Involvement of the N protein in (−)‐strand RNA synthesis

It appeared that (−)‐strand DI RNA can be produced from DI RNA with no poly(A) tail (Fig. 1B) or with a non‐poly(A) sequence (i.e., poly(U), (C) or (G)) (Fig. 1C), although the efficiency was reduced. These findings led to the hypothesis that in addition to poly(A) tail length and sequence, other factors such as proteins interacting with the 3′‐terminal sequence of DI RNA may also play an important role in (−)‐strand RNA synthesis. We speculated that the CoV N protein is a candidate factor involved in this interaction because it can bind to CoV genome 21, 22, 23, 24 including poly(A) tail 25 and is required for CoV replication 30, 31, 32. Because similar efficiencies of (−)‐strand RNA synthesis were observed for DI RNA with a poly(U), poly(C) or poly(G) tail (Fig. 1C), we hypothesized that the interaction of N protein with the DI RNA 3′‐terminal sequence may correlate with (−)‐strand RNA synthesis if N protein binds to poly(U), poly(C) and poly(G) tails with similar affinities. To test the hypothesis, ³²P‐labeled RNA containing the CoV 3′‐terminal 55 nt plus a 25‐nt poly(A), poly(U), poly(C) or poly(G) tail was generated (Fig. 2A, left panel) and the binding affinity between BCoV N protein (if not specified, the following N protein indicates BCoV N protein) and RNA was determined by the dissociation constant (K_d) derived from electrophoretic mobility shift assays (EMSAs). As shown in Fig. 2B–E, K_d values for the RNA containing CoV 3′‐terminal 55 nt plus a 25‐nt poly(A), poly(U), poly(C) or poly(G) tail were calculated to be 31.1 ± 1.2, 32.2 ± 2.1, 31.7 ± 3.7 and 30.2 ± 2.2 nm, respectively, suggesting similar binding affinities (Fig. 2F). To highlight the biological relevance of such binding between the N protein and these RNA elements, we tested further whether the N protein exhibits different binding affinities with other RNA species. To this end, 80‐nt RNA probes derived from the 3′‐terminus of the β‐actin mRNA and TOPO‐XL plasmid (Invitrogen, Carlsbad, CA, USA) were synthesized. As shown in Fig. 2G, H, K_d for the two RNA species was ~ 3–5‐fold that of the RNA containing the CoV 3′‐terminal 55 nt plus a 25‐nt poly(A), poly(U), poly(C) or poly(G) tail. Since BCoV DI RNA used for (−)‐strand RNA synthesis assay contained MHV 3′ UTR (Fig. 1), the binding affinity between MHV N protein and MHV 3′‐terminal 55 nt plus a 25‐nt poly(A) was also determined. As shown in Fig. 2I, K_d for MHV N protein was 35.6 ± 9.2 nm and similar to that (31.1 ± 1.2 nm) for BCoV N protein, suggesting BCoV and MHV N proteins have similar binding affinity with MHV 3′ terminus sequence. Together, the result supports our hypothesis that the N protein may be involved in (−)‐strand RNA synthesis by interacting with DI RNA 3′‐terminal sequence. In addition, as it is well characterized that poly(A)‐binding protein (PABP) binds to poly(A) tails with high affinity, the same RNA probes were also examined for their ability to interact with PABP (Fig. 3A) to evaluate whether PABP is also involved in the (−)‐strand RNA synthesis. As shown in Fig. 3B‐E, we calculated K_d values of 14.9 ± 2.0 nm, 30.1 ± 2.2 nm and 93.4 ± 4.3 for RNA containing the CoV 3′‐terminal 55 nt plus the 25‐nt poly(A), poly(U) and poly(C), respectively. However, we were unable to determine the K_d for RNA containing the CoV 3′‐terminal 55 nt plus the 25‐nt poly(G), suggesting that the value may be greater than 150 nm. The results suggest that the binding of PABP to the CoV 3′‐terminal sequence is less likely correlated with (−)‐strand RNA synthesis. Because (a) N protein but not PABP binds with similar affinity to the CoV 3′‐terminal 55 nt plus a 25‐nt poly(U), poly(C) or poly(G) tail, (b) the efficiency of (−)‐strand RNA synthesis is similar between DI RNA with a poly(U), poly(C) or poly(G) tail and (c) N protein binds with various affinities to other RNA probes containing different sequences (Fig. 2G,H), the results (Figs 1, ​,2,2, ​,3)3) together suggest that the N protein is involved in (−)‐strand RNA synthesis.

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Figure 2

Determination of binding affinity between N protein and RNA elements by K_d. (A) Schematic diagrams depicting the structure of RNA probes (left panel) and E. coli‐expressed BCoV N protein (right panel) for determination of N protein‐binding affinities. (B–E) Upper panel: EMSA showing binding experiments using a fixed concentration of 0.2 nm ³²P‐labeled RNA probe with increasing amount of N protein. Lower panel: a plot of a fraction of bound RNA against protein concentration based on the EMSA results shown in the upper panel, which was used for K_d determination using the Hill equation. (F) K_d values for the RNA probes shown in (A) with N protein. (G and H) Determination of K_d for RNA probes derived from the 3′‐terminus of the β‐actin mRNA and TOPO‐XL plasmid, respectively, using the same methods described for (B–E). (I) Determination of the bonding affinity between MHV N protein and RNA probe containing the CoV 3′‐terminal 55 nt plus a 25‐nt poly(A) tail by EMSA and K_d. The values in (B–I) represent the mean ± SD (n = 3) of three independent experiments. b, protein‐bound RNA; ub, unbound RNA.

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Figure 3

Determination of binding affinity between PABP and RNA elements by K_d. (A) Schematic diagrams depicting the structure of RNA probes used for determination of PABP binding affinities. (B–E) Upper panel: EMSA showing binding experiments using a fixed concentration of 0.2 nm ³²P‐labeled RNA probe with increasing amount of PABP. Lower panel: a plot of a fraction of bound RNA against protein concentration based on the EMSA results shown in the upper panel, which was used for K_d determination using the Hill equation. (F) K_d values for the RNA probes shown in (A) with PABP. The values in (B–F) represent the mean ± SD (n = 3) of three independent experiments. b, protein‐bound RNA; ub, unbound RNA; N/A, not available.

Binding affinity between the N protein and the 3′‐terminal structure correlates with the efficiency of (−)‐strand RNA synthesis

It has been shown that the CoV poly(A) tail length (Fig. 1B) and the 3′‐terminal 55 nt 16 are factors affecting (−)‐strand RNA synthesis. To further examine whether the binding of N protein to the 3′‐terminal sequence is associated with (−)‐strand RNA synthesis, RNA probes (Fig. 4A) containing the 3′‐terminal 55 nt and various poly(A) tail lengths or only the 25‐nt poly(A) tail were constructed. In comparison with the binding affinity between the N protein and the 3′‐terminal 55 nt plus a 25‐nt poly(A) tail (K_d: ~ 33 nm) (Fig. 4E), the binding affinity between N protein and the 3′‐terminal 55 nt plus a 15‐nt poly(A) tail (K_d: ~ 40 nm) was slightly decreased (Fig. 4D). In addition, the binding affinity between N protein and only the 3′‐terminal 55 nt, 3′‐terminal 55 nt plus a 5‐nt poly(A) tail, or only the 25‐nt poly(A) tail was also decreased (K_d: ~ 87, ~67 and 48 nm, respectively) (Figs. 4B,C,F). These results are in agreement with our hypothesis that the efficiency of (−)‐strand RNA synthesis (Fig. 1B and 16) correlates with the binding affinity between the N protein and poly(A) tail length (Fig. 4B‐E,G) and 3′‐terminal 55 nt (Fig. 4F,G).

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Figure 4

Correlation of binding affinity between the N protein and 3′‐terminal sequences with efficiency of the (−)‐strand RNA synthesis. (A) RNA probes with poly(A) tails of various lengths or deletion of 3′‐terminal 55 nt. (B–F) Determination of K_d between N and RNA probes illustrated in (A). Upper panel: EMSA showing binding experiments using a fixed concentration of 0.2 nm ³²P‐labeled RNA probe with increasing amounts of N protein. Lower panel: plot of a fraction of bound RNA against protein concentration based on the EMSA results shown in the upper panel, which was used for the determination of K_d using the Hill equation. Lower panel: K_d values for the RNA probe and the N protein. (G) Values of K_d based on the results shown in (B–F). The values in (B–G) represent the mean ± SD (n = 3) of three independent experiments. b, protein‐bound RNA; ub, unbound RNA.

Züst et al. 19 proposed that two helical stem structures (designated S3 and S4, Fig. 5A) form at the end of the MHV 3′ UTR, and S3 is predicted to be important for initiating (−)‐strand RNA synthesis. Using reverse‐genetic approaches, Liu et al. 20 demonstrated that mutation in the 3′‐side of S3 (designated construct E in that study) to disrupt the stem structure produced a lethal phenotype; however, the data regarding the relative level of (−)‐strand RNA synthesis between the mutant (construct E) and wild‐type (wt) virus are not available. Conversely, mutation in the 3′‐side of S4 to disrupt the stem structure resulted in a viable virus. To experimentally determine the effect of the two structures on (−)‐strand RNA synthesis, sequence substitution based on the aforementioned study by Liu et al. 20 at the 3′‐side of S3 and S4 to disrupt the stem structures was performed using DI RNA BM25A; these constructs were designated BMmtS3 and BMmtS4, respectively (Fig. 5B). As shown in Fig. 5C, the efficiency of (−)‐strand RNA synthesis from BMmtS3 was significantly impaired, whereas the efficiency of (−)‐strand RNA synthesis from BMmtS4 was not altered in comparison with that from BM25A. Note that no recombination occurred between the helper virus BCoV and the three DI RNA constructs, and no sequence in these DI RNA constructs was altered (data not shown). The results, therefore, suggest that the S3 structure is critical for (−)‐strand RNA synthesis. Accordingly, the results are in agreement with those of the study by Liu et al. 20 in terms of virus viability and also support the model proposed by Züst et al. 19. Next, we performed EMSA to determine the binding affinity between the N protein and the two helical stems, S3 and S4, and to evaluate whether binding affinity is also linked to the efficiency of (−)‐strand RNA synthesis. For this aim, RNA probes (Fig. 5D) predicted by the Mfold algorithm and containing intact S3 and S4 structures (wtS3S4), disrupted S3 and intact S4 structures (mtS3, by mutation at the 3′‐side of S3) and intact S3 and disrupted S4 structures (mtS4, by mutation at the 3′‐side of S4) were generated, and EMSA was performed to determine K_d. As shown in Fig. 5E,G, the binding affinity between the N protein and wtS3S4 (K_d: 70.3 nm) was similar to that between N protein and mtS4 (K_d: 66.3 nm). Conversely, the K_d value for the N protein and mtS3 could not be calculated (Fig. 5F), suggesting that the value is higher than 500 nm and that binding affinity is very low. Because the higher binding affinity (wtS3S4 and mtS4 with N) corresponds to more efficient (−)‐strand RNA synthesis (BM25A and BMmtS4) and lower binding affinity (mtS3 with N protein) corresponds to less efficient (−)‐strand RNA synthesis (BMmtS3) (Figs. 5C,H), the binding affinity of N protein to S3 structure is linked to the efficiency of (−)‐strand RNA synthesis. Therefore, the results suggest the involvement of N protein and S3 structure in (−)‐strand RNA synthesis and thus support our hypothesis and the model proposed by Züst et al. 19.

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Figure 5

Correlation of binding affinity between the N protein and 3′‐terminal structures S3 and S4 with efficiency of the (−)‐strand RNA synthesis. (A) Schematic diagram depicting RNA structures in the 3′‐UTR of MHV including the bulged stem‐loop, BSL; PK, HVR and two helical stems, S3 and S4. (B) BM‐DI RNA constructs with mutations to disrupt the stem structure of S3 (BMmtS3) or S4 (BMmtS4). (C) Relative level of (−)‐strand RNA synthesis from BM‐DI RNA constructs illustrated in (B), as measured by RT‐qPCR. Control 1 – total cellular RNA from mock‐infected cells. Control 2 – total cellular RNA from BCoV‐infected cells. Control 3 – total cellular RNA from BM25A‐transfected mock‐infected cells. Control 4 – a mixture of BCoV‐infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript. (D) RNA probes with an intact structure of S3 and S4 (wtS3S4) or a disrupted structure of S3 (mtS3) or S4 (mtS4). (E–G) Determination of K_d between N and RNA probes illustrated in (D). Upper panel: EMSA showing binding experiments using a fixed concentration of 0.2 nm ³²P‐labeled RNA probe with increasing amounts of N protein. Lower panel: plot of a fraction of bound RNA against protein concentration based on the EMSA results shown in the upper panel, which was used for the determination of K_d using the Hill equation. (H) Values of K_d based on the results shown in (E–G). The values in (C) and (E–H) represent the mean ± SD (n = 3) of three independent experiments. b, protein‐bound RNA; ub, unbound RNA; N/A, not available. The statistical significance was evaluated using a t‐test versus BM25A: ***P < 0.001.

Binding of the N protein to the 5′‐terminal sequence is involved in (−)‐strand RNA synthesis

It has been shown that (−)‐strand N sgmRNA can be synthesized using (+)‐strand N sgmRNA as a template 39, though the efficiency is decreased in comparison with that of DI RNA 10, a surrogate for the CoV genome. The discrepancy between the structure of N sgmRNA and DI RNA lies in the 5′‐terminal sequence: DI RNA contains the 5′‐terminal 498 nt of the CoV genome, but N sgmRNA contains only the 5′‐terminal 77 nt of the genome. It was, therefore, speculated that such a structural difference is the main factor leading to various efficiencies of (−)‐strand RNA synthesis. In the current study, as the interaction between N protein and 3′‐terminal sequence was correlated with (−)‐strand RNA synthesis (Figs. 1, ​,2,2, ​,3,3, ​,4,4, ​,5),5), we hypothesized that the binding affinity of N protein with the 5′‐terminal structures of N sgmRNA and DI RNA may also explain the discrepancy in the efficiency of (−)‐strand RNA synthesis between the two viral RNA species. To test the hypothesis, we first repeated the experiment by detecting (−)‐strand RNA synthesis from N sgmRNA (sBM25A) and DI RNA (BM25A) 10. Note that, similar to BM25A, sBM25A was also engineered to contain the MHV 3′ UTR for subsequent analysis of (−)‐strand RNA detection. As shown in Fig. 6A, the efficiency of (−)‐strand RNA synthesis from sBM25A was decreased by ~ 50% when compared with that from DI RNA BM25A. In addition, no recombination was found between the helper virus BCoV and these DI RNA constructs and no sequence in these DI RNA constructs was altered (data not shown). Thus, the results are consistent with a previously published study 10. To evaluate whether such a decrease in (−)‐strand RNA synthesis correlates with the binding affinity between the N protein and the 5′‐terminal sequence, 120 nt of RNA probes BM5′‐120 nts and sBM5′‐120 nts (Fig. 6B), which are derived from the 5′‐terminus of BM25A and sBM25A, respectively, and thus have different structures, were subjected to EMSA to determine the binding affinities with N protein. The K_d for BM5′‐120 nt was 66.1 nm; however, that for sBM5′‐120 nt could not be calculated (Figs. 6C,D), suggesting that the N protein binds to the 5′‐terminal sequence of DI RNA BM25A with higher affinity than that of N sgmRNA. Because the efficiency of (−)‐strand RNA synthesis from DI RNA is better than that from N sgmRNA and because N binds to the 5′‐terminal sequence of DI RNA BM25A with better efficiency than that of N sgmRNA, the results also suggest the involvement of N protein in (−)‐strand RNA synthesis and provide further evidence explaining how the discrepancy between 5′ terminal structure of DI RNA and N sgmRNA affects the efficiency of (−)‐strand synthesis in the previously published study 10.

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Figure 6

Correlation of binding affinity between the N protein and 5′‐terminal structures with efficiency of (−)‐strand RNA synthesis. (A) Upper panel: BM‐DI RNA construct containing the 5′‐terminal 498 nt of the CoV genome (BM25A) or only the 5′‐terminal 77 nt of the genome (sBM25A). Lower panel: relative level of (−)‐strand RNA between BM25A and sBM25A, as measured by RT‐qPCR. (B) Schematic diagram depicting the RNA probe containing the 5′‐terminal 120 nt of the genome (BM 5′‐120 nts) or N subgenome (sBM 5′‐120 nts). (C) Upper panel: EMSA showing binding experiments using a fixed concentration of 0.2 nm ³²P‐labeled RNA probe with increasing amount of N protein. Lower panel: plot of a fraction of bound RNA against protein concentration based on the EMSA results shown in the upper panel, which was used for K_d determination using the Hill equation. (D) K_d values based on the results shown in (C). Controls in (A): Control 1 – total cellular RNA from mock‐infected cells. Control 2 – total cellular RNA from BCoV‐infected cells. Control 3 – total cellular RNA from BM25A‐transfected mock‐infected cells. Control 4 – a mixture of BCoV‐infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript. The values in (A) (C) and (D) represent the mean ± SD (n = 3) of three independent experiments. b, protein‐bound RNA; ub, unbound RNA; N/A, not available. The statistical significance was evaluated using a t‐test versus BM25A: ***P < 0.001.

Plasmid constructs

Constructs pBM0A, pBM5A, pBM15A, pBM25U, pBM25C, pBM25G, pBMΔ55, pBMmst3 and pBMmst4 were generated as described previously 10, 16, 62; briefly, pBM25A DNA was used as the template with appropriate oligonucleotides containing various mutated 3′‐terminal sequences corresponding to the constructs for PCR. The resulting PCR product was digested with SpeI and MluI and cloned into SpeI‐ and MluI‐linearized pBM‐25A to create the aforementioned constructs. For RNA probes, PCR was performed using the oligonucleotides to generate DNA fragments, which were then used as templates for synthesizing RNA probes 3′‐55ntsA₂₅, 3′‐55ntsU₂₅, 3′‐55ntsC₂₅, 3′‐55ntsG₂₅, 3′‐55ntsA₀, mtS3, mtS4 and A₂₅. The DNA fragments for producing the RNA probes BM 5′‐120nts and sBM‐120nt were also generated by the PCR, but with pBM25A and psBM25A as the template, respectively 10. Note that each RNA probe contains additional three guanosine residues at its 5′ end after in vitro transcription.

Expression of recombinant proteins

pET32aN carrying the BCoV N protein gene, pET32aN1‐300 carrying the BCoV N protein gene 1‐900, pET32aN300‐448 carrying the BCoV N protein gene 901–1344, pET32aMHVN carrying the MHV N protein gene (GenBank accession no. AY700211.1) and pET28aPABP carrying human cytoplasmic PABP1 (PABPC1) gene (GenBank accession no. NM_002568) were constructed as described previously 25. Expression of His‐tagged N protein and PABP was also performed as described previously 25. Briefly, to express MHV N protein, BCoV N protein and its mutants, Escherichia coli BL21 (DE3) cells containing pET32aMHVN, pET32aN, pET32aN1‐300 or pET32aN301‐448 were inoculated into LB medium supplemented with 50 μg·mL⁻¹ ampicillin, induced with isopropyl thio‐β‐D‐galactoside, collected by centrifugation and resuspended in PBS. The resuspended cells were sonicated and the supernatant was collected by removing cell debris with centrifugation. The recombinant protein in collected supernatant was purified through the 6xHis tag by immobilized metal ion affinity chromatography with EDTA‐resistant Ni Sepharose excel resin (GE Healthcare, Houston, TX, USA) and loaded on a nickel‐chelating column (GE Healthcare). Fractions which contain recombinant N protein were dialyzed and collected. To obtain PABP, pET28aPABP‐containing E. coli BL21 (DE3) pLysS cells were inoculated into LB medium supplemented with 50 μg·mL⁻¹ kanamycin. The cells were also induced with isopropyl thio‐β‐D‐galactoside, centrifuged and resuspended in 20 mm Tris–HCl (pH 7.6) buffer. The resuspended cells were sonicated and centrifuged, and the supernatant was collected. The supernatant containing the recombinant PABP was loaded onto a Ni SepharoseTM 6 Fast Flow column (GE Healthcare) followed by washing with buffer containing 20 mm Tris–HCl, 0.5 m NaCl, pH 7.4 and 40, 100, 200, 300 and 500 mm imidazole. Fractions containing recombinant PABP were pooled and dialyzed. The purified recombinant N protein and PABP were stored in buffer (50 mm Tris‐Glycine, pH 7.4, 0.15 m NaCl, 30% Glycerol, and 0.01% NaN3) at −20 °C until use.

Evaluation of (−)‐strand RNA synthesis

To evaluate the efficiency of (−)‐strand RNA synthesis 8, 10, 16, MluI‐linearized plasmid DNA construct was transcribed in vitro with mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 90 min according to the manufacturer's instructions and then chromatographed through a Biospin 6 column (Bio‐Rad, Hercules, CA, USA). HRT‐18 cells were infected with BCoV and after 2 h of infection, 3 μg of DI RNA transcript was transfected into BCoV‐infected HRT‐18 cells. RNA was extracted with TRIzol (Thermo Fisher Scientific) at 8 h post‐transfection (hpt) and 6 μg of RNA was treated with tobacco acid pyrophosphatase (TAP; Epicentre, Madison, WI, USA) to generate monophosphate RNA. Head‐to‐tail ligation of RNA was performed using T4 RNA ligase I (New England Biolabs, Ipswich, MA, USA) followed by phenol‐chloroform extraction and ethanol precipitation. For RT reaction, 1 μg of TAP‐treated and ligated RNA was used with oligonucleotide MHV3′UTR3(−) and SuperScript III reverse transcriptase (Invitrogen) 8, 10, 16. To detect the (−)‐strand RNA without head‐to‐tail ligation, RT‐PCR was performed with 1 μg of RNA with oligonucleotides MHV3′UTR‐DR(−) (for RT) and MHV3UTR‐DR(+) 10. Real‐time PCR amplification was performed using TagMan^® Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's recommendations with a LightCycler^® 480 instrument (Roche Applied Science, Penzberg, Germany) with the primers MHV3′UTR6(−) and BCV23‐40(+) 8, 10, 16. Dilutions of plasmids containing the same sequence as the detected RT‐PCR product of (−) –strand DI RNA were run in parallel with quantitated cDNA for use as standard curves (dilutions ranged from 10⁸ to 10 copies of each plasmid). In addition, the levels of 18S rRNA, (+)‐strand reporter‐containing DI RNA or sgmRNA and helper virus M sgmRNA were applied as internal controls for normalization of (−)‐strand DI RNA synthesis 8, 10, 16. The reactions were conducted with an initial preincubation at 95 °C for 5 min, followed by 35 amplification cycles as follows: 95 °C for 15 s and 60 °C for 60 s.

EMSA and K_d

Electrophoretic mobility shift assays were performed essentially as described previously 18, 25, 63, 64. Briefly, ³²P‐labeled RNA was generated as specified by the manufacturer (Promega, Fitchburg, WI, USA) and separated on 6% sequencing gels. Passive elution was performed at 4 °C followed by phenol‐chloroform extraction. To determine binding affinity, a fixed concentration of 0.2 nm ³²P‐labeled RNA was titrated with the protein of interest in binding buffer [20 mm HEPES (pH 7.5), 6 mm MgCl₂, 1.5 μm EGTA, 22.5 mm NaCl, 330 mm KCl, 36% glycerol, 3.6 mm DTT, 82.5 μg·mL⁻¹ BSA] for 15 min at 37 °C with 1 U·mL⁻¹ RNasin (Promega). The bound RNA‐protein complexes were separated from unbound RNA by 8% native polyacrylamide gel electrophoresis with crosslink ratio 2.7%, dried, and analyzed by autoradiography. Free and bound RNAs were quantitated and fit to the Hill equation: $\text{RNAbound}=b\ast [\mathrm{P}]n/({\mathrm{K}}_{\mathrm{d}}^n+[{\mathrm{P}}^n])$, where b is the upper binding limit, [P] is the protein concentration, n is the Hill coefficient. graphpad prism (GraphPad Software, Inc., La Jolla, CA, USA) was then used to derive K_d, and the K_d values presented were determined based on at least three independent experiments.

Assay for interaction between the 5′‐ and 3′‐ends of the genome bridged by the N protein

The assay was based on the methods of Huang et al. 36, with some modifications. DNA templates pBM25A and psBM25A were used for in vitro transcription with T7 polymerase (Promega) in the presence of a biotin‐UTP labeling NTP mixture (Roche Applied Science), as recommended by the manufacturer, to synthesize biotinylated RNA with 5′‐terminal 120 nt of genome or N subgenome, respectively. After purification, the biotinylated RNA was incubated with recombinant N protein and ³²P‐labeled RNA containing 3′‐terminal 55 nt plus the poly(A) tail in a reaction buffer containing 5 U of RNasin (Promega), 25 mm KCl, 5 mm HEPES (pH 7.6), 2 mm MgCl₂, 0.1 mm EDTA, 0.2% glycerol, and 2 mm DTT. After incubation at room temperature for 30 min, a streptavidin suspension (MagQu, Taipei, Taiwan) was added to the mixture and incubated for 30 min at room temperature followed by three washes with binding buffer. RNAs bound to streptavidin agarose beads were eluted by a buffer [7 m urea, 350 mm NaCl, 10 mm Tris‐HCl (pH 8.0), 10 mm EDTA, and 1% SDS] followed by phenol‐chloroform extraction and ethanol precipitation. The samples were resolved on a 6% polyacrylamide gel.

We thank Dr Wei‐Li Hsu and Chih‐Jung Kuo at National Chung Hsing University, Taiwan, for the assistance of protein preparation and RNA‐protein interaction experiment. This work was supported by grants 105‐2320‐B‐005‐004‐ and 106‐2313‐B‐005‐046‐MY3 from the Ministry of Science and Technology (MOST), R.O.C. (https://www.most.gov.tw/?l=ch).