Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

Abstract

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA replication intermediates in infected cells and directly interacted with high affinity with DENV RNA in the 3’ untranslated region in vitro (KD = 48–62 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells

“…Silencing of AAEL004419 , AAEL008728 , and AAEL004859 , but not AAEL001216 resulted in a profound increase of dengue virus titers, to similar levels as silencing of Ago2 ( Fig 1F ). Similarly, knockdown of AAEL004419 , AAEL008728 , and AAEL004859 in an Aag2-derived clonal cell line (Aag2 C3PC12) that has been cleared from persistent virus infections [ 34 ] enhanced RNA replication of CHIKV by > 2-fold ( Fig 1G ), suggesting a broad antiviral activity of these helicases. Importantly, silencing of the identified RNA helicases in Aag2 C3PC12 cells also resulted in increased SINV levels as observed in the initial knockdown screen, which had been performed in the parental Aag2 cell line ( S1C and S1D Fig ).…”

“…How the latter two helicases control virus replication in mosquito cells remains to be established. Interestingly, Besson and colleagues recently identified AAEL004859 to strongly bind to the proviral host factor Loquacious in Aag2 cells [ 34 ]. Whether AAEL004859 plays an inhibitory function in this context warrants further investigation.…”

“…Ae . aegypti Aag2 cells and the Aag2 C3PC12 clone [ 34 ] derived from these cells (cleared of the persistently infecting viruses Cell fusing agent virus, Phasi Charoen-like virus and Culex Y virus) were maintained at 28°C in Leibovitz’s L-15 medium (Invitrogen: catalogue number: 21083027) supplemented with 10% foetal bovine serum (Gibco), 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco), 2% tryptose phosphate broth (Sigma), and 1% non-essential amino acids (Gibco). For lactate assays, Aag2 C3PC12 cells were cultured in Schneider’s Drosophila medium (Invitrogen, catalogue number 21720024) containing 11.11 mM D-glucose and 12.32 mM L-glutamine.…”

The costs of transgenerational immune priming for homologous and heterologous infections with different serotypes of dengue virus in Aedes aegypti mosquitoes

“…However, in the time window evaluated in the TGIP groups, the viral load was decreased in the priming groups, suggesting that the siRNA pathway has a limited participation in viral regulation in the TGIP. However, it is not ruled out that other pathway cofactors, such as Loquacious (Loqs), could intervene in immune regulation through maternal challenges ( 69 ).…”

Competition between two Usutu virus isolates in cell culture and in the common house mosquito Culex pipiens

“…Mosquito cell lines Aedes albopictus C6/36 (ATCC CRL-1660), U4.4 (Singh, 1967 ), U4.4 Argonaut 2 k/o (Ago 2 k/o) (Besson et al, 2022 ), and Culex tarsalis Chao Ball (C.B) cells (provided by Dr. Roy Hall, University of Queensland, Australia) (Chao and Ball, 1976 ) were cultured in Leibovitz's medium (Invitrogen) and supplemented with 10% FBS, 2% tryptose phosphate (Invitrogen), and 1% non-essential amino acids. All mosquito cells were passaged two times a week and kept at 27°C.…”