©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence and Structural Determinants of the Interaction between the 5`-Noncoding Region of Picornavirus RNA and Rhinovirus Protease 3C (*)

Philip A. Walker (§) , Louis E.-C. Leong (¶) , Alan G. Porter

From the (1)Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511, Republic of Singapore

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

It has previously been established that human rhinovirus 14 protease 3C binds specifically to the 5`-noncoding region of the viral RNA. A series of mutants of protease 3C and deletion or point mutants of the 5`-noncoding region of the viral RNA were analyzed to elucidate the sites of interaction between the protease and the RNA. Amino acids in protease 3C essential for RNA binding were found to be discontinuous in the amino acid sequence, and mutations which destroyed RNA binding did not affect the catalytic (proteolytic) activity of protease 3C. Based on the three-dimensional structure of rhinovirus 14 protease 3C, the RNA binding region is located in an extended area distinct from the catalytic triad. A single stem-loop structure of 27 nucleotides (stem-loop d) in the 5`-noncoding region was necessary and sufficient to bind protease 3C. Mutagenesis of either the base-paired stem or unpaired loop or bulge regions of stem-loop d suggested that the base-paired stem, but not the loop or bulge, carries important determinants of protease 3C binding. This conclusion is strengthened by the observation that rhinovirus 14 protease 3C bound specifically to the 5`-noncoding region of poliovirus RNA, and only the base-paired stem of stem-loop d is conserved between poliovirus and rhinovirus RNAs.


INTRODUCTION

The protease 3C (3C)()from human rhinovirus serotype 14 (HRV-14), in common with 3C proteases from other members of the picornavirus family (e.g. poliovirus), is classified as a cysteine protease but is structurally homologous to the cellular, trypsin-like serine proteases(1, 2) . 3C plays an indispensable role in the correct cleavage maturation of the viral proteins encoded by the single-stranded, positive sense genomic RNA(3, 4, 5) . HRV-14 viral RNA is 7,208 nucleotides in length and is composed of a 5`-noncoding region (5`-NCR) of 624 nucleotides, a continuous open reading frame of 6,537 nucleotides, and a 3`-NCR of 47 nucleotides(6) . The 5`-terminal 100 nucleotides of the long picornavirus 5`-NCR are essential for viral RNA replication, and much of the remainder of the 5`-NCR promotes cap-independent initiation of translation(7, 8, 9, 10, 11, 12) .

Certain mutations in amino acids 154-156 of poliovirus (PV) 3C were able to rescue the viability of the virus with a small insertion mutation in the 5`-NCR near the 5` terminus of the viral RNA, implying a second nonproteolytic function for 3C(8) . A second function of 3C was revealed when poliovirus 3C, either as mature 3C or in the form of the precursor polypeptide 3CD, was able to interact specifically with the 5`-terminal 90 nucleotides of the poliovirus plus strand 5`-NCR(7) . Poliovirus 3C, or more likely 3CD, together with a 36-kDa host protein formed a ribonucleoprotein (RNP) complex with the 5`-terminal 90 nucleotides of poliovirus RNA folded into a cloverleaf-like structure(7, 9) . As the RNP complex was shown to be essential for virus RNA replication, a model was proposed in which the complex catalyzes and initiates new viral plus strands in trans on a minus strand RNA template (9). A separate study showed that mutations in the 82-87 region of PV 3C destroyed virus viability without affecting maturation cleavages, further indicating that 3C has a nonproteolytic function(13) .

In a different approach, purified recombinant HRV-14 3C bound efficiently and specifically to the 5`-terminal 126 nucleotides of HRV-14 plus strand viral RNA in vitro, demonstrating conclusively that 3C alone bound to RNA (14) and suggesting that in the case of poliovirus the binding of 3CD to the 5`-NCR was mediated by 3C(7) .

Here we show that, together with complementary studies with poliovirus 3C, there are at least two regions in 3C distinct from the catalytic triad which are essential for its binding to the picornavirus 5`-NCR. These regions together define a putative RNA binding site in the recently solved three-dimensional structure of HRV-14 3C(15) . One of these regions is well conserved among diverse picornaviruses, and we suggest it contains a potential RNA binding motif. We also show that a short base-paired stem-loop structure near the 5` terminus of HRV-14 viral RNA is necessary and sufficient for binding to 3C.


EXPERIMENTAL PROCEDURES

Materials

The Escherichia coli strain JM109 was used for all cloning and expression work. Plasmids pGEM 4Z and pGEM 7Zf(+), and the plasmid pGEM-T for direct cloning of polymerase chain reaction (PCR) products, were all purchased from Promega. Plasmid pUC18 and glutathione-Sepharose 4B were purchased from Pharmacia Biotech Inc., and plasmid pGEX-1N was from AMRAD Corp., Australia. [-P]UTP and [-P]ATP were products of DuPont NEN, and bovine plasma thrombin was obtained from Sigma.

Cloning, Subcloning, and Site-directed Mutagenesis

The cloning and subcloning of the HRV-14 3C gene has been described previously(6, 14, 16) . PCR was used to amplify the 3C coding region from full-length HRV-14 cDNA(6) , which was first cloned into pGEM 7Zf(+) for DNA sequencing and site-directed mutagenesis(14) , and subsequently into plasmid pGEX-1N to generate the expression plasmid pLJ111(14) .

The procedure used for site-directed mutagenesis of the 3C coding region has been described previously(14) . The DNA sequence of all mutated 3C genes was confirmed before subcloning the BamHI- and EcoRI-digested gene segment from the pGEM 7Zf(+) vector into BamHI- and EcoRI-digested pGEX-1N for expression. lists all the codon and amino acid changes in the mutant proteins. All oligonucleotide primer sequences used to generate the mutant 3C genes, as well as those for the generation of the various 5`-NCR constructs (see below) are available upon request.

Expression, Purification, and Determination of Proteolytic Activity of 3C

The expression and purification of parental and mutant 3C proteins was done exactly as described(14) . The proteolytic activity of all the mutant proteases was determined by analyzing the results of a peptide cleavage assay (14, 16).

Cloning of Truncated 5`-NCRs and Synthesis of Run-off RNA Transcripts

The plasmid pLJ5NCR1 (14) was the template for cloning by PCR of all the truncated 5`-NCR fragments of HRV-14 virus RNA. Five new truncated 5`-NCRs coding for the phage T7 promoter and nucleotides 1-65, 1-72, 1-81, 1-90, or 1-98 at the 5` terminus of HRV-14 virus RNA were amplified by PCR from plasmid pLJ5NCR1 (14) (Fig. 3). These PCR products were first cloned into plasmid pGEM-T to verify their DNA sequences and, except for clone H (nucleotides 46-72; see below and Fig. 3), were digested with BamHI and SphI, then subcloned into BamHI/SphI-digested pUC18 vector. Following linearization of each plasmid with AflII upstream of the SphI site, run-off transcripts were synthesized using the T7 Riboprobe Gemini II transcription kit either using unlabeled ribonucleoside triphosphates as described by the manufacturer (Promega Corp.) or 50 µCi of [-P]UTP to radiolabel the RNA. The 1-47 RNA (clone G) was transcribed from pLJ5NCR1 after restriction with ScaI(14) .


Figure 3: Schematic representations of truncated HRV-14 5`-terminal RNA fragments. The restriction enzyme sites used to clone the fragments are shown. H = HindIII, B = BamHI, Sp = SphI, Sc = ScaI, and R = EcoRI. The length in nucleotides of each RNA is indicated (e.g. 98 RNA). Alongside each RNA is the secondary structure of the RNA predicted using the MFOLD program (see ``Experimental Procedures''). The predicted secondary structures of the 126 RNA, 98 RNA, 90 RNA, 81 RNA, and 72 RNA all contain stem-loops b, c, and d, but these three stem-loops are only depicted once alongside the 72 RNA.



The sense and antisense stem-loop d constructs (T7 promoter and 5`-NCR nucleotides 46-72 in sense or antisense orientation; Figs. 2 and 3) were synthesized by PCR and cloned into pGEM-T for sequencing prior to subcloning into BamHI- and EcoRI-digested pUC18. Mutants of stem-loop d (Fig. 5) were generated by PCR exactly as for the 72 RNA (see above) except that downstream primers of increasing length incorporated the appropriate nucleotide substitutions, and the downstream restriction site was EcoRI (not SphI).


Figure 5: Mutagenesis of stem-loop d in the 72 RNA. A-D, schematic representations of secondary structures of HRV-14 72 RNA and various mutants predicted by the MFOLD program (see ``Experimental Procedures''). The seven sets of mutants are indicated as I-VII.



The 5`-terminal 90 nucleotides of the poliovirus (PV) type 1 Mahoney strain 5`-NCR were amplified by PCR using an oligonucleotide primer corresponding to the 5`-terminal 90 nucleotides of the PV plus strand 5`-NCR and a downstream oligonucleotide primer corresponding to the complementary sequence of nucleotides 70 to 90 of the PV 5`-NCR(17) . Additional sequences were added to each oligonucleotide primer to allow cloning of the PCR-amplified fragment downstream of the T7 promoter in HindIII- and EcoRI-digested plasmid pGEM 4Z. The resultant plasmid, PV90, was linearized with EcoRI to generate a template suitable for transcription of the PV 5`-NCR using T7 RNA polymerase.

RNA Electrophoretic Mobility (Band Shift) Assay

The procedure used to detect the RNP complexes between 3C (or 3C mutants) and in vitro transcribed RNAs has been described previously(14) . Briefly, 1 µl of run-off RNA transcript (0.25 ng of RNA) was incubated with 5 µg of parental or mutant 3C at 25 °C for 15 min in 15 µl of buffer containing 3 mM spermidine, 30 mM KCl, 3 mM MgCl, 50 mM Tris-HCl, pH 8.0. The RNP complexes were separated from free RNA by nondenaturing PAGE (5% polyacrylamide gel) at 10 V/cm for 5 h. The gel was dried and autoradiographed. In competition experiments with unlabeled RNAs, the standard binding reaction was performed as above, then various amounts of unlabeled RNAs were introduced (see legend to Fig. 8for details) and the incubation was continued at 25 °C for 10 min before polyacrylamide gel electrophoresis.


Figure 8: Binding of HRV-14 3C to 5`-terminal 90 nucleotides of poliovirus 5`-NCR. Autoradiograph of native 5% polyacrylamide gel. Lane 1, free radiolabeled PV 90 RNA; lane 2, radiolabeled PV 90 RNA plus HRV-14 3C; lane 3, as lane 2 except in the presence of 300- to 500-fold molar excess of unlabeled HRV-14 126 RNA; lane 4, as lane 2 except in the presence of 300- to 500-fold molar excess of unlabeled HRV-14 65 RNA; lane 5, as lane 2 except in the presence of 300- to 500-fold molar excess of unlabeled HRV-14 stem-loop d RNA.



Computer Analysis

The RNA secondary structure predictions were made using the MFOLD program from the Genetics Computer Group(27) .


RESULTS

Proteolytic and 5`-NCR Binding Activity of 3CMutants

Several independent mutations were engineered within 3C, and the effects of the resultant single amino acid substitutions on the peptide cleavage and RNA binding activities of 3C were analyzed (). Changes in the 82 to 86 region of HRV-14 3C were chosen, since certain amino acid substitutions in the homologous region of PV 3C reduced virus viability without affecting the proteolytic activity of 3C(13) . We reasoned that RNA binding activity might be impaired in these PV mutants(13) . The ``TGK'' region (amino acids 153-155) of HRV-14 3C was chosen () as mutations in the corresponding TGK region of PV 3C were able to rescue a lethal insertion in the 5`-NCR (8), implying an involvement of TGK in RNA binding.

The mutant 3C proteases with single amino acid substitutions, K82Q, R84Q, R84K, I86A, T153S, G154A, and K155Q were all purified to near-homogeneity from glutathione S-transferase fusion proteins following thrombin cleavage (Fig. 1A). All the proteins were assayed for proteolytic activity with a synthetic peptide corresponding to the in vivo sequence between HRV-14 viral polypeptides 2C and 3A (14, 16). The results, summarized in , demonstrate that the catalytic activity of none of these mutants was impaired in a 2-h assay, although a significant reduction in proteolytic activity of the I86A mutant was observed in the 30-min assay (). In contrast, unlike parental 3C, all seven mutants with the possible exception of the R84K mutant 3C were unable to form a detectable RNP complex with the 126 RNA derived from the 5`-NCR (Fig. 1B and ). The highly conservative R84K substitution in 3C gave a mutant which bound weakly with <10% of the efficiency of parental 3C, but the R84Q mutant failed to bind detectably to 126 RNA (Fig. 1B). These results, which are very similar to those we previously reported for the D85E and D85N mutants of HRV-14 3C(14) , suggest that amino acids 82-86 and 153-155 are very important for binding of 3C to the 5`-NCR, but are not essential for its catalytic (proteolytic) activity.


Figure 1: Purification and RNA binding activity of 3C mutants. A, Coomassie Blue-stained SDS-polyacrylamide gel showing purified 3C and mutants. Molecular mass markers in kilodaltons are shown at the left. Lane 1, unmodified 3C; lane 2, R82Q 3C; lane 3, R84Q 3C; lane 4, R84K 3C; lane 5, I86A 3C; lane 6, T153S 3C; lane 7, G154A 3C; lane 8, K155Q 3C. B, binding of mutants of 3C to the 126 RNA detected by RNA band shift analysis. Autoradiograph of a native 5% polyacrylamide gel. All lanes have 0.25 ng of radiolabeled, unpurified 126 RNA. Lanes 2-9 each contain 5 µg of 3C (lane 2) or appropriate 3C mutant (lanes 3-9). Lane 1, no protein; lane 2, unmodified 3C; lane 3, R82Q mutant; lane 4, R84Q mutant; lane 5, R84K mutant; lane 6, I86A mutant; lane 7, T153S mutant; lane 8, G154A mutant; lane 9, K155Q mutant.



Ribonucleoprotein Complex Formation with 3C and Deleted 5`-NCRs

Theoretical and experimental evidence has been obtained for the existence of a specific cloverleaf-like structure near the 5` end of PV RNA(7, 18) . Despite extensive sequence divergence, the 5`-terminal 100 nucleotides of the plus strands of rhinoviruses and other enteroviruses have been widely predicted to fold into similar cloverleaf-like structures (7, 9, 12, 18, 19) (Fig. 2). To delineate the region(s) in the predicted HRV-14 5`-terminal cloverleaf (Fig. 2A) directly involved in the formation of the RNP complex with 3C, a series of truncated 5`-NCRs was constructed. Deletions were made progessively from the 3`-end of the 5`-terminal 126 nucleotide segment of HRV-14 viral RNA (the 126 RNA), since it had previously been shown that nucleotides 1-126, but not nucleotides 1-47, bind to 3C(14) . The exact position of each 3`-nucleotide (Fig. 3) was carefully chosen to minimize disruption to the predicted cloverleaf fold of the RNA (Fig. 2A).


Figure 2: Schematic representations of 5`-terminal regions of HRV-14 and PV RNAs. The secondary structures were predicted with the MFOLD program (see ``Experimental Procedures'' and Ref. 27). A, HRV-14 126 RNA. B, PV 5`-terminal 90 nucleotides. The stem-loop d in each viral RNA is encircled with a dashed line. Stem-loops b and c are also shown.



Six truncated 5`-NCR transcripts (1-98, 1-90, 1-81, 1-72, 1-65, and 1-47) were predicted to have the secondary structures shown in Fig. 3. Note that all transcripts comprising nucleotides 1 to 72 or larger retained stem-loops b, c, and d (Fig. 3). These RNAs were synthesized in vitro, and their binding to HRV-14 3C was compared with the binding of the 126 and 47 RNAs. Fig. 4, A and B, shows that the 98 RNA, 90 RNA, 81 RNA, and 72 RNA bound to 3C as efficiently as the 126 RNA, but the 65 RNA (like the 47 RNA) failed to bind to 3C. Thus, the deletion of only 7 nucleotides from the 3`-end of the 72 RNA leads to a complete loss of 3C binding (Fig. 3, compare E and F). To test the specificity of the RNP complexes formed between 3C and the truncated RNAs, competition experiments were performed using excess unlabeled 72 RNA or 65 RNA. A 300- to 500-fold molar excess of 72 RNA completely prevented complex formation between the 98 RNA, 90 RNA, 81 RNA, or the 72 RNA and 3C, whereas a similar molar excess of the 65 RNA did not prevent complex formation (data not shown).


Figure 4: Binding of 3C to truncated 5`-terminal fragments of HRV-14 RNA. Autoradiographs of native 5% polyacrylamide gels. A: lane 1, free 126 RNA; lane 2, 126 RNA plus 3C; lane 3, free 98 RNA; lane 4, 98 RNA plus 3C; lane 5, free 90 RNA; lane 6, 90 RNA plus 3C; lane 7, free 81 RNA; lane 8, 81 RNA plus 3C. B: lane 1, free 72 RNA; lane 2, 72 RNA plus 3C; lane 3, free 65 RNA; lane 4, 65 RNA plus 3C; lane 5, free 47 RNA; lane 6, 47 RNA plus 3C; lane 7, free stem-loop d; lane 8, stem-loop d plus 3C.



Examination of the truncated RNA species (Fig. 3, B-G) shows that they all retain stem-loops b and c present in the original 126 RNA (Fig. 2A), but only the 65 and 47 RNAs, which failed to bind to 3C, lack stem-loop d. The 65 RNA is predicted to have a new stem-loop which does not, however, bear any resemblance to stem-loop d in the sequence of either the stem or the loop (Fig. 3F). This is a clear indication that stem-loop d contains determinants of 3C binding.

Ribonucleoprotein Complex Formation with 3C and Stem-Loop d

A RNA transcript comprising nucleotides 46 to 72 (Fig. 2A) corresponding to the majority of the stem-loop d region of the 5`-NCR (Fig. 3H) was synthesized and shown to bind 3C as efficiently as the 126 RNA (Fig. 4B, compare lanes 7 and 8). When the mobilities in polyacrylamide of all the RNP complexes were compared (Fig. 4), it is clear that the RNP complex containing only stem-loop d was retarded the most (Fig. 4B, lane 8), despite the fact that stem-loop d was the smallest RNA tested (lane 7). We do not have an explanation for this surprising result.

In Vitro Mutagenesis of Stem-Loop din the 72 RNA

A variety of double and multiple nucleotide substitutions were engineered into stem-loop d within the 72 RNA (Fig. 5) in order to assess their participation in RNP complex formation (Fig. 6). The choice of substitutions was limited by the need to minimize disruption to the predicted cloverleaf structure in the 72 RNA (Fig. 3E), so that as far as possible the contributions of individual unpaired or base-paired nucleotides could be evaluated. Each set of mutations was designed to test the contribution to binding of the two stem regions d1 and d2, the central bulge, and the top loop (Fig. 5). Replacement of the sequence UAU in the top loop with AUA (Fig. 5A) or GGG (data not shown) and mutagenizing or ``closing'' the central bulge (Fig. 5, A and B) did not affect complex formation (Fig. 6). Similarly, replacement of a T:A with a C:G base pair in stem d2 (Fig. 5A) was totally without effect on RNP complex formation (Fig. 6). In contrast, more drastic changes to stems d1 or d2, such as eradication of stem d2 or conversion of the two A:U base pairs to G:C base pairs in stem d1 (Fig. 5, C and D) abolished or significantly impaired 3C binding (Fig. 6).


Figure 6: Binding of 3C to mutant 72 RNAs detected by RNA band shift analysis. Autoradiograph of a 5% native polyacrylamide gel. Lanes 3-14 show the mutant RNAs I-IV and VI and VII (Fig. 5). Lanes 1, 3, 5, 7, 9, 11, and 13 show the free RNAs (minus 3C) while lanes 2, 4, 6, 8, 10, 12, and 14 show the RNAs in the presence of 3C. Lanes 1 and 2, unmodified 72 RNA; lanes 3 and 4, mutant I; lanes 5 and 6, mutant II; lanes 7 and 8, mutant III; lanes 9 and 10, mutant IV; lanes 11 and 12, mutant VI; lanes 13 and 14, mutant VII (Fig. 5).



Finally, the cDNA coding for stem-loop d was inverted, and the antisense RNA was transcribed (Fig. 7B). Both sense and antisense RNAs have the same sequence and predicted base pairing in stems d1 and d2, but the loop and bulge regions are different (Fig. 7, A and B). Surprisingly, the antisense stem-loop d repeatedly failed to form a detectable complex with 3C (Fig. 7C).


Figure 7: Lack of binding of antisense ``stem-loop d'' to 3C. A and B, predicted secondary structures of sense and antisense stem-loop d, respectively. C, autoradiograph of a native 5% polyacrylamide gel. Lanes 1 and 2, sense stem-loop d minus and plus 3C, respectively; lanes 3 and 4, antisense stem-loop d minus and plus 3C, respectively.



HRV-14 3C Binds to Poliovirus 5`-NCR

As mentioned above, the 5`-terminal 90 nucleotides of poliovirus RNA are structurally homologous to the corresponding region in HRV-14 RNA including the presence of stem-loop d (Fig. 2B) (7, 8, 18, 19). Many nucleotides, particularly in stems d1 and d2 are identical in PV type 1 and HRV-14 stem-loop d (Fig. 2, A and B). Therefore, it was of interest to determine whether the 5`-terminal 90 nucleotides of PV RNA would form a specific RNP complex with HRV-14 3C. It may be seen from Fig. 8that the 90 RNA of poliovirus indeed formed a complex with HRV-14 3C, although, in contrast to HRV-14 RNA, some of the radiolabeled PV RNA remained unbound even in the presence of excess HRV-14 3C (Fig. 8, lane 2). The specificity of the HRV-14 3CPV RNA complex was demonstrated by using excess unlabeled HRV-14 126 RNA and stem-loop d to successfully compete with radiolabeled PV 90 RNA for binding to HRV-14 3C (Fig. 8, lanes 3 and 5). The 65 RNA failed to compete with PV RNA for binding to 3C (lane 4).


DISCUSSION

It has previously been established that the 5`-terminal 90 and 126 nucleotides of the PV and HRV-14 genomes, respectively, are important for interacting specifically with 3C(7, 14) . In the case of PV, this interaction was found to be essential for viral plus strand RNA synthesis and virus viability, and the protease-polymerase precursor polypeptide 3CD bound more tightly than 3C (7). Moreover, in an in vitro binding assay, the binding of PV 3CD to the PV 5`-NCR depended on a 36-kDa ``host factor,'' and 3C did not bind at all even in the presence of the host factor(20) . Perhaps the affinity of HRV-14 3C for the HRV-14 5`-NCR is higher than that of PV 3C for the PV 5`-NCR, thus obviating the need for a host factor in vitro. Because the 5`-terminal regions of PV and HRV-14 RNA are homologous both in sequence and structure(7, 12, 18) , it is highly likely that the specific binding of HRV-14 3C to the 126 RNA is a reflection of the requirement for 3CD binding in HRV-14 virus replication.

Using site-directed mutagenesis of amino acid residues in HRV-14 3C suspected of being involved in its binding to the viral 5`-NCR, we identified two distinct regions, amino acids 82-86 and 153-155, where even conservative substitutions abolished binding to the HRV-14 126 RNA. The catalytic (proteolytic) activity of 3C was hardly affected by these substitutions, indicating that gross structural alterations or misfolding of the mutant proteins is unlikely to be the explanation for their lack of RNA binding activity. Similarly, substitutions of PV 3C amino acids 84 and 85 and 154-156 (the TGK region equivalent to HRV-14 3C amino acids 153-155) gave mutant proteases with selective loss of RNA binding function(9) .

In light of the recently published x-ray crystallographic structures of both the hepatitis A virus and HRV-14 3C proteases(15, 21) , it is not surprising that substitutions made in the 82-86 and 153-155 regions have little or no effect on the catalytic activity of picornavirus 3C proteases. Examination of both crystal structures clearly shows that these two regions are located on the surface in or close to the ``interdomain'' connecting loop(15, 21) . In HRV-14 3C, this loop connects strands F and A and is positioned far away from the active site residues which are located on the opposite side of the 3C molecule (see Fig. 1of Ref. 15).

Which amino acids in the 82-86 region and 153-155 (TGK) region of picornavirus 3C proteases make contact with the 5`-NCR in the RNP complex? The K155E (HRV-14) and equivalent K156E (PV) mutants with nonconservative substitutions were able to bind to the respective 5`-NCRs(9, 14) , but the K155Q (HRV-14) mutant lacked detectable binding activity (). These results, coupled with the fact that the TGK motif and surrounding sequence are not well conserved among picornaviruses, imply that this region may play only an accessory role in RNA binding.

On the other hand, the 82-86 and surrounding region is well conserved among picornavirus 3C proteases (4) having the consensus sequence -74-LIXLXRNEKFRDIRXXI-90-. There is no significant primary sequence homology between this sequence and the two consensus sequences found within the 80-amino acid RNA recognition motif present in the large family of cellular RNA binding proteins(22, 23) . The main distinctive feature of RNA recognition motifs is the presence of basic and solvent-exposed hydrophobic amino acids which make ionic and stacking interactions with RNA(23, 24) . The 82-86 and surrounding conserved region of picornavirus 3C proteases, -74-LIXLXRNEKFRDIRXXI-90-, is also largely basic and hydrophobic in character, suggesting that particular basic and hydrophobic amino acids in this region interact directly with RNA. Our results in the context of the three-dimensional structure of HRV-14 3C, together with the PV data, suggest that Lys-82, Arg-84, and Arg-87 may well interact directly with phosphate oxygens in the RNA backbone(9, 13, 15) .

We were also interested in defining the minimum RNA structure in the 5`-terminal region of HRV-14 RNA that efficiently binds 3C. Our deletion analysis clearly pointed to a sequence between nucleotides 65 and 72 which is essential for RNA binding (Figs. 3 and 4). The deletion of this sequence from the 72 RNA resulted in the loss of the d1 stem region in stem-loop d and the creation of a new predicted RNA secondary structure without stem d2 as well (Fig. 3F), indicating that both stems (particularly d1) are critical for binding. In fact, the isolated stem-loop d (27 nucleotides) bound to 3C as efficiently as the 126 RNA, showing that stem-loop d contains all the determinants of 3C binding.

To elucidate the relative importance of the central bulge, the top loop, and the two stems (d1 and d2) in 3C binding, several types of nucleotide substitutions were made with the aid of the MFOLD program with a view to perturbing the structure of either the single- or double-stranded regions of stem-loop d in a predictable manner. Changing the sequence of the top loop to AUA or GGG and altering or ``closing'' the central bulge (Fig. 5) had no effect on 3C binding (Fig. 6). Likewise, a mutant with only a T:A to C:G substitution in stem d2 bound to 3C as efficiently as the parental RNA. More extensive changes such as completely preventing base pairing in stem d2 or conversion of two A:U base pairs in stem d1 to G:C base pairs, impaired 3C binding ( Fig. 5and Fig. 6). These results indicate that the base-paired stems d1 and d2, but not the loop or bulge regions, are important for protease binding. Why, then, did antisense stem-loop d fail to bind 3C when it has identical base-paired stems to sense stem-loop d (Fig. 7)? A closer examination of the sequences shows that the sense RNA has six pyrimidines in the central bulge whereas the antisense RNA has six purines in this bulge (Fig. 7). It is well known that purines have a much greater tendency to stack than pyrimidines(25) , which would result in destabilization of base pairing in stems d1 and d2. Thus, antisense stem-loop d may have little or no base pairing, consistent with the evidence that the stems are important in complex formation. A different approach provided further evidence for the importance of the stem regions. PV stem-loop d bound efficiently to HRV-14 3C. A comparison of the nucleotide sequences within stem-loop d of PV type 1 and HRV-14 (Fig. 2) shows that the stems d1 and d2 are almost totally conserved, whereas the top loops and bulges have diverged.

Overall, the pattern of results from PV and HRV-14 suggests that the double-stranded stems in stem-loop d are much more important for RNP complex formation than the single-stranded bulge or loop regions, but the precise nucleotide sequences in the stems may not always be important provided that base pairing is not disrupted. In general, loops, bulges, or base-paired stems (or any combination) have previously been found to interact specifically with particular proteins (23, 24, 26), but 3C appears to belong to a type of RNA-binding protein which recognizes base-paired stems. Only when the three-dimensional structure of 3C complexed with stem-loop d has been solved will it be possible to confirm the major RNA-protein interactions and perhaps also define the class of RNA-binding protein to which 3C belongs.

  
Table: Amino acid substitutions in 3C and their effects on proteolysis and RNA binding



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore. Tel.: 65-772-3384; Fax: 65-779-1117; E-mail: mcbauld@nus.sg.

Present address: Dept. of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92717-4025.

The abbreviations used are: 3C, protease 3C; 5`-NCR, 5`-noncoding region; HRV-14, human rhinovirus type 14; 126 RNA, 98 RNA, 90 RNA, 81 RNA, 72 RNA, 65 RNA, and 47 RNA, the 5`-terminal 126 to 47 nucleotides of HRV-14 plus strand viral RNA; plus strand, coding strand; minus strand, noncoding strand; PCR, polymerase chain reaction; RNP, ribonucleoprotein; PV, poliovirus.


ACKNOWLEDGEMENTS

We wish to thank Robin Philps for help with the peptide chemistry and Ben Li for discussions and oligonucleotide synthesis, M. N. G. James, University of Alberta for providing the coordinates of the structure of hepatitis A virus 3C, and Pearly Aw for typing the manuscript.


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