Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26–27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.
References
[1]
Brackney DE, Beane JE, Ebel GD (2009) RNAi Targeting of West Nile Virus in Mosquito Midguts Promotes Virus Diversification. PLoS Pathog 5: e1000502. doi: 10.1371/journal.ppat.1000502
[2]
Campbell C, Keene K, Brackney D, Olson K, Blair C, et al. (2008) Aedes aegypti uses RNA interference in defense against Sindbis virus infection. BMC Microbiol 8: 47. doi: 10.1186/1471-2180-8-47
[3]
Cirimotich CM, Scott JC, Phillips AT, Geiss BJ, Olson KE (2009) Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes. BMC Microbiol 9: 13. doi: 10.1186/1471-2180-9-13
[4]
Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, et al. (2004) RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae. Proc Nat Acad Sci 101: 17240–17245. doi: 10.1073/pnas.0406983101
[5]
Myles KM, Morazzani EM, Adelman ZN (2009) Origins of alphavirus-derived small RNAs in mosquitoes. RNA Biol 6: 387–391. doi: 10.4161/rna.6.4.8946
[6]
Myles KM, Wiley MR, Morazzani EM, Adelman ZN (2008) Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes. Proc Nat Acad Sci 105: 19938–19943. doi: 10.1073/pnas.0803408105
[7]
Sanchez-Vargas I, Scott JC, Poole-Smith BK, Franz AWE, Barbosa-Solomieu V, et al. (2009) Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway. PLoS Pathog 5: e1000299. doi: 10.1371/journal.ppat.1000299
[8]
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–366. doi: 10.1038/35053110
[9]
Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev 15: 188–200. doi: 10.1101/gad.862301
[10]
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293–296. doi: 10.1038/35005107
[11]
Liu Q, Rand T, Kalidas S, Du F, Kim H, et al. (2003) R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301: 1921–1925. doi: 10.1126/science.1088710
[12]
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146–1150. doi: 10.1126/science.1064023
[13]
Horwich MD, Li C, Matranga C, Vagin V, Farley G, et al. (2007) The Drosophila RNA Methyltransferase, DmHen1, Modifies Germline piRNAs and Single-Stranded siRNAs in RISC. Cur Biol 17: 1265–1272. doi: 10.1016/j.cub.2007.06.030
[14]
Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, et al. (2004) Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs. Mol Cell 15: 185–197. doi: 10.1016/j.molcel.2004.07.007
[15]
Okamura K, Ishizuka A, Siomi H, Siomi MC (2004) Distinct roles for argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev 18: 1655–1666. doi: 10.1101/gad.1210204
[16]
Aliyari R, Wu Q, Li H-W, Wang X-H, Li F, et al. (2008) Mechanism of Induction and Suppression of Antiviral Immunity Directed by Virus-Derived Small RNAs in Drosophila. Cell Host Microbe 4: 387–397. doi: 10.1016/j.chom.2008.09.001
[17]
Parameswaran P, Sklan E, Wilkins C, Burgon T, Samuel MA, et al. Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems. PLoS Pathog 6(2): e1000764. doi: 10.1371/journal.ppat.1000764
[18]
Fragkoudis R, Attarzadeh-Yazdi G, Nash AA, Fazakerley JK, Kohl A (2009) Advances in dissecting mosquito innate immune responses to arbovirus infection. J Gen Virol 90: 2061–2072. doi: 10.1099/vir.0.013201-0
[19]
Hardy JL, Houk EJ, Kramer LD, Reeves WC (1983) Intrinsic Factors Affecting Vector Competence of Mosquitoes for Arboviruses. Annu Rev Entomol 28: 229–262. doi: 10.1146/annurev.en.28.010183.001305
[20]
Lambrechts L, Scott TW (2009) Mode of transmission and the evolution of arbovirus virulence in mosquito vectors. Proc R Soc Biol Sci 276: 1369–1378. doi: 10.1098/rspb.2008.1709
[21]
Sabin LR, Hanna SL, Cherry SInnate antiviral immunity in Drosophila. Curr Opin Immunol 22: 4–9. doi: 10.1016/j.coi.2010.01.007
[22]
Souza-Neto JA, Sim S, Dimopoulos G (2009) An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense. Proc Nat Acad Sci 106: 17841–17846. doi: 10.1073/pnas.0905006106
[23]
Xi ZY, Ramirez JL, Dimopoulos G (2008) The Aedes aegypti Toll pathway controls dengue virus infection. PLoS Pathog 4(7): e1000098. doi: 10.1371/journal.ppat.1000098
[24]
Galiana-Arnoux D, Dostert C, Schneemann A, Hoffmann JA, Imler J-L (2006) Essential function in vivo for Dicer-2 in host defense against RNA viruses in Drosophila. Nat Immunol 7: 590–597. doi: 10.1038/ni1335
[25]
Li H, Li WX, Ding SW (2002) Induction and suppression of RNA silencing by an animal virus. Science 296: 1319–1321. doi: 10.1126/science.1070948
[26]
van Rij R, Saleh M, Berry B, Foo C, Houk A, et al. (2006) The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. Genes Dev 20: 2985–2995. doi: 10.1101/gad.1482006
[27]
Wang XH, Aliyari R, Li WX, Li HW, Kim K, et al. (2006) RNA interference directs innate immunity against viruses in adult Drosophila. Science 312: 452–454. doi: 10.1126/science.1125694
[28]
Desmyter J, Melnick JL, Rawls WE (1968) Defectiveness of interferon production and of rubella virus interference in a line of African Green Monkey kidney cells (Vero)I. J Virol 2: 955–961.
[29]
Chotkowski HL, Ciota AT, Jia Y, Puig-Basagoiti F, Kramer LD, et al. (2008) West Nile virus infection of Drosophila melanogaster induces a protective RNAi response. Virology 377: 197–206. doi: 10.1016/j.virol.2008.04.021
[30]
Shi PY, Tilgner M, Lo MK, Kent KA, Bernard KA (2002) Infectious cDNA clone of the epidemic West Nile virus from New York City. J Virol 76: 5847–5856. doi: 10.1128/JVI.76.12.5847-5856.2002
[31]
Hahn CS, Hahn YS, Braciale TJ, Rice CM (1992) Infectious Sindbis Virus Transient Expression Vectors for Studying Antigen Processing and Presentation. P Natl Acad Sci USA 89: 2679–2683. doi: 10.1073/pnas.89.7.2679
[32]
Gabitzsch ES, Blair CD, Beaty BJ (2006) Effect of La Crosse virus infection on insemination rates in female Aedes triseriatus (Diptera: Culicidae). J Med Entomol 43: 850–852. doi: 10.1603/0022-2585(2006)43[850:EOLCVI]2.0.CO;2
[33]
Hutchinson KL, Peters CJ, Nichol ST (1996) Sin Nombre Virus mRNA Synthesis. Virology 224: 139–149. doi: 10.1006/viro.1996.0515
[34]
Murata R, Eshita Y, Maeda A, Maeda J, Akita S, et al. Glycosylation of the West Nile Virus Envelope Protein Increases In Vivo and In Vitro Viral Multiplication in Birds. Am J Trop Med Hyg 82: 696–704. doi: 10.4269/ajtmh.2010.09-0262
[35]
Powers AM, Olson KE, Higgs S, Carlson JO, Beaty BJ (1994) Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector. Virus Res 32: 57–67. doi: 10.1016/0168-1702(94)90061-2
[36]
Vasilakis N, Deardorff ER, Kenney JL, Rossi SL, Hanley KA, et al. (2009) Mosquitoes Put the Brake on Arbovirus Evolution: Experimental Evolution Reveals Slower Mutation Accumulation in Mosquito Than Vertebrate Cells. PLoS Pathog 5: e1000467. doi: 10.1371/journal.ppat.1000467
[37]
Li F, Ding S-W (2006) Virus Counterdefense: Diverse Strategies for Evading the RNA-Silencing Immunity. Annu Rev Microbiol 60: 503–531. doi: 10.1146/annurev.micro.60.080805.142205
[38]
Uchil PD, Satchidanandam V (2003) Architecture of the flaviviral replication complex - Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. J Biol Chem 278: 24388–24398. doi: 10.1074/jbc.M301717200
[39]
Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CKE, et al. (2009) Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites. Cell Host Microbe 5: 365–375. doi: 10.1016/j.chom.2009.03.007
[40]
Scott JC, Brackney DE, Campbell CL, Bondu-Hawkins V, Hjelle B, et al. (2010) Comparison of Dengue Virus Type-2-Specific Smalls from RNA Interference-Competent and –Incompetent Mosquito Cells. PLoS NTD (in press).
[41]
Nishida K, Saito K, Mori T, Kawamura Y, Nagami-Okada T, et al. (2007) Gene silencing mechanisms mediated by Aubergine piRNA complexes in Drosophila male gonad. RNA 13: 1911–1922. doi: 10.1261/rna.744307
[42]
Saito K, Nishida K, Mori T, Kawamura Y, Miyoshi K, et al. (2006) Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20: 2214–2222. doi: 10.1101/gad.1454806
[43]
Vagin V, Sigova A, Li C, Seitz H, Gvozdev V, et al. (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313: 320–324. doi: 10.1126/science.1129333
[44]
Wu Q, Luo Y, Lu R, Lau N, Li WX, et al. (2010) Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs. Proc Nat Acad Sci 107: 1606–1611. doi: 10.1073/pnas.0911353107
[45]
Patterson JL, Kolakofsky D (1984) Characterization of LaCrosse virus small-genome transcripts. J Virol 49: 680–685.
[46]
Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR (2006) Double-Stranded RNA Is Produced by Positive-Strand RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Strand RNA Viruses. J Virol 80: 5059–5064. doi: 10.1128/JVI.80.10.5059-5064.2006
[47]
Cullen BR (2006) Is RNA interference involved in intrinsic antiviral immunity in mammals? Nat Immunol 7: 563–567. doi: 10.1038/ni1352
[48]
Opyrchal M, Anderson JR, Sokoloski KJ, Wilusz CJ, Wilusz J (2005) A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines. Insect Biochem Molec 35: 1321–1334. doi: 10.1016/j.ibmb.2005.08.004
[49]
Taylor MJ, Peculis BA (2008) Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme. Nucl Acids Res 36: 6021–6034. doi: 10.1093/nar/gkn605
