West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.

To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of approximately 25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response.

[1]  C. Mello,et al.  Revealing the world of RNA interference , 2004, Nature.

[2]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[3]  T. Tuschl,et al.  Mechanisms of gene silencing by double-stranded RNA , 2004, Nature.

[4]  Didier Raoult,et al.  Chikungunya outbreaks--the globalization of vectorborne diseases. , 2007, The New England journal of medicine.

[5]  C. Berg,et al.  Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases. , 1995, Genes & development.

[6]  J. Carlson,et al.  Short‐hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells , 2007, Insect molecular biology.

[7]  Munir Ahmad,et al.  Epidemiology of foot-and-mouth disease in Landhi Dairy Colony, Pakistan, the world largest Buffalo colony , 2008, Virology Journal.

[8]  J. Mackenzie,et al.  Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures , 1997, Journal of virology.

[9]  D. Beasley.  Recent advances in the molecular biology of west nile virus. , 2005, Current molecular medicine.

[10]  A. Schneemann,et al.  Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila , 2006, Nature Immunology.

[11]  G. Vriend,et al.  Conserved Role of a Complement-like Protein in Phagocytosis Revealed by dsRNA Knockout in Cultured Cells of the Mosquito, Anopheles gambiae , 2001, Cell.

[12]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[13]  D. Marks,et al.  The small RNA profile during Drosophila melanogaster development. , 2003, Developmental cell.

[14]  D. J. White,et al.  West Nile virus infection in birds and mosquitoes, New York State, 2000. , 2001, Emerging infectious diseases.

[15]  R. Russell,et al.  bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila , 2003, Cell.

[16]  F. Catteruccia,et al.  Stable and heritable gene silencing in the malaria vector Anopheles stephensi. , 2003, Nucleic acids research.

[17]  P. Schedl,et al.  Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation. , 2005, Genes & development.

[18]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[19]  W. Theurkauf,et al.  Biogenesis and germline functions of piRNAs , 2007, Development.

[20]  V. Vakharia,et al.  RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster , 2006, Cellular microbiology.

[21]  P. Uchil,et al.  Architecture of the Flaviviral Replication Complex , 2003, Journal of Biological Chemistry.

[22]  A. Spradling,et al.  A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. , 1997, Development.

[23]  A. James.  Control of Disease Transmission through Genetic Modification of Mosquitoes , 2000 .

[24]  A. Fire,et al.  dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. , 2000, Gene.

[25]  S. Ding,et al.  Antiviral silencing in animals , 2005, FEBS letters.

[26]  L. Kramer,et al.  Quantitation of flaviviruses by fluorescent focus assay. , 2006, Journal of virological methods.

[27]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[28]  Z. Adelman,et al.  RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted-Repeat RNA Derived from the Virus Genome , 2002, Journal of Virology.

[29]  R. Carthew,et al.  Methods and Materials , 1956, Eco-Art Therapy in Practice.

[30]  Morris F. Maduro,et al.  Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans , 2005, Nature.

[31]  F. Catteruccia,et al.  Comparative analysis of DNA vectors at mediating RNAi in Anopheles mosquito cells and larvae , 2003, Journal of Experimental Biology.

[32]  Shou-Wei Ding,et al.  Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Kuniaki Saito,et al.  Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. , 2006, Genes & development.

[34]  S. Akira,et al.  Recognition of viruses by innate immunity , 2007, Immunological reviews.

[35]  E. G. Westaway,et al.  Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. , 1985, Virology.

[36]  S. Higgs,et al.  RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  D. Gubler,et al.  Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses , 2004, Nature Medicine.

[38]  C. Sander,et al.  A novel class of small RNAs bind to MILI protein in mouse testes , 2006, Nature.

[39]  Daniel R. O’Leary,et al.  Epidemiology and Transmission Dynamics of West Nile Virus Disease , 2005, Emerging infectious diseases.

[40]  E. Weiss.  Arthropod Cell Cultures and Their Application to the Study of Viruses , 1971, Current Topics in Microbiology and Immunology.

[41]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[42]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[43]  Yi Guo,et al.  Overlapping Functions of Argonaute Proteins in Patterning and Morphogenesis of Drosophila Embryos , 2006, PLoS genetics.

[44]  T. Köcher,et al.  Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene , 2002, EMBO reports.

[45]  E. Gould,et al.  Ixodid and Argasid Tick Species and West Nile Virus , 2004, Emerging infectious diseases.

[46]  Shinji Yamaguchi,et al.  RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E. , 2002, Genes & development.

[47]  Khaled Machaca,et al.  RNA interference is an antiviral defence mechanism in Caenorhabditis elegans , 2005, Nature.

[48]  Ravi Sachidanandam,et al.  A germline-specific class of small RNAs binds mammalian Piwi proteins , 2006, Nature.

[49]  C. Banet-Noach,et al.  Argasid ticks as possible vectors of West Nile virus in Israel. , 2005, Vector borne and zoonotic diseases.

[50]  R. Carthew,et al.  Use of dsRNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway , 1998, Cell.

[51]  E. Sontheimer,et al.  Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways , 2004, Cell.

[52]  R. Andino,et al.  The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. , 2006, Genes & development.

[53]  B. McKee,et al.  A role of the Drosophila homeless gene in repression of Stellate in male meiosis , 2001, Chromosoma.

[54]  T. Pierson,et al.  Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA , 2005, Virology Journal.

[55]  G. Echalier,et al.  3. Arbovirus multiplication in an established diploid cell line of Drosophila melanogaster. , 1971, Current topics in microbiology and immunology.

[56]  A. Pélisson,et al.  Evidence for a piwi-Dependent RNA Silencing of the gypsy Endogenous Retrovirus by the Drosophila melanogaster flamenco Gene , 2004, Genetics.

[57]  J. Mackenzie,et al.  Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells , 1999, Journal of Virology.

[58]  J. H. Strauss,et al.  Recombination in Alphaviruses , 1997 .

[59]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[60]  D. Gubler,et al.  The use of mosquitoes to detect and propagate dengue viruses. , 1974, The American journal of tropical medicine and hygiene.

[61]  M J Morgan,et al.  Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. , 1979, The Journal of general virology.

[62]  S. Ding,et al.  Induction and Suppression of RNA Silencing by an Animal Virus , 2002, Science.

[63]  Brigitte Bogert,et al.  The fragile X-related Gene Affects the Crawling Behavior of Drosophila Larvae by Regulating the mRNA Level of the DEG/ENaC Protein Pickpocket1 , 2004, Current Biology.

[64]  N. Caplen,et al.  Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. , 2002, Molecular therapy : the journal of the American Society of Gene Therapy.

[65]  A. Aravin,et al.  Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline , 2001, Current Biology.

[66]  James A. Birchler,et al.  Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery , 2004, Science.

[67]  L Misquitta,et al.  Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[68]  S. Ding,et al.  RNA silencing: a conserved antiviral immunity of plants and animals. , 2004, Virus research.

[69]  D. Gubler,et al.  West Nile virus: epidemiology and clinical features of an emerging epidemic in the United States. , 2006, Annual review of medicine.

[70]  E. Holmes,et al.  The extent of homologous recombination in members of the genus Flavivirus. , 2003, The Journal of general virology.

[71]  G. Ebel,et al.  Partial genetic characterization of West Nile virus strains, New York State, 2000. , 2001, Emerging infectious diseases.

[72]  K. Olson,et al.  Characterization of RNA interference in an Anopheles gambiae cell line. , 2003, Insect biochemistry and molecular biology.

[73]  S. Whelan,et al.  An antiviral role for the RNA interference machinery in Caenorhabditis elegans. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[74]  B. Foy,et al.  RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[75]  J. Birchler,et al.  RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. , 2002, Molecular cell.

[76]  B. Bass,et al.  A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans , 2001, Science.

[77]  A. Raikhel,et al.  Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[78]  R. Carthew,et al.  A call to arms: coevolution of animal viruses and host innate immune responses. , 2007, Trends in genetics : TIG.

[79]  Susan A. Jones,et al.  High-Throughput Detection of West Nile Virus RNA , 2001, Journal of Clinical Microbiology.

[80]  N. Perrimon,et al.  Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells , 2004, Science.

[81]  Haifan Lin,et al.  A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. , 1998, Genes & development.

[82]  Peer Bork,et al.  Comparative Genome and Proteome Analysis of Anopheles gambiae and Drosophila melanogaster , 2002, Science.

[83]  A. James,et al.  Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[84]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[85]  G. Mudur.  Failure to control mosquitoes has led to two fever epidemics in India , 2006, BMJ : British Medical Journal.

[86]  D. Gubler.  The global emergence/resurgence of arboviral diseases as public health problems. , 2002, Archives of medical research.

[87]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[88]  B. Williams,et al.  The response of mammalian cells to double-stranded RNA. , 2007, Cytokine & growth factor reviews.

[89]  Akira Ishizuka,et al.  Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. , 2004, Genes & development.

[90]  Robert H. Silverman,et al.  Activation of the interferon system by short-interfering RNAs , 2003, Nature Cell Biology.