Suppression of RNA Interference by Adenovirus Virus-Associated RNA

ABSTRACT We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3′ strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.

[1]  E. Finnegan,et al.  The small RNA world , 2003, Journal of Cell Science.

[2]  M. Mathews,et al.  Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector. , 1990, Nucleic acids research.

[3]  O. Voinnet,et al.  RNA silencing: no mercy for viruses? , 2004, Immunological reviews.

[4]  T. Shenk,et al.  Adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection , 1982, Cell.

[5]  M. Katze,et al.  Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. , 1998, Pharmacology & therapeutics.

[6]  J. Manley,et al.  Primary structure of the human splicing factor asf reveals similarities with drosophila regulators , 1991, Cell.

[7]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[8]  B. Cullen,et al.  Adenovirus VA1 Noncoding RNA Can Inhibit Small Interfering RNA and MicroRNA Biogenesis , 2004, Journal of Virology.

[9]  Edouard Bertrand,et al.  Exportin-5 Mediates Nuclear Export of Minihelix-containing RNAs* , 2003, The Journal of Biological Chemistry.

[10]  S. J. Flint,et al.  Principles of Virology: Molecular Biology, Pathogenesis, and Control , 1999 .

[11]  Edwards Allen,et al.  P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. , 2003, Developmental cell.

[12]  M. Esteban,et al.  Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): Mechanism of action , 2000, Apoptosis.

[13]  J. Burgyán,et al.  Effects and side-effects of viral RNA silencing suppressors on short RNAs. , 2004, Trends in plant science.

[14]  B. Berkhout,et al.  Inhibition of virus replication by RNA interference. , 2003, Journal of biomedical science.

[15]  A. Fire,et al.  Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Frendewey,et al.  Pac1 ribonuclease of Schizosaccharomyces pombe. , 2001, Methods in enzymology.

[17]  M. Mathews.  Structure, function, and evolution of adenovirus virus-associated RNAs. , 1995, Current topics in microbiology and immunology.

[18]  R. Lehmann,et al.  Targeted mRNA degradation by double-stranded RNA in vitro. , 1999, Genes & development.

[19]  R. Duggleby,et al.  Quantitative analysis of the time courses of enzyme-catalyzed reactions. , 2001, Methods.

[20]  M. Mathews,et al.  Structure of genes for virus-associated RNAI and RNAII of adenovirus type 2. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Anton J. Enright,et al.  Identification of Virus-Encoded MicroRNAs , 2004, Science.

[22]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

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

[24]  E. Moss,et al.  Small-interfering RNAs in the radar of the interferon system , 2003, Nature Cell Biology.

[25]  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.

[26]  B. Cullen,et al.  Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. , 2003, Genes & development.

[27]  G. Hannon,et al.  RNAi: an ever-growing puzzle. , 2003, Trends in biochemical sciences.

[28]  G. Akusjärvi,et al.  Remodelling of the host cell RNA splicing machinery during an adenovirus infection. , 2003, Current topics in microbiology and immunology.

[29]  C. Samuel,et al.  Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2α kinase , 1986, Cell.

[30]  J. Gui,et al.  Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Mathews,et al.  A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2 , 1976, Cell.

[32]  A. Ganser,et al.  Specific inhibition of bcr-abl gene expression by small interfering RNA. , 2003, Blood.

[33]  G. Akusjärvi,et al.  Adenovirus VA RNAI: a positive regulator of mRNA translation , 1984, Molecular and cellular biology.

[34]  M. Mathews,et al.  Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA. , 1995, RNA.

[35]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

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

[37]  S. Goodbourn,et al.  Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. , 2000, The Journal of general virology.

[38]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[39]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[40]  P. Cohen,et al.  Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity , 1997, FEBS letters.

[41]  M. Mathews,et al.  Characterization of the double-stranded RNA implicated in the inhibition of protein synthesis in cells infected with a mutant adenovirus defective for VA RNA. , 1988, Virology.

[42]  S. Ding,et al.  Viral suppressors of RNA silencing. , 2001, Current opinion in biotechnology.

[43]  R. Bhat,et al.  Adenovirus mutants with DNA sequence perturbations in the intragenic promoter of VAI RNA gene allow the enhanced transcription of VAII RNA gene in HeLa cells. , 1984, Nucleic acids research.

[44]  Ben Berkhout,et al.  HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome , 2005, Nucleic acids research.

[45]  G. Akusjärvi,et al.  A novel adenovirus‐2 E1A mRNA encoding a protein with transcription activation properties. , 1987, The EMBO journal.

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

[47]  M. Mathews,et al.  Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  G. Akusjärvi,et al.  A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay , 1987, Molecular and cellular biology.

[49]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

[50]  A. Das,et al.  A Hairpin Structure in the R Region of the Human Immunodeficiency Virus Type 1 RNA Genome Is Instrumental in Polyadenylation Site Selection , 1999, Journal of Virology.

[51]  G. Akusjärvi,et al.  Control of adenovirus major late gene expression at multiple levels. , 1992, Journal of molecular biology.