From the
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.
The protease 3C (3C
Certain mutations in amino acids 154-156 of poliovirus (PV)
3C
In a different approach, purified recombinant
HRV-14 3C
Here we show that, together with
complementary studies with poliovirus 3C
The procedure used for site-directed mutagenesis
of the 3C
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
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
Using site-directed mutagenesis of
amino acid residues in HRV-14 3C
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
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
We were also
interested in defining the minimum RNA structure in the 5`-terminal
region of HRV-14 RNA that efficiently binds 3C
To elucidate the relative importance of the central
bulge, the top loop, and the two stems (d1 and d2) in 3C
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
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
(
)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) .
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) .
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) .
, 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
.
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) .
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) .
Proteolytic and 5`-NCR Binding Activity of
3C
Several independent mutations
were engineered within 3CMutants
, 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.
,
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
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 and Deleted 5`-NCRs
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
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 and Stem-Loop d
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
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 Binds to Poliovirus
5`-NCR
. 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 3C
PV 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).
(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.
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) .
, 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).
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) .
. 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.
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.
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
,
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.
, and
Pearly Aw for typing the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.