and PIWI domains are shown by green, blue and magenta ovals, respectively. Exon numbers correspond to lane numbers. B) Amplification of each exon of Piwi5 and Piwi7 on genomic DNA. Exon numbers correspond to lane numbers. C) Northern-blot results of Piwi5 indicate the presence of a transcript of 3 kb.
Fig 2
Fig 2. Venn diagrams showing the number of positions harbouring synonymous and non-synonymous mutations in tested samples for each Piwi gene.
Fig 3
Fig 3. Volcano plot.
Level of polymorphism (LoP) comparison between slow-evolving genes (SGs), fast-evolving genes (FGs) and Piwi genes by population. Genes on the right side of the panel have LoP values greater than those of SGs, while genes on the left side have smaller LoPs than SGs. The y-axis represents the -log10 p-values of the Kolmogorov-Smirnov test. Faint datapoints are not significant after Bonferroni correction for multiple testing (-log10 0.0024 (0.05/21 genes) = 2.62).
Fig 4
Fig 4. Computational homology models of the Ae. Albopictus Piwi proteins.
Homology models were generated for the seven Piwi genes as described in the methods section. A) Superposition of cartoon representations of Piwi homology models, with highlight of domain organization: the N-terminal domain is shown in orange, the PAZ domain in green, the MID domain in blue and the PIWI domain in magenta. B) CONSURF [46]overview of the aminoacid sequence conservation mapped on three-dimensional homology models in a putative RNA-bound arrangement based on the structure of human Argonaute bound to a target RNA (PDB ID 4Z4D), colored from teal (very low conservation) to dark magenta (highly conserved).
Fig 5
Fig 5. Expression profile of Piwi genes.
Heatmap representations of log10 transformed fold-change expression values of each Piwi gene. A) Developmental expression pattern of the Piwi genes normalized on the expression in sugar fed females. B) Expression pattern of Piwi genes following viral infection normalized with respect to sugar-fed samples. Expression was verified in ovaries and carcasses separately, during the early and late stages of infections, that is 4 dpi for both viruses and 14 or 21 dpi for CHIKV and DENV, respectively. Each day post-infection was analysed with respect to sugar and blood-fed controls of the same day. * indicates significant difference (P<0.05) between infected samples and the corresponding blood-fed control.

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Grants and funding

This research was funded by a European Research Council Consolidator Grant (ERCCoG) under the European Union’s Horizon 2020 Programme (Grant Number ERCCoG 682394) to M.B.; by the Italian Ministry of Education, University and Research FARE-MIUR project R1623HZAH5 to M.B.; by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti Eccellenza Program (2018–2022) Dept. of Biology and Biotechnology “L. Spallanzani”, University of Pavia. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.