Microbiol Immunol 2015; 59: 299–304 doi: 10.1111/1348-0421.12253
ORIGINAL ARTICLE
Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA P. Shaik Syed Ali1,2, Jasmine John3, Manikandan Selvaraj2, Teh Lay Kek2 and Mohd Zaki Salleh2 1
Institute of Biophysical Chemistry, Goethe University, Max-von-Laue-Str. 9, Frankfurt 60438, Germany, 2Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi, MARA, 42300 Puncak Alam, Selangor, Malaysia and 3Institute of General Microbiology and Microbe Genetics, Friedrich-Schiller University Jena, Neugasse 24, D-07743 Jena, Germany
ABSTRACT Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations. Key words
Dicer, Nodamura virus B2, RNA interference suppressor, small interfering RNA.
RNA interference, a conserved RNA-guided genesilencing mechanism, is widespread in eukaryotes. The innate roles of RNAi comprise maintenance of gene regulation (1) and antiviral defense (2, 3). RNAi occurs at the post-transcriptional level and is triggered by dsRNA. siRNA and miRNA generated from dsRNA by Dicer are the central molecules of RNAi. Exogenous dsRNA is cleaved by Dicer, a RNAse III homolog, into 2125 nt siRNAs with characteristic 2 nt overhangs at the 30 end (4, 5). One of the single strands of siRNA incorporates into RISC (6) to base pair perfectly to its target mRNA and subsequently cleaves the mRNA (7). RISC is a multiprotein complex and the essential catalytic component of the complex is AGO (6), which has a PAZ domain that recognizes and binds 30 end 2 nt overhangs of siRNA (8, 9) and a PIWI domain with significant homology to RNAse H (10). In addition, Dicer is also involved in miRNA biogenesis by processing 70 nt precursor miRNA derived from primary miRNA (endogenous) to produce mature miRNA that incorporates into RISC. In
contrast to siRNA, miRNA base pairs imperfectly to its target mRNA resulting in translational repression (11). To overcome the host cell RNAi defense mechanism and undergo complete replication, as a counter defense mechanism, viruses produce proteins that are capable of suppressing RNAi. RNA silencing suppressor proteins encoded by viruses suppress RNAi across plant (12, 13) and animal kingdoms (14, 15). RNAi is suppressed by these proteins at different steps of Dicer (pre-Dicer/postDicer) through various strategies. Among the plant viruses, tombusvirus p19 acts by sequestering siRNA into RISC complex (16), whereas the cucumovirus suppressor protein 2B binds AGO1 and inhibits its enzymatic activity (17). Parallel to plant viruses, some animal viruses have also evolved to encode RNA silencing suppressor proteins that suppress RNAi. NoV belongs to the Alphanodavirus genus of the Nodaviridae family. NoV is unique that it is the only known arthropod-borne virus able to infect insects and vertebrate animals. NoV
Correspondence P. Shaik Syed Ali, iPROMISE, Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia. Tel.: þ603 3258 4652; Fax: þ603 3258 4658; email: [email protected] Received 22 December 2014; revised 17 February 2015; accepted 3 March 2015. List of Abbreviations: AGO, argonaute; Ala, alanine; CD, circular dichroism, Cys, cysteine; dsRNA, double stranded RNA; EMSA, electrophoretic mobility shift assay; FHV, flock house virus; Met, methionine; mRNA, messenger RNA; miRNA, microRNA; nm, nanometer; NoV, Nodamura virus; Nterminal, amino terminal; PAZ, Piwi Argonaute and Zwille; Phe, phenylalanine; RISC, RNA induced silencing complex; RNAi, RNA interference; RT, room temperature; siRNA, small interfering RNA.
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
299
P. Shaik Syed Ali et al.
possesses two positive sense single stranded RNA (RNA1 and RNA2). Protein B2 from NoV, a suppressor of RNAi, is encoded by RNA3 that is a subgenomic RNA transcribed from RNA1 (18). The full length protein B2 of NoV binds both dsRNA and siRNA, prevents the Dicer processing of dsRNA and sequesters siRNA into RISC, acting at pre- and post-Dicer steps, respectively to suppress RNAi (19). In this study we investigated whether the amino (N) terminal domain of NoV B2, which consists of residues 179 (NoV B2 79), binds both dsRNA and siRNA similar to full length NoV B2. This investigation contributes to better understand whether the Nterminal domain is the minimal domain of NoV B2 that is indispensable to bind both dsRNA and siRNA. Moreover, it may help to understand whether the NoV B2 N-terminal domain is involved at either steps of Dicer or specifically at the pre-Dicer step (by binding dsRNA/Dicer substrates) or the subsequent post-Dicer step (by binding siRNA/Dicer products) to suppress RNAi.
Site directed mutagenesis of NoV B2 79 The pET 16bþ plasmid carrying the NoV B2 79 gene was used as a template. The mutations were generated using a Quick Change site directed mutagenesis kit (Stratagene, Germany). The mutagenic primers containing the mutation were synthesized (Biomers, Germany). The primer sequences used to generate the mutants are shown in Table 2. Site directed mutagenesis was performed as described by the manufacturer and the mutations were confirmed by DNA sequencing. Electrophoretic mobility shift assay
Truncated NoV B2 gene (1-79) was cloned into modified pET16bþ plasmid containing a N-terminal His tag. For expression, Escherichia coli BL21 DE3 cells transformed with the plasmid were grown at 37°C in LB broth until an OD600 of 0.60.8 was reached. The cells were then induced with 1 mM isopropyl-b-thiogalactopyranoside and grown overnight at 25°C. One liter of harvested cells suspended in 20 mM Hepes, pH 7.4, 300 mM NaCl was sonicated and the protein was purified from the supernatant by Ni-NTA affinity chromatography in the same buffer containing imidazole. Further, the protein dimer was purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 preparative scale column (GE Healthcare, Germany), in 20 mM Hepes, pH 7.4, 300 mM NaCl, as previously described (20).
Electrophoretic mobility shift assays to analyze the interaction of NoV B2 79 with siRNAs and dsRNA were performed as follows: 50 fluorescein labeled siRNAs and dsRNA were synthesized (MWG Biotech, Germany). 75 nM of labeled siRNAs and dsRNA were titrated with 2 mM of NoV B2 79 and incubated for 15 min at RT. The total volume of binding reaction was 20 mL and the equilibration buffer contained 20 mM Hepes, pH 7.4, 120 mM NaCl, 150 mg/mL of yeast tRNA, 0.1 mM EDTA, 1 mM dithiothreitol and 1 mM MgCl2. The reaction mixtures were loaded on to a native 4% polyacrylamide gel and electrophoresed at 100 V for 45 min in 1X tris-borate-EDTA. After electrophoresis the gel was transferred on to a Lumi-Imager F1 (Roche, Switzerland) and exposed for 5 seconds at 520 nm. Electrophoretic mobility shift assays to compare the RNA binding affinity of wild type and double mutant (Phe49Cys/Ala60Met) NoV B2 79 were performed as follows: 75 nM of labeled 21 nt siRNA was titrated with two fold serial dilutions (3 mM90 nM) of wild type and double mutant NoV B2 79 and incubated at RT for 15 min. The binding reaction volume, equilibration buffer composition, electrophoresis conditions and gel exposure time were as described above. From two independent experiments (conducted with different batches of purified protein), the band intensities of the bound complexes were quantified by ImageJ software and histogram was plotted by using Graphpad Prism5.
RNA oligomers
Analytical gel filtration chromatography
The sequences of siRNA and dsRNA used in this study are shown in Table 1.
Analytical gel filtration chromatography was performed on a Superdex 200 10/300 GL column (GE Healthcare,
MATERIALS AND METHODS NoV B2 79 protein purification
Table 1. RNA oligomers used in this study RNA oligomers 21 25 27 30
nt nt nt nt
300
siRNA siRNA siRNA dsRNA
Sequence 0
0
Sense 5 -CGU ACG CGG AAU ACU UCG ATT-3 and antisense Sense 50 -CGU ACG CGG AAU ACU UCG ACG GATT-30 and antisense Sense 50 -CGU ACG CGG AAU ACU UCG ACG GAU ATT-30 and antisense 50 -CGU ACG CGG AAU ACU UCG ACG GAU AUU CGU-30 and 30 -GCA
30 -TTG CAU GCG CCU UAU GAA GCU-50 30 -TTG CAU GCG CCU UAU GAA GCU GCCU-50 30 -TTG CAU GCG CCU UAU GAA GCU GCC UAU-50 UGC GCC UUA UGA AGC UGC CUA UAA GCA-50
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
NoV B2 N-terminus binds siRNA only
Table 2. Sequences of primers used in this study Mutation Single/point mutants Phe49Cys Ala60Met Double mutant Phe49Cys/Ala60Met
Forward primer
Reverse primer
50 -GAT CTC CGC GCC TTT TGT GAA TGT TTA ACC G-30 50 -GGC TAT TAG TCA TAC GAT AGG CAC GTT G-30
50 -CGG TTA AAC ATT CAC AAA AGG CGC GGA GAT C-30 50 -CAA CGT GCC TAT CGT ATG ACT AAT AGC C-30
50 -GAT CTC CGC GCC TTT TGT GAA TGT TTA ACC G-30
50 -CAA CGT GCC TAT CGT ATG ACT AAT AGC C-30
Germany). The column was equilibrated and the chromatography was carried out in a buffer containing 20 mM Hepes, pH 7.4, 120 mM NaCl and 1 mM MgCl2 at RT with a flow rate of 0.4 mL/min and a sample loop volume of 500 mL. The siRNA was titrated to NoV B2 79 in a 1:10 molar ratio. The mixtures were incubated at RT for 15 min before applying on the column and the complex formation was analyzed by monitoring the absorption at 280 nm. Far-UV CD spectroscopic analyses Far-UV CD spectra were acquired on a Jasco-810 spectropolarimeter (Jasco, USA) equipped with a peltier temperature controller, using a 0.1 cm path length cuvette at a scan speed of 50 nm/min. The spectra were recorded at 20°C. CD spectra were averaged from four scans. Buffer baselines were recorded and subtracted from the spectra.
RESULTS NoV B2 N-terminal domain preferentially binds siRNA NoV B2 (full length) binds both dsRNAs and siRNAs (19) that are the substrates and products of Dicer, respectively. However, it has not yet been determined whether the Nterminal domain of NoV B2 binds both dsRNAs and siRNAs. To investigate this, 2 mM of NoV B2 79 was incubated with 75 nM of 50 fluorescein labeled 21 nt, 25 nt and 27 nt siRNAs and 30 nt dsRNA and analyzed by EMSA (Fig. 1). NoV B2 79 showed binding to 21 nt, 25 nt and 27 nt siRNAs (Fig. 1, lanes 2, 4 and 6) but not to 30 nt dsRNA, albeit having a similar sequence to siRNAs except lacking the 3'end 2 nt overhangs (Fig. 1, lane 8). These findings demonstrate that the NoV B2 N-terminal domain specifically recognizes and binds only siRNAs (Dicer products) but not dsRNAs (Dicer substrates). Oligomerization of NoV B2 N-terminal domain Oligomerization of NoV B2 N-terminal domain was investigated by incubating 1 mM of NoV B2 79 with © 2015 The Societies and Wiley Publishing Asia Pty Ltd
10 mM of 21 nt, 25 nt and 27 nt siRNAs and analyzed by gel filtration chromatography. The 21 nt and 27 nt siRNA alone eluted at 17.0 mL (Fig. 2a) and 16.7 mL (Fig. 2b), respectively. The NoV B2 79 alone eluted at 18.5 mL (Fig. 2a,b and Supp. Fig. S1), whereas as a complex with 21 nt siRNA, it eluted at 16.5 mL (Fig. 2a). However, the NoV B2 79/27nt siRNA complex showed a remarkable difference in the shift by eluting more rapidly at 15.5 mL (Fig. 2b). Surprisingly, a similar significant difference in the shift was not observed with NoV B2 79/25 nt siRNA complex that eluted at 16.4 mL, whereas the 25 nt siRNA alone eluted at 16.9 mL (Supp. Fig. S1). The unbound NoV B2 79 eluted at 18.5 mL analogous to NoV B2 79 alone (Fig. 2a,b and Supp. Fig. S1). NoV B2 79/27 nt siRNA complex displayed a 1 mL difference in the shift compared to NoV B2 79/25 nt and NoV B2 79/21 nt siRNA complexes, which is indicative of oligomeric NoV B2 79 binding to 27 nt siRNA. Effect of Phe49 and Ala60 substitutions on RNA binding In NoV B2, the two residues that contact RNA via van der Waals interactions are Phe49 and Ala60 that correspond to Cys44 and Met55 in FHV B2 (20). In order to determine the importance of Phe49 and Ala60
Fig. 1. NoV B2 79 interaction with siRNA and dsRNA. EMSA experiments were carried out by titrating 75 nM of 50 fluoresceinlabeled 21 nt, 25 nt and 27 nt siRNAs and 30 nt dsRNA with (þ) and without () 2 mM of NoV B2 79 protein.
301
P. Shaik Syed Ali et al.
binding affinity to siRNA than the wild type. Binding affinity experiments for the single mutants, Phe49Cys and Ala60Met, could not be conducted because of irreversible protein aggregation. The double mutant (Phe49Cys/Ala60Met) NoV B2 79 may have assumed a different structural conformation that contributed to higher RNA binding affinity. To investigate this CD spectra were recorded for both the wild type and double mutant NoV B2 79. CD spectra of the wild type and the double mutant were considerably similar with very negligible changes, suggesting that overall they assumed a similar structural conformation (Supp. Fig. S2).
DISCUSSION
Fig. 2. Oligomerization of NoV B2 79. Gel filtration chromatography of (a) 21 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 21 nt siRNA. (b) 27 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 27nt siRNA. The primary and secondary black peaks correspond to NoV B2 79/siRNA complex and unbound NoV B2 79.
of NoV B2 in binding siRNA, the residues were substituted with Cys and Met (corresponding residues in FHV B2) by site directed mutagenesis. Two point mutants and one double mutant were generated, namely Phe49Cys, Ala60Met and Phe49Cys/Ala60Met, respectively. The mutant B2 proteins were expressed and purified. 75 nM of 50 fluorescein labeled 21 nt siRNA was incubated with two fold serial dilutions (3 mM90 nM) of wild type and double mutant (Phe49Cys/ Ala60Met) NoV B2 79 and analyzed by EMSA (Fig. 3a). The double mutant showed significantly increased bound complex intensities (Fig. 3a, lanes 813) than the wild type NoV B2 79 (Fig. 3a, lanes 27). The bound complex intensities quantified from two independent experiments are presented as histograms (Fig. 3b). The above result demonstrates that the double mutant (Phe49Cys/Ala60Met) NoV B2 79 has a higher 302
Previous studies have shown that the full length NoV B2 binds both dsRNA and siRNA with dual modes of RNAi suppression (19). However, preferential binding of the NoV B2 N-terminal domain to siRNA demonstrates that it may be sensitive to the 30 end 2 nt overhangs that is present in the Dicer products (siRNA) but absent in the Dicer substrates (dsRNA). The NoV B2 N-terminal domain may specifically act at the post-Dicer step (after the Dicer processes dsRNA into siRNA), by sequestering siRNA into RISC or it may compete with the AGO PAZ domain that selectively recognizes and binds the 2 nt overhangs of siRNA (8, 9), to suppress RNAi. The inability of the NoV B2 N-terminal domain to bind dsRNA indicates that the co-operation of carboxy terminal residues may be required to achieve binding to dsRNA that are the siRNA precursors/Dicer substrates. Alternatively, the carboxy terminal domain of NoV B2 may directly interact with the PAZ domain of Dicer to prevent dsRNA processing, similar to FHV B2 (21). NoV B2 79 is a dimer; therefore a lone dimer may bind 21 nt siRNA, which is in agreement with 20 nt dsRNA binding a single dimer of N-terminal domain of FHV B2 (22) that adopts a similar structure to NoV B2 79 (20). However, considering the length of siRNA involved, in 27 nt siRNA/NoV B2 79 complex possibly a dimer of NoV B2 79 dimer may bind 27 nt siRNA. Oligomerization of NoV B2 on longer siRNAs such as a 27 nt siRNA suggests that the binding of additional NoV B2 suppressors of RNAi may aid in efficiently sequestering the siRNA into RISC. Computational molecular dynamics simulations showed that the Phe49Cys mutation in NoV B2 reduces its affinity to bind RNA (23). However, our study through in vitro RNA binding assay showed that the mutation Phe49Cys (with a co-mutation Ala60Met) did not reduce the RNA binding affinity, but rather favored a © 2015 The Societies and Wiley Publishing Asia Pty Ltd
NoV B2 N-terminus binds siRNA only
Fig. 3. Interaction of NoV B2 79 wild type and double mutant (phenylalanine 49 cysteine/alanine 60 methionine) to 21 nt siRNA. (a) EMSA experiments were carried out by titrating 75 nM of 50 fluorescein labeled 21 nt siRNA with the indicated protein concentrations of wild type and double mutant NoV B2 79. (b) The bound complex intensities were quantified and are presented as histogram. White and black color bars represent the bound complex intensities of wild type and double mutant NoV B2 79, respectively.
higher affinity to bind RNA. In protein-RNA complexes, van der Waals contacts play a significant role in binding RNA (24). All the four residues Phe, Ala, Cys and Met were found to contact RNA via van der Waals interactions in RNA-protein complexes (20, 24). Our study demonstrates that the residues Cys and Met cooperatively show a stronger interaction with RNA than Phe and Ala in NoV B2.
ACKNOWLEDGEMENTS This work was funded by the Hessian Ministry for Science and Culture. Support by Professor Dr. Volker D€otsch (Institute of Biophysical Chemistry, Goethe University Frankfurt, Germany) and Dr. Julian Chen (Goethe University Frankfurt, Germany) is gratefully acknowledged. © 2015 The Societies and Wiley Publishing Asia Pty Ltd
DISCLOSURE The authors declare no competing interests.
REFERENCES 1. Ruvkun G. (2001) Molecular biology. Glimpses of a tiny RNA world. Science 294: 797–9. 2. Agius C., Eamens A.L., Millar A.A., Watson J.M., Wang M.B. (2012) RNA silencing and antiviral defense in plants. Methods Mol Biol 894: 17–38. 3. Umbach J.L., Cullen B.R. (2009) The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev 23: 1151–64. 4. Bernstein E., Caudy A.A., Hammond S.M., Hannon G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–6. 5. Zamore P.D., Tuschl T., Sharp P.A., Bartel D.P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33.
303
P. Shaik Syed Ali et al.
6. Macrae I.J., Ma E., Zhou M., Robinson C.V., Doudna J.A. (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A 105: 512–7. 7. Wilson R.C., Doudna J.A. (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42: 217–39. 8. Ma J.B., Ye K., Patel D.J. (2004) Structural basis for overhangspecific small interfering RNA recognition by the PAZ domain. Nature 429: 318–22. 9. Lingel A., Simon B., Izaurralde E., Sattler M. (2004) Nucleic acid 3'-end recognition by the Argonaute2 PAZ domain. Nat Struct Mol Biol 11: 576–7. 10. Parker J.S., Roe S.M., Barford D. (2004) Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J 23: 4727–37. 11. Djuranovic S., Nahvi A., Green R. (2011) A parsimonious model for gene regulation by miRNAs. Science 331: 550–3. 12. Roth B.M., Pruss G.J., Vance V.B. (2004) Plant viral suppressors of RNA silencing. Virus Res 102: 97–108. 13. Voinnet O. (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6: 206–20. 14. Johnson K.L., Price B.D., Eckerle L.D., Ball L.A. (2004) Nodamura virus nonstructural protein B2 can enhance viral RNA accumulation in both mammalian and insect cells. J Virol 78: 6698–704. 15. Li H., Li W.X., Ding S.W. (2002) Induction and suppression of RNA silencing by an animal virus. Science 296: 1319–21. 16. Lakatos L., Szittya G., Silhavy D., Burgyan J. (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J 23: 876–84. 17. Zhang X., Yuan Y.R., Pei Y., Lin S.S., Tuschl T., Patel D.J., Chua N.H. (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20: 3255–68. 18. Johnson K.L., Price B.D., Ball L.A. (2003) Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins. Virology 305: 436–51. 19. Sullivan C.S., Ganem D. (2005) A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J Virol 79: 7371–9.
304
20. Korber S., Shaik Syed Ali P., Chen J.C. (2009) Structure of the RNA-binding domain of Nodamura virus protein B2, a suppressor of RNA interference. Biochemistry 48: 2307–9. 21. Singh G., Popli S., Hari Y., Malhotra P., Mukherjee S., Bhatnagar R.K. (2009) Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer. FASEB J 23: 1845–57. 22. Chao J.A., Lee J.H., Chapados B.R., Debler E.W., Schneemann A., Williamson J.R. (2005) Dual modes of RNAsilencing suppression by Flock House virus protein B2. Nat Struct Mol Biol 12: 952–7. 23. Allen W.J., Wiley M.R., Myles K.M., Adelman Z.N., Bevan D.R. (2014) Steered molecular dynamics identifies critical residues of the Nodamura virus B2 suppressor of RNAi. J Mol Model 20: 2092. 24. Jones S., Daley D.T., Luscombe N.M., Berman H.M., Thornton J.M. (2001) Protein-RNA interactions: a structural analysis. Nucleic Acids Res 29: 943–54.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher's web-site. Fig. S1. Gel filtration chromatography of NoV B2 79 with 25 nt siRNA. Gel filtration chromatography of 25 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 25 nt siRNA. The primary and secondary black peaks correspond to NoV B2 79/ 25nt siRNA complex and unbound NoV B2 79. Fig. S2. Secondary structures of wild type and double mutant (phenylalanine 49 cysteine/alanine 60 methionine) NoV B2 79. CD spectra of wild type (black) and double mutant NoV B2 79 (gray).
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
ORIGINAL ARTICLE
Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA P. Shaik Syed Ali1,2, Jasmine John3, Manikandan Selvaraj2, Teh Lay Kek2 and Mohd Zaki Salleh2 1
Institute of Biophysical Chemistry, Goethe University, Max-von-Laue-Str. 9, Frankfurt 60438, Germany, 2Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi, MARA, 42300 Puncak Alam, Selangor, Malaysia and 3Institute of General Microbiology and Microbe Genetics, Friedrich-Schiller University Jena, Neugasse 24, D-07743 Jena, Germany
ABSTRACT Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations. Key words
Dicer, Nodamura virus B2, RNA interference suppressor, small interfering RNA.
RNA interference, a conserved RNA-guided genesilencing mechanism, is widespread in eukaryotes. The innate roles of RNAi comprise maintenance of gene regulation (1) and antiviral defense (2, 3). RNAi occurs at the post-transcriptional level and is triggered by dsRNA. siRNA and miRNA generated from dsRNA by Dicer are the central molecules of RNAi. Exogenous dsRNA is cleaved by Dicer, a RNAse III homolog, into 2125 nt siRNAs with characteristic 2 nt overhangs at the 30 end (4, 5). One of the single strands of siRNA incorporates into RISC (6) to base pair perfectly to its target mRNA and subsequently cleaves the mRNA (7). RISC is a multiprotein complex and the essential catalytic component of the complex is AGO (6), which has a PAZ domain that recognizes and binds 30 end 2 nt overhangs of siRNA (8, 9) and a PIWI domain with significant homology to RNAse H (10). In addition, Dicer is also involved in miRNA biogenesis by processing 70 nt precursor miRNA derived from primary miRNA (endogenous) to produce mature miRNA that incorporates into RISC. In
contrast to siRNA, miRNA base pairs imperfectly to its target mRNA resulting in translational repression (11). To overcome the host cell RNAi defense mechanism and undergo complete replication, as a counter defense mechanism, viruses produce proteins that are capable of suppressing RNAi. RNA silencing suppressor proteins encoded by viruses suppress RNAi across plant (12, 13) and animal kingdoms (14, 15). RNAi is suppressed by these proteins at different steps of Dicer (pre-Dicer/postDicer) through various strategies. Among the plant viruses, tombusvirus p19 acts by sequestering siRNA into RISC complex (16), whereas the cucumovirus suppressor protein 2B binds AGO1 and inhibits its enzymatic activity (17). Parallel to plant viruses, some animal viruses have also evolved to encode RNA silencing suppressor proteins that suppress RNAi. NoV belongs to the Alphanodavirus genus of the Nodaviridae family. NoV is unique that it is the only known arthropod-borne virus able to infect insects and vertebrate animals. NoV
Correspondence P. Shaik Syed Ali, iPROMISE, Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia. Tel.: þ603 3258 4652; Fax: þ603 3258 4658; email: [email protected] Received 22 December 2014; revised 17 February 2015; accepted 3 March 2015. List of Abbreviations: AGO, argonaute; Ala, alanine; CD, circular dichroism, Cys, cysteine; dsRNA, double stranded RNA; EMSA, electrophoretic mobility shift assay; FHV, flock house virus; Met, methionine; mRNA, messenger RNA; miRNA, microRNA; nm, nanometer; NoV, Nodamura virus; Nterminal, amino terminal; PAZ, Piwi Argonaute and Zwille; Phe, phenylalanine; RISC, RNA induced silencing complex; RNAi, RNA interference; RT, room temperature; siRNA, small interfering RNA.
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
299
P. Shaik Syed Ali et al.
possesses two positive sense single stranded RNA (RNA1 and RNA2). Protein B2 from NoV, a suppressor of RNAi, is encoded by RNA3 that is a subgenomic RNA transcribed from RNA1 (18). The full length protein B2 of NoV binds both dsRNA and siRNA, prevents the Dicer processing of dsRNA and sequesters siRNA into RISC, acting at pre- and post-Dicer steps, respectively to suppress RNAi (19). In this study we investigated whether the amino (N) terminal domain of NoV B2, which consists of residues 179 (NoV B2 79), binds both dsRNA and siRNA similar to full length NoV B2. This investigation contributes to better understand whether the Nterminal domain is the minimal domain of NoV B2 that is indispensable to bind both dsRNA and siRNA. Moreover, it may help to understand whether the NoV B2 N-terminal domain is involved at either steps of Dicer or specifically at the pre-Dicer step (by binding dsRNA/Dicer substrates) or the subsequent post-Dicer step (by binding siRNA/Dicer products) to suppress RNAi.
Site directed mutagenesis of NoV B2 79 The pET 16bþ plasmid carrying the NoV B2 79 gene was used as a template. The mutations were generated using a Quick Change site directed mutagenesis kit (Stratagene, Germany). The mutagenic primers containing the mutation were synthesized (Biomers, Germany). The primer sequences used to generate the mutants are shown in Table 2. Site directed mutagenesis was performed as described by the manufacturer and the mutations were confirmed by DNA sequencing. Electrophoretic mobility shift assay
Truncated NoV B2 gene (1-79) was cloned into modified pET16bþ plasmid containing a N-terminal His tag. For expression, Escherichia coli BL21 DE3 cells transformed with the plasmid were grown at 37°C in LB broth until an OD600 of 0.60.8 was reached. The cells were then induced with 1 mM isopropyl-b-thiogalactopyranoside and grown overnight at 25°C. One liter of harvested cells suspended in 20 mM Hepes, pH 7.4, 300 mM NaCl was sonicated and the protein was purified from the supernatant by Ni-NTA affinity chromatography in the same buffer containing imidazole. Further, the protein dimer was purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 preparative scale column (GE Healthcare, Germany), in 20 mM Hepes, pH 7.4, 300 mM NaCl, as previously described (20).
Electrophoretic mobility shift assays to analyze the interaction of NoV B2 79 with siRNAs and dsRNA were performed as follows: 50 fluorescein labeled siRNAs and dsRNA were synthesized (MWG Biotech, Germany). 75 nM of labeled siRNAs and dsRNA were titrated with 2 mM of NoV B2 79 and incubated for 15 min at RT. The total volume of binding reaction was 20 mL and the equilibration buffer contained 20 mM Hepes, pH 7.4, 120 mM NaCl, 150 mg/mL of yeast tRNA, 0.1 mM EDTA, 1 mM dithiothreitol and 1 mM MgCl2. The reaction mixtures were loaded on to a native 4% polyacrylamide gel and electrophoresed at 100 V for 45 min in 1X tris-borate-EDTA. After electrophoresis the gel was transferred on to a Lumi-Imager F1 (Roche, Switzerland) and exposed for 5 seconds at 520 nm. Electrophoretic mobility shift assays to compare the RNA binding affinity of wild type and double mutant (Phe49Cys/Ala60Met) NoV B2 79 were performed as follows: 75 nM of labeled 21 nt siRNA was titrated with two fold serial dilutions (3 mM90 nM) of wild type and double mutant NoV B2 79 and incubated at RT for 15 min. The binding reaction volume, equilibration buffer composition, electrophoresis conditions and gel exposure time were as described above. From two independent experiments (conducted with different batches of purified protein), the band intensities of the bound complexes were quantified by ImageJ software and histogram was plotted by using Graphpad Prism5.
RNA oligomers
Analytical gel filtration chromatography
The sequences of siRNA and dsRNA used in this study are shown in Table 1.
Analytical gel filtration chromatography was performed on a Superdex 200 10/300 GL column (GE Healthcare,
MATERIALS AND METHODS NoV B2 79 protein purification
Table 1. RNA oligomers used in this study RNA oligomers 21 25 27 30
nt nt nt nt
300
siRNA siRNA siRNA dsRNA
Sequence 0
0
Sense 5 -CGU ACG CGG AAU ACU UCG ATT-3 and antisense Sense 50 -CGU ACG CGG AAU ACU UCG ACG GATT-30 and antisense Sense 50 -CGU ACG CGG AAU ACU UCG ACG GAU ATT-30 and antisense 50 -CGU ACG CGG AAU ACU UCG ACG GAU AUU CGU-30 and 30 -GCA
30 -TTG CAU GCG CCU UAU GAA GCU-50 30 -TTG CAU GCG CCU UAU GAA GCU GCCU-50 30 -TTG CAU GCG CCU UAU GAA GCU GCC UAU-50 UGC GCC UUA UGA AGC UGC CUA UAA GCA-50
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
NoV B2 N-terminus binds siRNA only
Table 2. Sequences of primers used in this study Mutation Single/point mutants Phe49Cys Ala60Met Double mutant Phe49Cys/Ala60Met
Forward primer
Reverse primer
50 -GAT CTC CGC GCC TTT TGT GAA TGT TTA ACC G-30 50 -GGC TAT TAG TCA TAC GAT AGG CAC GTT G-30
50 -CGG TTA AAC ATT CAC AAA AGG CGC GGA GAT C-30 50 -CAA CGT GCC TAT CGT ATG ACT AAT AGC C-30
50 -GAT CTC CGC GCC TTT TGT GAA TGT TTA ACC G-30
50 -CAA CGT GCC TAT CGT ATG ACT AAT AGC C-30
Germany). The column was equilibrated and the chromatography was carried out in a buffer containing 20 mM Hepes, pH 7.4, 120 mM NaCl and 1 mM MgCl2 at RT with a flow rate of 0.4 mL/min and a sample loop volume of 500 mL. The siRNA was titrated to NoV B2 79 in a 1:10 molar ratio. The mixtures were incubated at RT for 15 min before applying on the column and the complex formation was analyzed by monitoring the absorption at 280 nm. Far-UV CD spectroscopic analyses Far-UV CD spectra were acquired on a Jasco-810 spectropolarimeter (Jasco, USA) equipped with a peltier temperature controller, using a 0.1 cm path length cuvette at a scan speed of 50 nm/min. The spectra were recorded at 20°C. CD spectra were averaged from four scans. Buffer baselines were recorded and subtracted from the spectra.
RESULTS NoV B2 N-terminal domain preferentially binds siRNA NoV B2 (full length) binds both dsRNAs and siRNAs (19) that are the substrates and products of Dicer, respectively. However, it has not yet been determined whether the Nterminal domain of NoV B2 binds both dsRNAs and siRNAs. To investigate this, 2 mM of NoV B2 79 was incubated with 75 nM of 50 fluorescein labeled 21 nt, 25 nt and 27 nt siRNAs and 30 nt dsRNA and analyzed by EMSA (Fig. 1). NoV B2 79 showed binding to 21 nt, 25 nt and 27 nt siRNAs (Fig. 1, lanes 2, 4 and 6) but not to 30 nt dsRNA, albeit having a similar sequence to siRNAs except lacking the 3'end 2 nt overhangs (Fig. 1, lane 8). These findings demonstrate that the NoV B2 N-terminal domain specifically recognizes and binds only siRNAs (Dicer products) but not dsRNAs (Dicer substrates). Oligomerization of NoV B2 N-terminal domain Oligomerization of NoV B2 N-terminal domain was investigated by incubating 1 mM of NoV B2 79 with © 2015 The Societies and Wiley Publishing Asia Pty Ltd
10 mM of 21 nt, 25 nt and 27 nt siRNAs and analyzed by gel filtration chromatography. The 21 nt and 27 nt siRNA alone eluted at 17.0 mL (Fig. 2a) and 16.7 mL (Fig. 2b), respectively. The NoV B2 79 alone eluted at 18.5 mL (Fig. 2a,b and Supp. Fig. S1), whereas as a complex with 21 nt siRNA, it eluted at 16.5 mL (Fig. 2a). However, the NoV B2 79/27nt siRNA complex showed a remarkable difference in the shift by eluting more rapidly at 15.5 mL (Fig. 2b). Surprisingly, a similar significant difference in the shift was not observed with NoV B2 79/25 nt siRNA complex that eluted at 16.4 mL, whereas the 25 nt siRNA alone eluted at 16.9 mL (Supp. Fig. S1). The unbound NoV B2 79 eluted at 18.5 mL analogous to NoV B2 79 alone (Fig. 2a,b and Supp. Fig. S1). NoV B2 79/27 nt siRNA complex displayed a 1 mL difference in the shift compared to NoV B2 79/25 nt and NoV B2 79/21 nt siRNA complexes, which is indicative of oligomeric NoV B2 79 binding to 27 nt siRNA. Effect of Phe49 and Ala60 substitutions on RNA binding In NoV B2, the two residues that contact RNA via van der Waals interactions are Phe49 and Ala60 that correspond to Cys44 and Met55 in FHV B2 (20). In order to determine the importance of Phe49 and Ala60
Fig. 1. NoV B2 79 interaction with siRNA and dsRNA. EMSA experiments were carried out by titrating 75 nM of 50 fluoresceinlabeled 21 nt, 25 nt and 27 nt siRNAs and 30 nt dsRNA with (þ) and without () 2 mM of NoV B2 79 protein.
301
P. Shaik Syed Ali et al.
binding affinity to siRNA than the wild type. Binding affinity experiments for the single mutants, Phe49Cys and Ala60Met, could not be conducted because of irreversible protein aggregation. The double mutant (Phe49Cys/Ala60Met) NoV B2 79 may have assumed a different structural conformation that contributed to higher RNA binding affinity. To investigate this CD spectra were recorded for both the wild type and double mutant NoV B2 79. CD spectra of the wild type and the double mutant were considerably similar with very negligible changes, suggesting that overall they assumed a similar structural conformation (Supp. Fig. S2).
DISCUSSION
Fig. 2. Oligomerization of NoV B2 79. Gel filtration chromatography of (a) 21 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 21 nt siRNA. (b) 27 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 27nt siRNA. The primary and secondary black peaks correspond to NoV B2 79/siRNA complex and unbound NoV B2 79.
of NoV B2 in binding siRNA, the residues were substituted with Cys and Met (corresponding residues in FHV B2) by site directed mutagenesis. Two point mutants and one double mutant were generated, namely Phe49Cys, Ala60Met and Phe49Cys/Ala60Met, respectively. The mutant B2 proteins were expressed and purified. 75 nM of 50 fluorescein labeled 21 nt siRNA was incubated with two fold serial dilutions (3 mM90 nM) of wild type and double mutant (Phe49Cys/ Ala60Met) NoV B2 79 and analyzed by EMSA (Fig. 3a). The double mutant showed significantly increased bound complex intensities (Fig. 3a, lanes 813) than the wild type NoV B2 79 (Fig. 3a, lanes 27). The bound complex intensities quantified from two independent experiments are presented as histograms (Fig. 3b). The above result demonstrates that the double mutant (Phe49Cys/Ala60Met) NoV B2 79 has a higher 302
Previous studies have shown that the full length NoV B2 binds both dsRNA and siRNA with dual modes of RNAi suppression (19). However, preferential binding of the NoV B2 N-terminal domain to siRNA demonstrates that it may be sensitive to the 30 end 2 nt overhangs that is present in the Dicer products (siRNA) but absent in the Dicer substrates (dsRNA). The NoV B2 N-terminal domain may specifically act at the post-Dicer step (after the Dicer processes dsRNA into siRNA), by sequestering siRNA into RISC or it may compete with the AGO PAZ domain that selectively recognizes and binds the 2 nt overhangs of siRNA (8, 9), to suppress RNAi. The inability of the NoV B2 N-terminal domain to bind dsRNA indicates that the co-operation of carboxy terminal residues may be required to achieve binding to dsRNA that are the siRNA precursors/Dicer substrates. Alternatively, the carboxy terminal domain of NoV B2 may directly interact with the PAZ domain of Dicer to prevent dsRNA processing, similar to FHV B2 (21). NoV B2 79 is a dimer; therefore a lone dimer may bind 21 nt siRNA, which is in agreement with 20 nt dsRNA binding a single dimer of N-terminal domain of FHV B2 (22) that adopts a similar structure to NoV B2 79 (20). However, considering the length of siRNA involved, in 27 nt siRNA/NoV B2 79 complex possibly a dimer of NoV B2 79 dimer may bind 27 nt siRNA. Oligomerization of NoV B2 on longer siRNAs such as a 27 nt siRNA suggests that the binding of additional NoV B2 suppressors of RNAi may aid in efficiently sequestering the siRNA into RISC. Computational molecular dynamics simulations showed that the Phe49Cys mutation in NoV B2 reduces its affinity to bind RNA (23). However, our study through in vitro RNA binding assay showed that the mutation Phe49Cys (with a co-mutation Ala60Met) did not reduce the RNA binding affinity, but rather favored a © 2015 The Societies and Wiley Publishing Asia Pty Ltd
NoV B2 N-terminus binds siRNA only
Fig. 3. Interaction of NoV B2 79 wild type and double mutant (phenylalanine 49 cysteine/alanine 60 methionine) to 21 nt siRNA. (a) EMSA experiments were carried out by titrating 75 nM of 50 fluorescein labeled 21 nt siRNA with the indicated protein concentrations of wild type and double mutant NoV B2 79. (b) The bound complex intensities were quantified and are presented as histogram. White and black color bars represent the bound complex intensities of wild type and double mutant NoV B2 79, respectively.
higher affinity to bind RNA. In protein-RNA complexes, van der Waals contacts play a significant role in binding RNA (24). All the four residues Phe, Ala, Cys and Met were found to contact RNA via van der Waals interactions in RNA-protein complexes (20, 24). Our study demonstrates that the residues Cys and Met cooperatively show a stronger interaction with RNA than Phe and Ala in NoV B2.
ACKNOWLEDGEMENTS This work was funded by the Hessian Ministry for Science and Culture. Support by Professor Dr. Volker D€otsch (Institute of Biophysical Chemistry, Goethe University Frankfurt, Germany) and Dr. Julian Chen (Goethe University Frankfurt, Germany) is gratefully acknowledged. © 2015 The Societies and Wiley Publishing Asia Pty Ltd
DISCLOSURE The authors declare no competing interests.
REFERENCES 1. Ruvkun G. (2001) Molecular biology. Glimpses of a tiny RNA world. Science 294: 797–9. 2. Agius C., Eamens A.L., Millar A.A., Watson J.M., Wang M.B. (2012) RNA silencing and antiviral defense in plants. Methods Mol Biol 894: 17–38. 3. Umbach J.L., Cullen B.R. (2009) The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev 23: 1151–64. 4. Bernstein E., Caudy A.A., Hammond S.M., Hannon G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–6. 5. Zamore P.D., Tuschl T., Sharp P.A., Bartel D.P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33.
303
P. Shaik Syed Ali et al.
6. Macrae I.J., Ma E., Zhou M., Robinson C.V., Doudna J.A. (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A 105: 512–7. 7. Wilson R.C., Doudna J.A. (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42: 217–39. 8. Ma J.B., Ye K., Patel D.J. (2004) Structural basis for overhangspecific small interfering RNA recognition by the PAZ domain. Nature 429: 318–22. 9. Lingel A., Simon B., Izaurralde E., Sattler M. (2004) Nucleic acid 3'-end recognition by the Argonaute2 PAZ domain. Nat Struct Mol Biol 11: 576–7. 10. Parker J.S., Roe S.M., Barford D. (2004) Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J 23: 4727–37. 11. Djuranovic S., Nahvi A., Green R. (2011) A parsimonious model for gene regulation by miRNAs. Science 331: 550–3. 12. Roth B.M., Pruss G.J., Vance V.B. (2004) Plant viral suppressors of RNA silencing. Virus Res 102: 97–108. 13. Voinnet O. (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6: 206–20. 14. Johnson K.L., Price B.D., Eckerle L.D., Ball L.A. (2004) Nodamura virus nonstructural protein B2 can enhance viral RNA accumulation in both mammalian and insect cells. J Virol 78: 6698–704. 15. Li H., Li W.X., Ding S.W. (2002) Induction and suppression of RNA silencing by an animal virus. Science 296: 1319–21. 16. Lakatos L., Szittya G., Silhavy D., Burgyan J. (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J 23: 876–84. 17. Zhang X., Yuan Y.R., Pei Y., Lin S.S., Tuschl T., Patel D.J., Chua N.H. (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20: 3255–68. 18. Johnson K.L., Price B.D., Ball L.A. (2003) Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins. Virology 305: 436–51. 19. Sullivan C.S., Ganem D. (2005) A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J Virol 79: 7371–9.
304
20. Korber S., Shaik Syed Ali P., Chen J.C. (2009) Structure of the RNA-binding domain of Nodamura virus protein B2, a suppressor of RNA interference. Biochemistry 48: 2307–9. 21. Singh G., Popli S., Hari Y., Malhotra P., Mukherjee S., Bhatnagar R.K. (2009) Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer. FASEB J 23: 1845–57. 22. Chao J.A., Lee J.H., Chapados B.R., Debler E.W., Schneemann A., Williamson J.R. (2005) Dual modes of RNAsilencing suppression by Flock House virus protein B2. Nat Struct Mol Biol 12: 952–7. 23. Allen W.J., Wiley M.R., Myles K.M., Adelman Z.N., Bevan D.R. (2014) Steered molecular dynamics identifies critical residues of the Nodamura virus B2 suppressor of RNAi. J Mol Model 20: 2092. 24. Jones S., Daley D.T., Luscombe N.M., Berman H.M., Thornton J.M. (2001) Protein-RNA interactions: a structural analysis. Nucleic Acids Res 29: 943–54.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher's web-site. Fig. S1. Gel filtration chromatography of NoV B2 79 with 25 nt siRNA. Gel filtration chromatography of 25 nt siRNA alone (dotted line) and NoV B2 79 in the absence (gray) and presence (black) of 25 nt siRNA. The primary and secondary black peaks correspond to NoV B2 79/ 25nt siRNA complex and unbound NoV B2 79. Fig. S2. Secondary structures of wild type and double mutant (phenylalanine 49 cysteine/alanine 60 methionine) NoV B2 79. CD spectra of wild type (black) and double mutant NoV B2 79 (gray).
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
