Characterization of dengue virus 3'UTR RNA binding proteins in mosquitoes reveals that AeStaufen reduces subgenomic flaviviral RNA in saliva

PLoS Pathog. 2022 Sep 19;18(9):e1010427. doi: 10.1371/journal.ppat.1010427. eCollection 2022 Sep.

Abstract

Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry-a technique initially developed in mammalian cells-to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3'UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3'UTR, whereas AeStaufen interacts with both 3'UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen's role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3'UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Aedes* / genetics
  • Animals
  • Carrier Proteins / genetics
  • Dengue Virus* / genetics
  • Dengue*
  • Humans
  • Mammals
  • Mosquito Vectors / genetics
  • RNA-Binding Proteins / genetics
  • Saliva

Substances

  • 3' Untranslated Regions
  • Carrier Proteins
  • RNA-Binding Proteins

Grants and funding

Funding for this research came from a National Medical Research Council ZRRF grant (ZRRF/007/2017) and a French Agence Nationale de la Recherche grant (ANR-20-CE15-0006) both awarded to JP, a Rubicon scholarship from the Dutch Research Council (NOW) awarded to MD, a PhD fellowship from the French Ministry of higher education and research awarded to FR and the Emerging Infectious Diseases (EID) Signature Research Programme at Duke-NUS funded by the Agency for Science Technology and Research (A*STAR). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.