Antiviral RNAi response in Culex quinquefasciatus-derived HSU cells

Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus

Semliki Forest -viruksen nsP1-proteiinin ja isäntäproteiinien vuorovaikutusten selvittäminen

Semliki Forest -virus (SFV) on hyttyslevitteinen paljon tutkittu mallivirus lääketieteellisesti merkittäville alfaviruksille kuten chikungunya virukselle ja Sindbis-virukselle. SFV:n genomi monistuu solunsisäisissä kalvokuromissa, sferuleissa, joiden kaulan viruksen koodaama nsP1-proteiini muodostaa. Tämän maisterintutkielman tavoitteena oli tunnistaa nsP1-proteiinin kanssa vuorovaikuttavia isäntäsolun proteiineja aiemman proteomiikkatutkimuksen perusteella ja kehittää seulontamenetelmä vuorovaikutusten tunnistamiseksi. Geenien hiljentämiskokeilla selvitettiin proviraalisesti tai antiviraalisesti SFV:n infektioon vaikuttavia isäntäproteiineja, minkä jälkeen varmistettiin, että solujen elinkyky ei laskenut merkittävästi. Useita tilastollisesti merkitseviä proviraalisia ja antiviraalisia isäntätekijöitä havaittiin. Merkittävien proviraalisten löydösten joukosta kloonattiin neljä lupaavaa ehdokasproteiinia nisäkkässoluekspressiota varten ja niihin lisättiin Myc-epitooppi. Biokemialliset kokeet eivät osoittaneet stabiileja vuorovaikutuksia, mikä viittaa hetkellisiin tai epäsuoriin vuorovaikutuksiin näiden isäntäproteiinien ja nsP1:n välillä. Myöskään kolokalisaatiota SFV:n replikaatiokompleksin kanssa ei havaittu, mikä tukee muita tuloksia. Seulontamenetelmä tunnisti tehokkaasti proteiini-proteiini-vuorovaikutukset ja sitä voidaan soveltaa jatkossa kandidaattiproteiinien seulontaan. Tulevaisuudessa olisi hyödynnettävä menetelmiä, jotka soveltuvat ohimenevien vuorovaikutusten tutkimiseen, jotta saisimme lisää tietoa tärkeimmistä isännän proteiineista SFV-sferulien muodostumisessa ja replikaatiossa. Tämä voi johtaa uusiin antiviraalisiin lääkeaineisiin, jotka kohdistuvat alphavirusten replikaatioon.Semliki Forest virus (SFV) is a well-studied model virus of medically important mosquito-borne alphaviruses, like chikungunya virus and Sindbis virus. SFV replicates within membrane invaginations called spherules at the host plasma membrane, facilitated by the virus-encoded nsP1 protein. The objectives of this MSc thesis were to identify candidate host proteins interacting with nsP1 based on previous proteomics work and develop a screening workflow to identify stable nsP1 interactors. The overarching aim is to improve our understanding of the roles of host proteins in spherule formation and replication complex functions. The screening workflow involved knockdown assays to assess the antiviral and proviral effects of potential host interactors, followed by cell viability assays for toxicity assessment. Selected promising hits were further investigated for protein interaction with nsP1, which required cloning for mammalian expression and tagging with Myc epitope. Knockdowns resulted in several statistically significantly proviral and antiviral host factors, and all the knockdowns were non-toxic to the cells. Among the significant proviral hits, four promising candidate host proteins were cloned and expressed. Pull-down assays did not reveal stable interactions, suggesting transient or indirect interactions between these host proteins and nsP1. The lack of co-localisation with SFV replication complex supported this finding. This work sheds light on possible transient or indirect interactors of nsP1. The screening workflow effectively identified protein-protein interactions and can be applied to screen additional proteins. Future studies should employ methods suitable for studying transient interactors to gain further insights. This would enhance our understanding of key host proteins in SFV spherule formation and replication, potentially leading to novel antiviral therapies targeting alphavirus replication

Antiviral RNAi Response in Culex quinquefasciatus-Derived HSU Cells

Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus