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Mosquito Antiviral piRNA Pathway

Periodic Reporting for period 1 - MAPP (Mosquito Antiviral piRNA Pathway)

Reporting period: 2015-09-01 to 2017-08-31

Chikungunya virus (CHIKV, genus Alpahvirus) and Zika virus (ZIKV, genus Flavivirus) belong to a group of viruses that are transmitted by Aedes aegypti mosquitoes and cause severe disease in humans including fever, headache, malaise, arthritis and in the case of ZIKV neurological disease and birth defects. Mosquito-transmitted viruses put a huge economical and medical burden on societies in high risk areas. The epidemic spread of chikungunya virus began approximately 10 years ago, in the Indian Ocean region and later reached Europe (France and Italy). In recent years, the disease reached South America and USA. The Zika virus epidemic began later, at the beginning of 2015 in Brazil and has now spread across South America.
Mosquitoes are not just the carrier but also become infected with the virus which spreads through the whole organism. Their immune system actively fights the virus; however, mosquitos do not have an antibody based immune system and yet the infection is asymptomatic and not pathogenic. In case of the human host, the activation of the adaptive immune system (e.g. antibodies, T-cells) is required to clear the organism of virus infection. On the other hand, mosquitoes have an efficient innate immune system that acts on the cellular level to control the virus replication but is unable to clear the virus from the organism. This persistent infection of the mosquito is thought to be important factor for an efficient viral transmission to humans.
The main antiviral response in mosquitoes is based on virus specific small RNA molecules (and their key interacting proteins) that are used to identify and silence the replicating virus. Over the years many studies have identified different classes of virus-specific small RNAs in infected mosquito cells, called small interfering (si)RNAs, micro (mi)RNAs and PIWI-interacting (pi)RNAs. Recent findings have suggested that the piRNAs (and the interacting key proteins: Piwi1-7 and Ago3), in addition to the rather well characterized siRNAs (and key proteins: Ago2 and Dcr2), have antiviral potential in mosquitoes. The aim of the project was to characterize if and how piRNAs are antiviral against mosquito-borne viruses in their main mosquito vector, Aedes aegypti: (i) dissecting the role of the piRNA pathway as antiviral response against CHIKV and ZIKV, (ii) identification of the piRNA pathway proteome and bound RNAs in Aedes aegypti and their changes during CHIKV and ZIKV, (iii) the role of novel piRNA related proteins in the antiviral response against CHIKV and ZIKV.
During the project it was found that although CHIKV and ZIKV infection results in the production of virus-specific piRNAs, these do not carry antiviral protective role. Only antiviral PIWI proteins was found to be Piw4, yet it does not bind piRNAs itself and is not involved in their production. Piwi4 also binds multiple other cellular protein, some of these are also antiviral.
The project initially focused on chikungunya virus, but Zika virus was included at a later stage for comparative purposes and due to the occurrence of the Zika virus epidemic.
(i) Our findings showed that the infection of Aedes aegypti mosquito derived Aag2 cells with either virus, CHIKV or ZIKV, resulted in the production of virus-specific siRNAs and piRNAs, as characterized by small RNA sequencing and bioinformatic analysis. In case of both viruses, the siRNAs with size of 21 nt mapped along the genomic and antigenomic strands. However, differences were observed between the produced virus specific piRNAs. Chikungunya virus specific piRNAs mapped to the subgenomic region of virus genome and these piRNAs carry characteristic signatures similar to other cellular piRNAs: sense piRNAs having nucleotide A at position 10 and antisense having U at position 1. Zika virus specific piRNAs were unique: piRNAs mapped to only one location on the genomic strand and these piRNAs lacked the characteristic signature, hence these are rather piRNA-like. By knocking down the primary proteins of the piRNA pathway individually, we found that the key proteins involved in the production of piRNAs did not affect chikungunya or Zika virus replication, unlike the case with siRNA producing proteins. Interestingly, one PIWI protein was found to be antiviral against both viruses: Piwi4.
(ii) It was further established that the key proteins for alphavirus-specific piRNA production are Ago3, Piwi5, and Piwi6. In case of ZIKV, Ago3 binds the ZIKV specific piRNA-like molecules, expanding the knowledge on flavivirus infection in mosquito cells in general. Additional studies on antiviral Piwi4 indicated that it interacts with key proteins in siRNA pathway (Ago2, Dcr2) and key proteins in piRNA pathway (Ago3, Piwi5, Piwi6), but its antiviral function is not directly linked to either classes of the virus specific small RNAs (siRNAs or piRNAs), indicating that mosquitoes have evolved additional layer of defence against viruses, which is different from the defence mechanisms of fruitfly, the model insect for molecular biology studies.
(iii) Moreover, quantitative proteomic study performed to find interaction partners of Piwi4 showed that it is complexed with multiple of proteins, including ribosomal proteins. Some of the Piwi4 partner do not have orthologues in other species. Further research indicated that some of the identified Piwi4 interactors are also antiviral and could be good targets to use for the control of vector-based control strategies.
Studies on mosquito innate immunity are important. The knowledge on how viruses keep the balance between sufficient infection to be transmitted to humans and not killing the vector mosquito offers the possibility to demolish this balance and thereby limit the spread of deadly diseases among the human (and animal) populations. It was established that piRNAs are not involved in the antiviral defence and that only antiviral PIWI protein in mosquitoes is Piwi4, which is not involved in piRNA protection. It offers protection against dangerous chikungunya and Zika virus. Thus, the research conducted has offered new ways to look at mosquito immune system and in a long-term perspective the findings can be used to generate genetically modified mosquitoes that are less capable of transmitting viral diseases.
Virus infected cells