Cell Host & Microbe, Volume 4 Supplemental Data Mechanism of Induction and Suppression of Antiviral Immunity Directed by Virus-Derived Small RNAs in Drosophila Roghiyh Aliyari, Qingfa Wu, Hong-Wei Li, Xiao-Hong Wang, Feng Li, Lance D. Green, Cliff S. Han, Wan-Xiang Li, and Shou-Wei Ding Supplemental Experimental Procedures GST Pull-Down Assay S2 cells were co-transfected with pmt-fr1δb2, dsrna of AGO2 and a plasmids expressing one of the following proteins: FB2, mfb2, NB2 or mnb2. Cells were collected three days after transfection and were subjected to pulldown assay as described in http://www.bioprotocol.com with some modifications. Briefly, cells were lysed in NETN (20 mm Tris-HCl, ph 7.5, 0.05% Nonidet P-40, 1 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF) with 1 M NaCl followed by sonication (50% pulsar, 15 seconds for 6 times and 30 seconds interval). Cellular extracts were incubated with Glutathione Sepharose 4B Beads (GSA4B, Amersham, Sweden) for 1 hour on an orbital shaker at 4 C. The pulled down complexes were collected through centrifugation at 1000g for 1 minute, washed successively with NETN containing decreasing concentrations of NaCl, and eluted by the elution buffer (4mM MgCl 2, 300mM KCl, 20 mm HEPES-KOH, ph 7.6, 0.05% Nonidet P-40, 1 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF). The pulled down complexes were treated with proteinase K at 65 C for 20 minutes. The RNAs in complexes were extracted by phenol/chloroform extraction and ethanol precipitation for the detection of virnas. For detecting the high molecular weight RNAs in the pulled down complexes, 1/3 of the RNAs obtained was treated with RNase I or RNase III (NEB) before they were subjected to Northern blot analysis to detect high or low molecular weight viral RNAs.
Coimmunoprecipitation 5x10 7 S2 cells grown in 100 mm dish were infected with FHV virions at a multiplicity of infection (MOI) 3, collected at 12 h post-inoculation (hpi), and lysed in the IP buffer containing 25 mm Tris-HCl, ph 7.5, 150mM NaCl, 1.5 mm MgCl2, 1% NP40, 1 mm DTT and the protease inhibitor cocktail (Roche, CA). For detecting B2-RdRP complexes, the lysates from mock-inoculated cells or FHV-infected cells were cleared by centrifugation for 15 minutes at 20,000g at 4 C and incubated with the pre-immune antibody, or rabbit polyclonal antibody against FB2-GST or FHV RdRP overnight on an orbital shaker. The immunocomplexes were captured by incubating the lysates with protein A agarose (Upstate, CA) for additional 4 h. The agarose beads were collected and washed four times with ice-cold IP buffer. The immune complexes bound to the agarose beads were subjected to RNase A treatment as described (Tacken et al., 2002) with minor modifications. Briefly, the immunocomplexes precipitated by the B2 antibody were treated with 700 μg/ml RNase A at 37 C for 45 minutes in 10 mm Tris-HCl (ph 7.5) containing either 10 mm of MgCl 2 to degrade both ssrna and dsrna or 100 mm of MgCl 2 to degrade ssrna only (Tacken et al., 2002). The agarose beads were washed three times with IP buffer before the immunocomplexes eluted and analyzed by Western blotting. Fractionation of B2 proteins was carried out in 15% SDS-PAGE and of protein A (RdRP) in 7.5% SDS-PAGE. Co-IP was carried out similarly from S2 cells mocktransfected, S2 cells transfected with pfr1 or pfr1δb2 using either the pre-immune antibody or FB2-GST antibody. To detect B2-dsRNA complexes, lysates were prepared from S2 cells 12 hpi with FHV. To use as controls, S2 cells were mock-inoculated and S2 cells pre-treated with dsrna of AGO2 (dsago2) were also infected with FHVΔB2 virions. Immunocomplexes were co-immunoprecipitated with the rabbit polyclonal antibody to FB2-GST as mentioned above and collected as described (Niranjanakumari et al., 2002) with some modifications. Briefly, beads were washed successively with high (1M NaCl) and low (250mM NaCl) salt IP buffer (25 mm Tirs-HCl, ph 7.5, 1% NP-40, 0.1% SDS, 1mM EDTA, 0.2 mm PMSF) and the immunocomplexes bound to beads were eluted in 100 ul of elution buffer (25 mm Tirs-HCl, ph 7.5, 5 mm EDTA, 10mM DTT and 1%SDS). RNA was extracted and subjected to Northern blot analysis as described above.
Co-immunoprecipitation with the monoclonal antibodies to AGO1 and AGO2 and peridate oxidation and beta elimination treatments of small RNAs were as described (Miyoshi et al., 2005; Kawamura et al., 2008). References Niranjanakumari, S., Lasda, E., Brazas, R., and Garcia-Blanco, M. A. (2002). Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26, 182-190. Tacken, M. G., Peeters, B. P., Thomas, A. A., Rottier, P. J., and Boot, H. J. (2002). Infectious bursal disease virus capsid protein VP3 interacts both with VP1, the RNAdependent RNA polymerase, and with viral double-stranded RNA. J Virol 76, 11301-11311.
Supplemental Figure 1 (A) The length distribution of FHV RNA1- and RNA2-specific virnas sequenced from S2 cells abortively infected with FHVΔB2. Only reads that are 100% identical or complementary to the viral genomic RNAs were included in the analysis. The percentages of (+) and (-) virnas specific to RNA1 or RNA2 are indicated by different colors.
(B) (C) FHV virnas have a preference for C at 5' terminal nucleotide. The cumulative percentage of virnas for both strands of RNA1 and RNA2. The plots for both positive and negative strands were drawn following the same 5 ->3 direction of positive strand.
Supplemental Figure 2 (A) Alignment of B2 proteins of FHV and NoV. Secondary structural elements of the N- terminal 70 amino acids of FHV B2 (Chao et al., 2005) were given and the conserved Arg at position 54 and 59 of FB2 and NB2, respectively, is located on α 2 of FHV B2. (B, C) The mutant of NoV B2 containing R Q substitution at position 59 is defective in binding to both a 44-bp dsrna (B) and a 21-nt sirna (C). The gel mobility shift electrophoresis assay was performed essentially as described (Lu et al., 2005). Duplex
RNA binding is required for the in vitro (D) and in vivo (E) activities of B2. (D) In vitro dicing of dsrna into sirna (lane 2) was inhibited by GST fused with B2 of FHV (lanes 4 and 9) and NoV (lanes 6 and 11), but not by GST (lane 3 and 8), or GST fused with the mutant B2 of FHV (lanes 5 and 10) and NoV (lanes 7 and 12), which carry the R Q substitution and are defective in binding to duplex RNA. (E) Induction of antiviral silencing in S2 cells was inhibited by B2 of FHV (lanes 1 and 4) and NoV (lanes 12, 15 and 17), but not by the R Q substitution mutant B2 of FHV (lanes 2, 5 and 9) and NoV (lanes 13, 16 and 20). The R Q substitution was introduced to either a B2 expression plasmid (lanes 5 and 16) or an RNA1 full-length infectious cdna clone (lane 2, 9, 10, 13, 20 and 21). By comparison, the single R Q substitution was more effective in eliminating the VSR activity of NB2 than that of FB2 (A). First, both FB2 and NB2 inhibited processing of long dsrna into sirnas, and addition of either mfb2 or mnb2 failed to prevent production of sirnas (D). However, the dsrna precursor was partially protected from dicing by mfb2 but not by mnb2 at either protein concentration (D, compare lanes 5 and 10 with lanes 7 and 12). Second, when this substitution was introduced into RNA1 of NoV (NR1), the resultant RNA1 mutant, NR1mB2, did not replicate to detectable levels in Drosophila S2 cells unless antiviral RNAi was suppressed by AGO2 depletion, and was thus similar to NR1DB2 that does not express B2 (C, lanes 18-21). By contrast, replication of a similar mutant of FHV RNA1 (FR1), FR1mB2, was greatly reduced as compared to FRI, but was detectable unlike FR1DB2, although AGO2 depletion significantly enhanced accumulation of both FR1mB2 and FR1DB2 (E, lanes 6-10). Third, expression of FB2 from a co-transfected plasmid can rescue replication of FR1DB2 (E, lanes 3-4) and similarly, NR1DB2 is also rescued by NB2 expressed in trans (E, lanes 14-15) as shown previously (Li et al., 2002; Li et al., 2004). Whereas mnb2 was completely inactive in the trans complementation assay (E, lane 16), mfb2 was partially active as low level replication of FR1DB2 was detected in S2 cells that expressed mfb2 (E, lane 5). These data indicate that B2 binding to dsrna is required for B2 inhibition of dsrna processing into sirnas and that the activities of B2 observed in vitro are correlated with the activity of B2 in the suppression of antiviral RNAi in the infected Drosophila cells.
Supplemental Figure 3 Wildtype B2 of NoV (NB2), but not its mutant containing R Q substitution at position 59 (mnb2) binds to virnas during replication of NoV RNA1 in Drosophila cells. (A) Western blot analysis of GST-tagged wildtype and mutant B2 proteins of FHV and NoV in S2 cells co-transfected with pfr1δb2 and dsrna of AGO2 GST specific antibody. (B) Pulldown of virnas but not endogenous mirnas by GST-tagged NB2. Note that mnb2 was unable to pull down virnas and that both (+) and (-) virnas were markedly enriched by GST-NB2 compared to the low levels of virnas in the input.
Supplemental Table 1: Summary of Small RNAs Annotation FHV genome 1177 complete match 834 1-2 mismatch # 343 mirna 106 complete match 98 1-2 mismatch 8 repeat 2370 complete match 1830 1-2 mismatch 540 others 718 Total 4371 # Many sequences contain 5' and/or 3' non-virus-derived sequence extensions.