Secondary structure of the panhandle RNA of influenza virus A studied by NMR spectroscopy

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1 1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No Secondary structure of the panhandle RNA of influenza virus A studied by NMR spectroscopy Hae-Kap Cheong, Chaejoon Cheong 1 and Byong-Seok Choi* Department of Chemistry, Korea Advanced Institute of Science and Technology, Kusung-dong 373-1, Yusung-gu, Taejon , Korea and 1 Magnetic Resonance Group, Korea Basic Science Institute, Eoun-dong 52, Yusung-gu, Taejon , Korea Received July 15, 1996; Revised and Accepted September 12, 1996 ABSTRACT The double-stranded panhandle structure of the influenza virus RNA is important for replication, transcription and packaging into the virion of the virion RNA. The solution structure of a 34 nt RNA which contains the conserved panhandle sequences has been investigated by one- and two-dimensional NMR spectroscopy. The partially complementary 5 - and 3 -ends of the RNA form a double helical structure which is, on average, close to A-form. The stem contains bulges at nucleotides A10, A12 and C26. In between these bulges, C11 and G25 form a Watson Crick base pair. The structural features of the panhandle provide a framework for the explanation of mutational analysis and for a better understanding of RNA polymerase interactions. INTRODUCTION The influenza A virus genome is composed of eight segments of single-stranded RNAs of negative polarity (1). During influenza virus infection, the negative sense virion RNA (vrna) serves as a template for both mrna synthesis and crna synthesis (2). Although control of transcription and replication of viral RNA is not well understood, both processes of RNA replication and RNA transcription are catalyzed by the same RNA-dependent RNA polymerase (2). Since these two modes of RNA synthesis occur at the same promoter in competition, there must be a kind of regulatory mechanism to ensure the control of both mrna and crna for optimal growth of the virus. Sequencing of influenza A virus RNA segments revealed that all the segments contain 13 and 12 conserved nt at their 5 - and 3 -ends respectively (3,4). It has been shown that the vrna promoter is located at the 3 -end of the vrna segment (5,6). A model for polymerase promoter recognition has been proposed in which four contiguous internal nucleotides are crucial for binding, located at or near the initiation site of the 3 -terminus (7). More recently, however, it has been shown that the 5 -end of the vrna plays an important role in regulation of transcription as well. The sequences of the 5 - and 3 -ends of the vrna are partially complementary to each other (3,4) and it has been suggested that a panhandle structure can form (8; Fig. 1A). The double-stranded panhandle structure appears to be required for polyadenylation and for transcription termination (9,10). In vitro studies using recombinant influenza virus polymerase have demonstrated that both viral 5 - and 3 -ends are required for polymerase activity and that the polymerase binds sequence-specifically to the partially double-stranded panhandle structure (11,12). Both an RNA polymerase binding assay and an in vitro transcription assay have shown that the panhandle structure is involved in initiation of transcription (13). Nuclease S1 mapping of the influenza virus RNA has indicated that the termini of the RNA segments form a 15 bp panhandle structure (8; Fig. 1A). Another alignment, which contains an internal loop, has been proposed as a model structure of the panhandle of the influenza virus RNA (14; Fig. 1B). Recently, however, by using different nucleases and chemical probes, Baudin et al. (15) have determined a somewhat different panhandle structure, in which the panhandle contains an internal loop and a bulge (Fig. 1C). In this paper, we report the structure of this panhandle RNA determined in solution by NMR spectroscopy. On average, the influenza panhandle structure is close to double helical A-form geometry, although it contains a G U base pair and internal bulges. From our studies we conclude that C11 and G25 ( Fig. 1D for nomenclature), which were considered part of an internal loop in the previously proposed panhandle structures, are base paired. We have changed the residue U at position 5 to C to increase the transcription yield. We do not think that this single base substitution can alter the overall secondary structure of the panhandle RNA. MATERIALS AND METHODS RNA synthesis and purification An RNA oligonucleotide, 5 -GGAGCAGAAACAAGGCUUCG- GCCUGCUUUUGCUC-3, was synthesized using T7 RNA polymerase and synthetic DNA templates (16). DNA templates were synthesized chemically on an Applied Biosystems 391 and were purified by 15% polyacrylamide gel electrophoresis under denaturing conditions (7 M urea). Synthesis and purification of the RNA oligonucleotide were done as previously reported (17,18). The purified sample was dialyzed extensively against 10 mm sodium phosphate, 0.01 mm EDTA, ph 6.5. It was then lyophilized and dissolved in 0.25 ml 90% H 2 O/10% D 2 O for exchangeable proton experiments. For non-exchangeable proton *To whom correspondence should be addressed. Tel: ; Fax: ; haekap@reflex.kaist.ac.kr

2 4198 Nucleic Acids Research, 1996, Vol. 24, No. 21 Figure 1. Secondary structures and numbering scheme of the panhandle structure of the influenza virus RNA. (A) Model for the structure of the panhandle proposed by Hsu et al. (8). (B) The shown alignment has been widely used as a model structure of the panhandle (14). (C) Baudin et al. (15) have proposed an alternative alignment which was determined using nucleases and chemical probes. (D) Another alignment determined by NMR spectroscopy in this work. experiments, the sample was lyophilized several times from 99.9% D 2 O and dissolved in 0.25 ml 99.96% D 2 O (Aldrich). A microtube from Shigemi was used to decrease the sample volume. NMR experiments All NMR experiments were done on a Bruker DMX-600 NMR spectrometer operating at 600 MHz proton frequency. Exchangeable proton spectra were obtained using the 1 1 (jump return) method (19). One-dimensional NOE experiments were performed with a pre-irradiation time of 600 ms. All two-dimensional NMR spectra were acquired in phase-sensitive mode using the TPPI method (20). The two-dimensional spectra were acquired with 400 or 512 FIDs of 2048 complex data points, using spectral widths of 3800 Hz for spectra in D 2 O and Hz for spectra in H 2 O. The repetition delay was set to 2 s and 64 scans were averaged for each FID; the total acquisition time was <24 h. Data were zero-filled to 1 K real points in t 1 and apodized by using phase-shifted squared sine bells in both dimensions. NOESY spectra in H 2 O with mixing times of 100 and 400 ms were obtained using a jump return pulse for solvent suppression (21). NOESY spectra in D 2 O were obtained at a mixing time of 400 ms. The residual HDO resonance was presaturated during the relaxation delay. DQF-COSY spectra were obtained using the standard pulse sequence (22). RESULTS Spectral assignments The imino proton spectra were assigned by standard methods using one- and two-dimensional NNR spectroscopy. Uracil imino protons were identified by their strong NOEs to the H2 protons of base paired adenines. The amino protons of the cytosines in the stem were assigned by NOEs to their own H5 protons and to the cross-strand guanine imino protons. We have introduced an RNA hairpin loop, 5 -CUUCGG-3, which possesses a unique conformation (23,24), to increase the stability of the panhandle and to help the assignment. Figure 2 shows the imino proton spectrum of the panhandle RNA studied here (Fig. 1D). The upfield shifted Figure 2. A section of a 400 ms NOESY spectrum of the panhandle structure of the influenza virus RNA in 10 mm sodium phosphate, ph 6.5, 0.01 mm EDTA, H 2 O at 14 C showing NOEs between imino protons. The assignments for the imino proton resonances are shown in a one-dimensional spectrum., connectivities from U27 U28 U29 U30 G31 G4 U33 G2; --->, connectivities from G20--->G21--->G15--->G14. resonance at 9.90 p.p.m., which shows high thermal stability, was assigned to the G20 imino proton in the loop from the previously studied results (24). Assignments of the imino protons of G14, G15 and G21 were by sequential imino imino NOE connectivities (Fig. 2). A strong NOE was observed between the two imino protons of the G7 U29 base pair. Sequential imino imino NOEs were observed from the G2 C34 to A9 U27 base pairs. The sharp resonance at p.p.m. was assigned to the imino proton of G25, since it gave NOEs to amino protons of cross-strand C11. A NOESY crosspeak from the G25 imino to C11 H5 proton was also observed at long mixing times (400 ms), providing further support once the non-exchangeable protons had been assigned. The non-exchangeable proton resonances were assigned by standard methods using DQF-COSY and NOESY (25). The H8/H2/H6 H1 /H5 region of the NOESY spectrum (400 ms mixing time) is shown in Figure 3. The positions of pyrimidine H5 H6 crosspeaks were confirmed by the DQF-COSY experiment, providing a convenient starting point for spectral assignments. From the NOESY spectrum, it was possible to sequentially walk from the H6 or H8 protons of bases to the H1 protons of riboses for most of the stem nucleotides (Fig. 3). Adenine H2 protons were identified by the NOEs to the H1 protons of the 3 -side nucleotides and the cross-strand nucleotides (Figs 3 and 4). The assignments of the loop protons (U17 G20) were based on previously studied results (23,24). The assignments are summarized in Table 1.

3 4199 Nucleic Acids Research, 1994, 1996, Vol. Vol. 22, 24, No. No Table 1. Resonance assignments for the panhandle RNA in 10 mm sodium phosphate, ph 6.5, 0.01 mm EDTA Base H8/H6 H5/H2 H1 H2 Imino Amino G1 na a G na A na G na C na 8.37/6.83 A na 7.74/6.93 G na A na 8.48/6.92 A na 7.96/6.85 A na C na 8.14/7.19 A na A na G na G na C na 8.62/6.95 U na U na C na G na G na /6.42 C na 8.80/6.90 C na 8.49/6.98 U na G na C na U na U na U na U na G na C na 8.53/6.90 U na C na 8.39/6.99 a na, not applicable. The non-exchangeable proton chemical shifts were measured at 30 C. Amino and imino chemical shifts were measured at 14 C. All chemical shifts (p.p.m.) are referenced relative to 3-(trimethylsilyl)-1-propanesulfonate. Structural features The imino proton data suggest that G2 A9 and A13 C16 form double helical stem regions by base pairing with U27 C34 and G21 U24 respectively. The H8/H6 H1 sequential connectivities as well as H8/H6 H2 connectivities confirm these results. The majority of nucleotides in these stem regions adopt N-type Figure 3. A section of a 400 ms, NOESY spectrum of the panhandle RNA in 10 mm sodium phosphate, ph 6.5, 0.01 mm EDTA, D 2 O at 30 C. All the crosspeaks shown here are identified. Sequential aromatic anomeric connectivities are shown by solid lines and pyrimidine H5 H6 crosspeaks are connected by dashed lines. The labeled crosspeaks are intranucleotide H6/H8 H1 and internucleotide H2 H1 crosspeaks. sugar conformation as judged by the absence of H1 H2 crosspeaks in the DQF-COSY spectrum, suggesting that the stems adopt A-form helical conformation. The observations of AH2 H1 crosspeaks across the minor groove further support the A-form geometry (17). Nucleotides G7 and U29 form a wobble-type base pair which is accommodated in the helix with little distortion from the regular A-helical geometry. NOEs from the G7 and U29 imino protons to the H2 protons of A6 and A8 were observed as well as imino protons of the A6 U30 and A8 U28 base pairs. The intensities of the sequential H8/H6 H1 /H2 NOEs are similar to those observed for the A-helix. The riboses of G7 and U29 have weak H1 H2 couplings (<2 Hz) and are, therefore, primarily in the N-type sugar conformation, as is usually found in an A-helix. Observations of NOEs from the G25 imino proton to the amino protons of C11 suggest that the nucleotides form a base pair. It is a Watson Crick base pair as judged by the chemical shift (12.64 p.p.m.) of the G25 imino proton. This is supported by the chemical modification experiment (15) where N3 of C11 is not reactive with DMS, like other Watson Crick base paired cytosines, although their conclusion on the secondary structure of this molecule was different from ours. The NOEs from the A10 and A12 H2 resonances to U27 and G25 H1 respectively show that the bulged nucleotides are stacked within the helix. Normal base stackings were observed through the bulged nucleotides, as judged by NOEs connecting A10 H8 to C11 H5 and A12 H2 to A13 H2, as well as strong NOEs from A10 H2 to C11 H1 and from A12 H2 to A13 H1. The riboses of A12, A13 and G25 exhibit H1 H2 couplings of 4 5 Hz, indicating that these are mixed N- and S-type sugar conformations (26).

4 4200 Nucleic Acids Research, 1996, Vol. 24, No. 21 Although it is evident that the 5 - and 3 -ends of the vrna forms base paired structure, the detailed secondary structure of the panhandle is controversial. Seong (14) has proposed an alignment which contains an internal loop and two G U base pairs (Fig. 1B). This has been generally used as a model structure of the panhandle of the influenza vrna. The results of Baudin et al. (15), which were obtained using a variety of enzymatic and chemical probes, suggest that A6 bulges out and the stretch GAAA 7 10 base pairs with UUUU (Fig. 1C). In our model, A6 base pairs with U30 and the stretch AGAA 6 9 base pairs with UUUU Our model is different from that proposed by Seong (14), which there forms a Watson Crick base pair between C11 and G25. The loop region consists of 4 nt (U17 G20) that cross the stem region over the C16 G21 base pair. The strong NOE between H8 and H1 of nt G20 suggests that the base is in the syn conformation. The chemical shift patterns and NOE connectivities in the loop region are the same as those of the results of Varani et al. (1991). This led us to conclude that the unique loop structure within the hairpin remains essentially unchanged when it is part of a longer panhandle. Figure 4 shows a schematic diagram summarizing internucleotide NOEs and hydrogen bonds in the influenza panhandle RNA. DISCUSSION Figure 4. Schematic diagram summarizing internucleotide NOEs and hydrogen bonds in the panhandle RNA. Base pairings confirmed by the observed imino resonances are indicated by double (G U wobble) or thick (Watson Crick) bars. Internucleotide NOEs are indicated by solid lines (aromatic sugar) and arrows (imino imino). Nucleotides with dynamic sugar conformations of the N-type and S-type are boxed with double lines and those with S-type sugar conformation are boxed with thick lines. In this paper we have described the panhandle structure of the influenza virus RNA. The influenza virus RNA contains 13 and 12 nt conserved sequences at the 5 - and 3 -ends respectively, which are partially complementary to each other (3,4). These sequences are known to form a unique panhandle structure in the RNP complex in virions as well as in infected cells, but not in the naked vrna (8). Baudin et al. (15) have shown an opposite result, where the 5 - and 3 -ends of the RNA form a base paired panhandle structure in the naked state, whereas in the RNP complex they are single-stranded. Our results clearly show that in the naked state the RNA forms a base paired panhandle structure which contains a G U base pair and internal bulges. Many detailed mutational analyses of various sequences of the panhandle RNAs have been performed to investigate the functional importance of specific sequences for both replication and transcription. The NMR data provide an additional framework for interpreting the mutation studies and for suggesting further biochemical and genetic studies. Modification by diethyl pyrocarbonate or hydrazine interference analyses have shown that the influenza virus RNA polymerase binds sequence-specifically to four residues, ACAA 10 13, which are located in a proposed bulge region in the 5 -side of the panhandle (Fig. 1; ref 12). Mutations of either C11 or G25 have been found to result in large decreases in the initiation of transcription (27). Our NMR data indicate that C11 and G25 form a Watson Crick base pair that may be important for recognition and binding of the virus RNA polymerase. Although mutation of A12 did not interfere with transcriptional activity (27), imidazole ring opening by diethyl pyrocarbonate of the residue decreased polymerase binding (12). Mutation of A10 inhibited transcriptional activity (27). Systematic mutational analyses have been carried out showing that three contiguous internal residues, GCU 25 27, are crucial for polymerase binding (28) and transcriptional activity (7). We observe that residues A10, A12 and C26 form bulges and all the residues are stacked into the helix. It is very likely that the backbone might be bent or kinked in this region, although the overall geometry of the stem conserves the A-form. Unlike other nucleotides in the stem, the riboses of A12, A13 and G25 adopt 40 60% N-type sugar conformation (26). The presence of bulges and dynamic sugar conformations suggest that this region is more flexible than the other parts of the panhandle and this flexibility might be important for protein RNA interaction as well as base-specific interactions. An RNA fork model for the initiation of influenza virus RNA has been proposed in which the RNA forked structure is partly double-stranded and partly single-stranded (13). It was suggested that polymerase binds to the single-stranded 5 -end of the vrna and the RNA polymerase complex binds to the single-stranded 3 -end to form an RNA fork. Although Fodor et al. (27) assumed that the 5 - and 3 -ends of the vrna would not form the panhandle structure, Baudin et al. (15) and we have shown that the 5 - and 3 -ends are base paired in the naked state. Thus, we suggest that

5 4201 Nucleic Acids Research, 1994, 1996, Vol. Vol. 22, 24, No. No initially the polymerase recognizes and binds to the proposed bulge region of the partially double-stranded vrna termini, followed by melting of the termini, generating the RNA fork. ACKNOWLEDGEMENTS The authors are indebted to Dr Baik L. Seong (Hanhyo Institutes of Technology) for suggesting this project and for useful discussions. This work was supported by the Center for Molecular Catalysis and the Korea Science and Engineering Foundation (to B.-S.C.) and by the Ministry of Science and Technology, the Republic of Korea (to C.C.). REFERENCES 1 Palese,P. and Kingsbury,D.W. (eds) (1983) Genetics of Influenza Virus. Springer Verlag, Vienna, Austria. 2 Ishihama,A. and Nagata,K. (1988) CRC Crit. Rev. Biochem., 23, Desselberger,U., Racaniello,V.R., Zazra,J.J. and Palese,P. (1980) Gene, 8, Robertson,J.S. (1979) Nucleic Acids Res., 6, Parvin,J.D., Palese,P., Honda,A., Ishanma,A. and Krystal,M. (1989) J. Virol., 63, Li,X. and Palese,P. (1992) J. Virol., 66, Fodor,E., Seong,B.L. and Brownlee,G.G. (1993) J. Gen. Virol., 74, Hsu,M.T., Parvin,J.D., Gupta,S., Krystal,M. and Palese,P. (1987) Proc. Natl. Acad. Sci. USA, 84, Li,X. and Palese,P. (1994) J. Virol., 68, Luo,G., Luytjes,W., Enami,M. and Palese,P. (1991) J. Virol., 65, Hagen,M.,Chung,T.D.Y., Butcher,J.A. and Krystal,M. (1994) J. Virol., 68, Tiley,L.S., Hagen,M., Matthews,J.T. and Krystal, M. (1994) J. Virol., 68, Fodor,E., Pritlove,D.C. and Brownlee,G.G. (1994) J. Virol., 68, Seong,B.L. (1993) Infectious Agents Dis., 2, Baudin,F., Bach,C., Cusack,S. and Ruigrok,R.W.H. (1994) EMBO J., 13, Milligan,J.F., Groebe,D.R., Witherell,G.W. and Uhlenbeck,O.C. (1987) Nucleic Acid Res., 15, Varani,G., Wimberly,B. and Tinoco,I.,Jr (1989) Biochemistry, 28, Puglisi,J.D., Wyatt,J.R. and Tinoco,I.,Jr (1990) Biochemistry, 29, Hore,P.J. (1983) J. Magn. Resonance, 55, Marion,D. and Wüthrich,K. (1983) Biochem. Biophys. Res. Commun., 113, Sklenar,V. and Bax,A. (1987) J. Magn. Resonance, 75, Müller,N., Ernst,R.R. and Wüthrich,K. (1986) J. Am. Chem. Soc., 108, Cheong,C., Varani,G. and Tinoco,I.,Jr (1990) Nature, 346, Varani,G., Cheong,C. and Tinoco,I.,Jr (1991) Biochemistry, 30, Wüthrich,K. (1986) NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York, NY. 26 de Leeuw,F.A.A.M. and Altona,C. (1982) J. Chem. Soc. Perkin Trans., 2, Fodor,E., Pritlove,D.C. and Brownlee,G.G. (1995) J. Virol., 69, Seong,B.L. and Brownlee,G.G. (1992) J. Gen. Viol., 111,