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

Abstract

Current knowledge of the piRNA pathway is based mainly on studies on the model organism 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 the Dengue and Chikungunya viruses, Piwi clade genes underwent expansion, are also expressed in the soma, and code for proteins that may elicit antiviral functions and crosstalk with other proteins of recognized antiviral mechanisms. These observations underline the importance of expanding our knowledge of the piRNA pathway beyond D. melanogaster.\n\nHere 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 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. Expression profiles of Piwi transcripts during mosquito development and after infection with the Dengue 1 virus showed a concerted elicitation of all Piwi transcripts during viral dissemination, while the maintenance of infection primarily relied on the expression of Piwi5. In contrast, establishment of persistent infection by the Chikungunya virus is accompanied by an 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, further provide useful knowledge to design tailored functional experiments.\n\nAuthor summaryArgonautes are ancient proteins involved in many cellular processes, including innate immunity. Early in eukaryote evolution, Argonautes separated into Ago and Piwi clades, which maintain a dynamic evolutionary history with frequent duplications and losses. The use of Drosophila melanogaster as a model organism proved fundamental to understand the function of Argonautes. However, recent studies showed that the patterns and observations made in D. melanogaster, including the number of Argonautes, their expression profile and their function, are a rarity among Dipterans.\n\nIn vectors of epidemic arboviruses such as Dengue and Chikungunya viruses, Piwi genes underwent expansion, are expressed in the soma, and some of them appear to have antiviral functions. Besides being an important basic question, the identification of which (and how) Piwi genes have antiviral functions may be used for the development of novel genetic-based strategies of vector control. Here we coupled population genetics models with computational predictions of protein structure and expression analyses to investigate the evolution and function of Piwi genes of the emerging vector Aedes albopictus. Our data support a general antiviral role for Piwi5. Instead, the detection of complex expression profiles with the presence of sites under positive selection in Piwi1/3, Piwi4 and Piwi6 requires tailored functional experiments to clarify their antiviral role.