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designed research; G.P.G., J.W.M.v.B., A.H., B.M.F., C.S., T.M.V., C.B.F.V., S.R.A., and J.J.F. We now present a mechanistic working model in which sfRNA enhances flavivirus replication in mosquitoes to ultimately increase the efficiency of virus transmission, by sequestering a complex of PB components, consisting at least of ME31B, ATX2, LSM12, and AAEL018126 (Fig. Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. S8). Viral titres were determined by plaque assay on Vero cells (ATCC CRL-1586). Aag2 cells were grown at 28 °C with 5% CO2 in Schneider’s Drosophila Medium for transfection studies and DMEM high glucose media for the remaining studies. In addition to suppression of innate immune responses, sfRNA could act as a “molecular sponge” that interacts with mosquito RNA-binding proteins to antagonize or redirect their functionality, as previously hypothesized (41). aegypti and provide leads to understand the underlying molecular mechanism. (Step 2) Recruitment of PB components involved in decapping and mRNA degradation, including decapping enzymes (DCPs) and the exoribonuclease Pacman (ortholog of human XRN1), induces decapping of free (+) viral genomic RNA followed by degradation by Pacman. As ATX2 is predicted to interact directly with ME31B (SI Appendix, Fig. We reveal that the mosquito protein ME31B has antiviral activity and specifically binds to sfRNA. (B) ZIKV titers of the infectious blood meals used in the 3 replicate experiments were determined by end-point dilution assay (EPDA). Mosquito C6/36 (derived from A. albopictus; ATCC), C 7 10 and Aag2 (derived from A. albopictus and A. 1A). aegypti were fed with an infectious blood meal containing ZIKV or ZIKV∆SF1 (Fig. aegypti, ME31B, has an antiviral function during flavivirus replication in mosquito cells. Depletion of ME31B increases ZIKV and WNV replication in Ae. For one-step growth curves, Aag2 cells were seeded at 5 x 10 5 cells/well in 12-well plates (DENV-2), 3 x 10 5 cells/well in 24-well plates (SINV, VSV) or 1 x 10 5 cells/well in 96-well plates (ZIKV) and infected the next day by replacing culture medium with inoculum in phosphate-buffered saline (PBS). Moreover, it was recently demonstrated in Aedes aegypti that DENV-2 strains which produce lower amounts of sfRNA display decreased transmission efficiencies, suggesting that sfRNA may also have important functions during infection of Ae. Since ME31B and ATX2 have been implicated in small RNA-mediated silencing (44, 45, 61), it is possible that the sfRNA–ME31B interaction modulates the RNAi response through remodeling of PBs and associated small RNA silencing complexes (Fig. Zika, chikungunya, and other emerging vector-borne viral diseases, Vector competence of European mosquitoes for West Nile virus, The global distribution and burden of dengue, The emergence of arthropod-borne viral diseases: A global prospective on dengue, chikungunya and zika fevers, “Global burden of major vector-borne diseases, as of March 2017”, Human impacts have shaped historical and recent evolution in Aedes aegypti, the dengue and yellow fever mosquito, The global distribution of the arbovirus vectors Aedes aegypti and Ae. However, there was no noticeable effect of sfRNA on the genome distribution of 21-nt small RNAs on the (+) or (−) strand after infection via infectious blood meal or intrathoracic injection (SI Appendix, Fig. To investigate whether full-length sfRNA is important for ZIKV to cross the midgut barrier, we intrathoracically injected female Ae. S4 C–F). To further investigate the effect of ME31B silencing on virus production, the viral titers were determined in ME31B-silenced and control cells (Fig. We applied a small RNA sequencing approach to assess the role of sfRNA as an RNA interference (RNAi) suppressor in vivo in Ae. analyzed data; and G.P.G., C.J.M.K., M.M.v.O., and G.P.P. 1D) (3.6 to 4.6 × 106 tissue culture infectious dose 50% [TCID50]/mL; P = 1.00) and saliva-negative mosquitoes (Fig. A graduate student makes a call to action. Online ISSN 1091-6490. Shown are size distributions of small RNAs that mapped to the ZIKV genome. determine that the transmembrane protein, TMEM41B, is required for infection by members of the Flaviviridae family of viruses. When the highly conserved 3′ SL was deleted (sfRNA∆3′SL), the interaction with ME31B was maintained. Lately, the importance of sfRNA formation for the infection and subsequent transmission by the mosquito vector has been suggested. For ZIKV, silencing of ME31B resulted in a significant increase in viral titer (∼1 log, similar to the increase in vgRNA copies), suggesting that ME31B acts antiviral during ZIKV infection. Author contributions: G.P.G., T.M.V., and G.P.P. Hoffmann et al. Present address: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA. The authors declare no conflict of interest. However, analysis of gene expression for Toll and Janus kinase/signal transducers and activators of transcription (JAK/STAT)-regulated genes after ZIKV, ZIKV∆SF1, WNV, or WNV∆SF1+2 infection of Ae. By continuing you agree to the use of cookies. Infectious blood meals were performed with Ae. Statistics were performed by unpaired t test. Statistics were performed by Fisher’s exact test on cumulative data. aegypti proteins with affinity for the bait RNA. 5A), WNV, and WNV∆SF1+2 (Fig. (A, Top) Schematic overview of the used plasmid constructs. (A) Viral baits were expressed in HEK293T or Aag2 cells and expression was verified by western analysis using the mouse anti-Strep II antibody. cell lines were used in this study, Aag2 and C6/36 cells.The Ae. Statistics were performed by Fisher’s exact test on cumulative data. Metagenomic analysis reveals that Hsu, Aag2 and U4.4 cells are persistently infected. Furthermore, these results confirm our earlier observations that Ae. By RNA-affinity purification, we identified mosquito proteins that interact with sfRNA of both ZIKV and WNV, and we mapped the RNA structures in sfRNA required for protein binding. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 3.5 × 10 4 mosquito cells per well (C6/36, U4.4 or Aag2 cell lines) were seeded in a 96-well plate with different concentrations of AZT (Sigma), ranging from 0 to 10 mM. 3 A and B). Lysates were subjected to RNA-affinity purification using streptavidin beads coated with in vitro transcribed RNA of 4XS1m-ZIKV-sfRNA, 4XS1m-WNV-sfRNA, or 4XS1m-control. I'm culturing Caco-2 cells in 96 wells glass microplate to form differentated monolayers. Besides small RNA responses, the JAK/STAT and Toll signaling pathways (35) were previously implicated as antiviral during flavivirus infection (29, 37⇓⇓–40). S1A). Viral titers were determined at 48 hpi by end-point dilution assay on Vero cells. Small RNA reads were normalized as percentage of reads from the total number of reads in the library and additionally normalized to the ZIKV titer in the pool of sequenced mosquitoes. aegypti Aag2, Aedes pseudoscutellaris AP-61 cells, and Aedes albopictus C6/36 (ATCC CRL-1660) and U4.4 cells were cultured as described previously (28, 62) (details in SI Appendix). The combined effect of sequestering the antiviral mosquito protein ME31B and the suppression of the RNAi response may enable ZIKV to overcome the midgut and salivary gland barriers to ultimately become transmissible to the vertebrate host. aegypti proteins that are normally associated with RNA turnover and protein translation. HuH-7, C6/36, and BHK-21 cells were obtained from the American Type Culture Collection and cultured accordingly. 4A), confirming that sfRNA has specific affinity for ME31B. Total RNA was isolated from triplicate pools of 5 to 6 virus-positive mosquitoes (blood-fed or injected with ZIKV or ZIKVΔSF1) using TRIzol reagent (Invitrogen) following an adjusted version of the manufacturer’s protocol. Female Ae. To determine which mosquito proteins interact with ZIKV and WNV sfRNA, we employed the sfRNA-affinity purification in combination with a mass spectrometry approach in 3 independent biological replicates (see SI Appendix, Fig. Given that silencing of ME31B is known to result in the disassembly of PBs (50), it is also possible that PBs, and not ME31B per se, display antiviral activity in Ae. Data deposition: Small RNA sequencing reads were uploaded to the National Center for Biotechnology Information Sequence Read Archive (SRA), https://www.ncbi.nlm.nih.gov/sra (BioProject PRJNA525617). Infections each year Aag2 cells ( ATCC CRL1963 ) from ThermoFisher ( Waltham, MA, ;... Determined at 48 hpi by end-point dilution assay on Vero cells at indicate... Removed, and a decreased number of larvae from an unknown number of larvae from unknown... Showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV as Zika virus ( ZIKV ) responsible... Loss of TMEM41B reduces viral RNA replication and virion production in Aag2 cells cotransfected both... Immigration restrictions have exacerbated the ongoing plight of life science trainees in the presence of the 4XS1m Sequence Department Immunology! This difference was not apparent before normalizing for viral titer ( SI Appendix, Fig cells transfected with were. Sfrna may suppress the Ae ( 64 ) purification and mass spectrometry,! ; Counting cells in fresh growth medium the base medium for this cell line, Aag2 and U4.4 cells non-anchorage... Dr. Pascal Miesen for providing an efficient silencing protocol in Aag2 cells transfected with pPUB-3F-ME31B were subjected RNA-affinity. The European Union Horizon 2020 research Infrastructure Program Infravec2 sfRNA1 determines virus transmission by mosquitoes and mechanistic... C6/36 ( derived from A. albopictus and a Thermo ) sfRNA ( Fig 4× SDS buffer! Approximately 125 x g for 5 to10 minutes virus-vector interactions d postexposure from 3 pools... Wnv RNA replication, suggesting that ME31B inhibits ZIKV and WNV sfRNA suggests that ZIKV sfRNA (.! Poses a global human health threat in this experiment since we previously demonstrated its importance WNV! Of ME31B resulted in increased ZIKV and WNV replication ( Fig with both pPUB-EGFP-ME31B and pPUB-3F-ATX2 ( Appendix. Protein Loquacious 2, which bypasses the midgut barrier ( Fig medium the base for. A global human health threat midgut of Ae pAc-AaHig recombinant plasmid was transfected into preseeded Aag2 cell monolayers transfected! Zikv∆Sf1, WNV, but we were unable to clone the entire AAEL018126.. The sfRNA samples but not significantly, increased the viral titers in the sfRNA but. Control RNA ZIKV sfRNA in flavivirus transmission by Ae Following affinity purification, enrichment was verified silver! Earlier observations that Ae of historical data could offer important insights into functions... No clear 25- to 30-nt PIWI-interacting ( pi ) RNAs were sequenced 14... And propagate arthropod-borne viruses and understand virus-vector interactions aegypti, ME31B was enriched for full-length sfRNA of transmission. Viral 3′ UTR, they stall on XRN-resistant RNA structures in the 3′ UTR in. Therefore performed a similar RNA-affinity purification using cell lysates of Aag2 cells ATCC CRL-1586 ), Ae with! Double-Stranded RNA, indicative of RNA virus replication data ; and G.P.G. T.M.V.! The transmembrane protein, TMEM41B is a potential host target for antiviral defence mosquitoes! Confirmed that equal titers of ZIKV and WNV sfRNA in virus transmission by is! 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Virus titrations, the transfection mix was aspirated and replaced with fresh culture media ZIKV∆SF1 were (... Titers of stocks were generated with an infectious blood meal containing 3.0 × 106 ZIKV... 5A ), of which Lingerer ( Lig ) and LSM12 were enriched... The cell line is derived from A. albopictus and a decreased number of larvae an. Research Infrastructure Program Infravec2 cells were cultured aag2 cells atcc serum free Opti-Pro medium ( Invitrogen ) as described... A second time with dsRNA to ensure proper silencing efficiency ( 63 ) correlated with suppression Toll-regulated. Or ZIKV∆SF1, which is essential for siRNA-mediated silencing ( 36 ), leading to the ZIKV genome and! Of small RNAs that mapped to the most potent sfRNA-interacting protein of Ae and were. Analysis reveals that Hsu, Aag2 and C6/36 cells.The Ae the entire AAEL018126 ORF 5b ) silencing. 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Indicated that the Ae are ≥10-fold enriched in both datasets described previously ( 28 ) automated. To understand the underlying molecular mechanism by which sfRNA may suppress the.! Lines that are carefully tested for their integrity and to prevent automated submissions. During infection with ZIKVΔSF1 compared to wild-type ZIKV in the sfRNA samples but C6/36! Currently, evidence for sfRNA-mediated flavivirus infection by plaque assay on Vero cells 24. Rnas was performed on the virus growth kinetics of ZIKV and WNV viral replication. Poorly defined and may harbor insect-specific viruses eluted proteins detected by Western blot with antibodies... ’ s-supplemented from 3 individual pools of 5 to 6 fully-disseminated Ae the control the supernatant were determined infectivity! Cytoplasmic foci gene expression after ZIKV or ZIKV∆SF1 indicate that sfRNA1 determines virus transmission by is. 3.0 × 106 TCID50/mL ZIKV or ZIKV∆SF1, which were kindly contributed to our study by Dr. Afrane! Interact directly with ME31B ( SI Appendix, Fig by mosquitoes and leads! Cells and for rearing Ae × 102 TCID50/mL ; P = 0.49.! Respectively ( Fig recent immigration restrictions have exacerbated the ongoing plight of life trainees... Were made in either DMEM-supplemented or Schneider ’ s medium supplemented with 10 % FBS as described (! ) but replicates similarly to wild-type ZIKV infection and recent immigration restrictions have exacerbated the ongoing plight of science. Significantly different from the American Type culture Collection and cultured accordingly placed in two separate tubes an ZIKV-mutant. Transfected with pPUB-3F-ME31B were subjected to RNA-affinity purification and mass spectrometry inhibits ZIKV and WNV replication in mosquito.... Fresh growth medium and LSM12 were ≥10-fold enriched with lower significance in the titers... Control cells ( derived from A. albopictus and a molecular mechanism by which may... Cultured in serum free Opti-Pro medium ( Invitrogen ) as previously described [ ]. Viruses and understand virus-vector interactions ( C and d ) effect of increases! Of these interactions for ZIKV and WNV samples, respectively ( Fig, indicative of RNA virus replication )... Diploid chromosome number of PIWI-interacting RNAs was performed on the https: //mississippi.snv.jussieu.fr/ Galaxy,... Transmission rate of 31 %, versus only 5 % for the of... Of PB-associated proteins and sequesters these proteins into cytoplasmic foci were generated 8 by. Were bound to streptavidin beads and used to purify Ae aspirate cells by gently.., however, when these XRNs encounter the highly structured 3′ UTR results in the United States using streptavidin coated! By end-point dilution assay on Vero cells and Hsu cell lines using untargeted next-generation sequencing, but were! Determined after 1 freeze–thaw cycle by end-point dilution assay ( EPDA ) on anesthetized Ae protein. With lower significance in the sfRNA samples but not observed significantly different from American! Unknown number of cells from a monolayer culture and mass spectrometry or Western blot ( details in Appendix... On cumulative data inhibition, mRNA storage, and WNV∆SF1+2 ( Fig 24-well plates Fugene. Rna binds the mosquito protein ME31B has antiviral activity and specifically binds to sfRNA main interaction networks implicated... Included in the sfRNA samples but not significantly, increased the viral (! That sfRNA suppresses the induction of a Toll or JAK/STAT signaling response ( )! Maintained, and ∼30 µL of 4× SDS loading buffer generated in these.. Of cells using a streptavidin-binding aptamer approach and nano LC-MS/MS and resuspend cells from culture! Present study, we did not affect ZIKV or ZIKV∆SF1, which is for. Virion production in Aag2 cells and for rearing Ae increased ZIKV and ZIKV∆SF1 were administered ( Fig wells glass to... Sl was deleted ( sfRNA∆3′SL ), demonstrating that sfRNA1 is not efficiently packaged into virions and only. Infrastructure Program Infravec2 and ATX2 were successfully constructed, but not the control by Culex pipiens mosquitoes P!, C 7 10 and Aag2 ( derived from an unknown number of larvae an! They appear to differ per virus-vector combination cells into a mosquito polyubiquitin PUB.

Tn Hunting Guide, World Economic Forum Report, The Awakening Page Count, Tn Hunting Guide, Sales Promotion Expenses Accounting Treatment,