From the Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the role of the RNA polymerase domain
(3D) in the proteinase substrate recognition and RNA binding properties
of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with
structural and non-structural viral polyprotein substrates, we found
that 3CD processes the poliovirus structural polyprotein precursor (P1)
100 to 1000 times more efficiently than 3C processes P1. We also found
that trans-cleavage of other 3CD molecules and sites within
the non-structural P3 precursor is more efficiently mediated by 3CD
than 3C. However, 3C and 3CD appear to be equally efficient in the
processing of a non-structural polyprotein precursor, 2C3AB. Four
mutated 3CD polyproteins with site-directed lesions in the 3D domain of
the proteinase were analyzed for their ability to process viral
polyprotein precursors and to form a ternary complex with RNA sequences
encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD
polypeptides revealed that specific mutations within the 3D amino acid
sequences of 3CD confer differential effects on 3CD activity. All four
mutated 3CD proteins tested were able to process the P1 structural
precursor with wild type or near wild type efficiency. However, three
of the mutated enzymes demonstrated an impaired ability to process some
sites within the P3 non-structural precursor, relative to wild type
3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with
poliovirus 5' RNA sequences.
The positive strand RNA genome of poliovirus contains a single
open reading frame that codes for a 247-kDa viral polyprotein. Proper
expression of poliovirus gene products requires specific protein
processing of the viral polyprotein by the enzymatic activities of
virally encoded proteinases. These enzymes cleave the polyprotein co-
and post-translationally into the structural and non-structural viral
gene products (Fig. 1). The majority of cleavage events within the
viral polyprotein occur at Q-G bonds and is mediated by a proteinase
function associated with the viral-encoded 3C protein (1-6). The rate
at which 3Cpro1-mediated
proteolytic processing occurs at various sites within the viral
polyprotein controls the temporal expression of viral gene products
(7). This regulation of gene expression is essential for viral
replication since polyprotein precursors have functions in replication
that are distinct from those of the mature cleavage products (8, 9). An
example of a molecule exhibiting these differential functions is the
viral polyprotein 3CD, which is an active proteinase containing the
entire amino acid sequences of the 3C proteinase and the
RNA-dependent RNA polymerase, 3D. Polypeptide 3CD is a
multi-functional protein required for both proteolytic processing of
the capsid precursor protein and, although it has no detectable
elongation activity in vitro, viral RNA replication. The
role of 3CD in viral RNA replication is dependent on its ability to
form a ternary ribonucleoprotein (RNP) complex with the 5'-terminal sequences of genomic RNA (the 5' cloverleaf structure) and a cellular RNA-binding protein termed poly(rC)-binding protein 2 (PCBP2) (10,
11).
Previous biochemical studies on poliovirus 3C and 3CD enzymes and
structural studies on poliovirus 3C have suggested that, whereas the
proteinase active site and RNA binding domains reside in 3C, processing
of the viral capsid precursor (P1) and RNP complex formation with the
5'-terminal RNA sequences are more efficiently mediated by 3CD than 3C
(10, 12-16). These findings have led to the hypothesis that structural
domains within the 3D portion of the 3CD polyprotein contribute to the
enhanced 3C activities. Although the exact biochemical roles of
specific 3D amino acid sequences and domains within 3CDpro
necessary for proteinase and RNA binding activities are poorly understood, the 3D domain is necessary for the complete processing of
the poliovirus capsid precursor polyprotein P1, as has been demonstrated using both in vitro synthesized 3CD enzymes
(13, 14) and purified 3C expressed in Escherichia coli (17).
In these previous studies, neither purified 3C nor carboxyl-terminal truncations of 3CD expressed in vitro were able to
efficiently mediate processing of the P1 precursor. Studies detailing
the precise function of specific residues or structural motifs in 3D
necessary for mediating 3CD processing of the P1 precursor have been
limited. An early report described a four-amino acid insertion in the
3D domain of the poliovirus polyprotein that eliminated cleavage of the
P1 precursor while still retaining 3C-mediated processing of the
non-structural polypeptides (12). Two reports describing the effects of
specific mutations in poliovirus 3D on RNA polymerase activity and P1
proteolytic processing found that whereas mutations in 3D had effects
on both processing and RNA replication, no single mutation in 3D
sequences affected processing without also affecting RNA replication
(18, 19). In this study, we have expressed and purified recombinant
poliovirus 3C and 3CD proteins. We have used these proteins in a
comparative biochemical analysis of their proteolytic processing
activities on viral polyprotein substrates and their RNA binding
properties in the presence of 5' noncoding region sequences of
poliovirus RNA and purified recombinant PCBP2 protein. In order to
determine the contribution of specific amino acid residues in 3D on 3CD
functions, this analysis was extended to a number of recombinant 3CD
polyproteins containing site-specific mutations in 3D previously shown
to have deleterious effects on viral RNA replication (19, 20).
Design of P1 "Stop" Plasmid and 3CD Expression
Plasmids--
pT7-PV1-P1(stop) was constructed by placing sequences
coding for tandem stop codons (in bold, below) at the 3' terminus of the P1 coding region in pT7-PV1 using oligonucleotide mismatch mutagenesis (21, 22) with the Stratagene QuickChange site-directed mutagenesis kit and oligonucleotides as follows: P1stop + (5'-CCACATATTGATAAGGACACC-3') and P1stop
All 3CD expression plasmids coding for mutations in 3D sequences were
sub-cloned into the pET15b-3CDµ10 expression plasmid. This plasmid
encodes a histidine-tagged 3CD protein containing a serine insertion
proximal to the 3C/3D junction that essentially eliminates
auto-processing at this site without affecting proteinase and RNA
binding activities (11, 23).
Site-directed lesions in 3D resulting in clustered charge to alanine
mutations at 3D amino acid positions Arg-136 and Asp-137 (pET15b-3CDµ10-AL10), and Glu-226 and Glu-227 (pET15b-3CDµ10-AL14) (19) were generated by incubating the gel-isolated 1110-bp
AvrII-AflII restriction fragment of plasmids AL10
and AL14 (kind gifts from Dr. Karla Kirkegaard) with the ~6700-bp
AvrII-AflII restriction fragment of
pET15b-3CDµ10 in the presence of T4 DNA ligase. Mutations in 3D
sequences coding for 3D-N424H were generated by incubating the
gel-isolated 1108-bp AflII-SacI restriction
fragment of plasmids pEXC-3CD-3DN424H (20) (a kind gift from Dr. Oliver
Richards and Dr. Ellie Ehrenfeld) with the ~6700-bp
AflII-SacI restriction fragment of
pET15b-3CDµ10 in the presence of T4 DNA ligase. The single site
mutation coding for 3D-K127A was generated using oligonucleotide mismatch mutagenesis (21, 22) with the Stratagene QuickChange site-directed mutagenesis kit and the following oligonucleotides: 3D
K127A+ (5'-GCAATGGGAAAGAAGGCGAGAGACATCTTGAACAA-3')
and 3D K127A Expression and Purification of Recombinant Poliovirus 3C and 3CD
Proteinases--
Wild type and mutated histidine-tagged 3CDµ10
proteins were purified as described previously (11) with the following
modification. Nickel affinity purified recombinant 3CD proteins were
subjected to gel filtration chromatography on an Amersham Pharmacia
Biotech Superdex 200 column with continuous buffer flow (20 mM HEPES, pH 7.4, 0.5 mM NaCl, 1 mM
DTT, 1 mM EDTA, and 5% (v/v) glycerol). When necessary,
gel filtration fractions were concentrated with Centricon 50 concentrators (Amicon).
Radiolabeled 3CD-C147A (a proteolytically inactive form of 3CD (24))
substrate was purified from E. coli BL21 (DE3) cells harboring the expression plasmid pET15b-3CD(C147A). Briefly,
transformed cells were grown to log phase and induced with 0.5 mM isopropyl-1-thio-
Recombinant histidine-tagged 3C protein was purified from bacterial
expression of pET15b-3CD. This expression plasmid (unlike the µ10
version) contains wild type sequences at the 3C/3D cleavage site and
thus auto-processing of 3CD to His-3C and -3D occurs during the course
of induction. Histidine-tagged 3C was purified from 2 liters of
isopropyl-1-thio-
Recombinant PCBP2 protein was purified as previously reported (11) with
the following modification. Peak nickel affinity purified fractions
were subjected to gel filtration on an Amersham Pharmacia Biotech
Superdex 200 column with constant buffer flow (5 mM Tris,
pH 7.4, 100 mM KCl, 0.05 mM EDTA, 1 mM DTT, 5% glycerol (25)). Peak fractions resulting from
gel filtration were then used in RNA binding assays. All purified
proteins were quantified using the Bio-Rad protein assay, and the
percent purity of each protein was calculated by densitometric analysis
of SDS-polyacrylamide gels stained with Coomassie Blue.
Protein Processing Assays, Enzyme Dilution Analysis--
For
enzyme dilution analysis, poliovirus P1 and 2C3AB precursor proteins
were synthesized in rabbit reticulocyte lysates programmed with
in vitro transcribed RNA from plasmids
pTM1-P12 and
pTM1-2C3AB3 in the presence
of [35S]methionine. 1 µl of either the P1 or 2C3AB
(~25 fmol) translation reaction was then incubated for 2 h at
30 °C in the absence or presence of decreasing amounts of either 3C
or 3CD proteinase in a 10-µl reaction containing 24 µg of protein
from a HeLa S10 extract and 1 µl of 10× cleavage buffer (final
concentration of 20 mM HEPES, pH 7.4, 1 mM DTT,
and 150 mM KOAc). Following incubation, 2× Laemmli sample
buffer (LSB) was added to each reaction; the mixtures were boiled, and
the cleavage products were resolved on a SDS-12.5% polyacrylamide gel.
For trans-cleavage analysis of 3CD substrate, 10 pmol of
purified radiolabeled 3CD(C147A) and 1 µl of 10× cleavage buffer
were incubated for 2 h at 30 °C in the absence or presence of
decreasing amounts of His-3CD or His-3C in a total reaction volume of
10 µl. Following incubation, 2× LSB was added to each reaction
mixture; the mixtures were boiled, and the cleavage products were
resolved on a SDS-12.5% polyacrylamide gel.
Protein Processing Assays, Time Course Analysis--
For time
course kinetic experiments, P1 and P3 polyprotein substrates were
translated in vitro in HeLa cell extract in the presence of
[35S]methionine (26). Briefly, plasmids pT7-PV1-P1(stop)
and pT7-5'NCR-P3(C147A) (27) were linearized, in vitro
transcribed with T7 RNA polymerase, and the resulting RNA was used to
program HeLa S10 translation extracts.
The in vitro translated substrate (50% (v/v) final volume)
was diluted in cleavage buffer and preincubated at 30 °C for 15 min.
Following preincubation, purified 3C or 3CD was added to a final enzyme
concentration of 100 nM, and incubation was continued at
30 °C. 8-µl aliquots were then removed from each reaction and mixed with 8 µl of 2× LSB at 15 and 30 min and 1, 2, 4, and 8 h. Additionally, substrate was incubated at 30 °C in the absence of
exogenous proteinase, and time points at 0 and 8 h were taken. Samples were boiled, and cleavage products were resolved on
SDS-polyacrylamide gels. Following electrophoresis, gels were subjected
to fluorography, dried, and exposed to XMR film (Eastman Kodak
Co.).
RNA Mobility Shift Assays--
The ability of recombinant 3C and
3CD proteins to form a ternary complex with RNA sequences representing
the first 108 nucleotides of the poliovirus genome (the 5' cloverleaf)
was assessed using modifications (11, 29) of a previously reported RNA
mobility shift assay (10). For this analysis, recombinant purified
PCBP2 (200 nM) was preincubated with 0.1 nM
radiolabeled RNA representing the first 108 nucleotides of the
poliovirus genome for 10 min at 30 °C in RNA binding buffer
containing 1 mg/ml E. coli tRNA and 0.5 mg/ml bovine serum
albumin. Following preincubation, recombinant histidine-tagged 3C or
3CD protein was added, and the incubation was continued for 5 min at
30 °C. 2.5 µl of 50% glycerol was then added, and complexes were
resolved on a non-denaturing 4% polyacrylamide gel at 4 °C.
Following electrophoresis, gels were dried and exposed to XAR film (Kodak).
Protein Processing Assays with Purified Recombinant Poliovirus 3C
and 3CD Proteinases--
Due to limitations in the quantities of
substrate produced using in vitro translation, we were
unable to perform kinetic experiments under conditions of saturating
substrate. Therefore, we used enzyme dilution and time course
experiments to assess the relative proteolytic activities of purified
recombinant 3C and 3CD proteinases to process viral polyprotein
precursors. Complete processing of the P1 structural precursor by
3C-containing enzymes results in the production of the capsid proteins
VP0, VP1, and VP3 (Fig. 1). For the first set of experiments, we used different enzyme dilutions and incubated the cleavage reactions for 2 h at 30 °C. The results of
incubation of an in vitro translated P1 precursor with
various concentrations of purified 3Cpro (authentic or
amino-terminally His-tagged) or 3CDpro (amino-terminally
His-tagged) are shown in Fig.
2A. These data demonstrate
that the two purified proteinases display differential activities in
the processing of P1, a result that is consistent with P1 processing
assays utilizing in vitro synthesized 3C and 3CD proteinases
(12-14). As can be seen in lanes 3-7, the P1 precursor was
faithfully processed into VP0, VP1, and VP3 capsid proteins by 3CD
proteinase, even at the lowest concentration of enzyme (lane
7). However, both the His-3C (lanes 8-12) and 3C
proteinases (lanes 13-17) were only able to process P1 at
the highest concentrations of enzyme (lanes 8 and
13), and processing of P1 by His-3C and 3C was incomplete,
as indicated by the presence of 1ABC (VP0-VP3, lanes 8, 9, and 13).
Results of this experiment are illustrated graphically in Fig.
2B, which shows the production of either VP1 or VP3 as a
function of enzyme concentration. The percentages of VP1 and VP3 were
determined by scanning the autoradiograph of Fig. 2A and
using calculations described in the legend to Fig. 2. As indicated by
the graph, both 3C enzymes are significantly impaired, relative to 3CD,
in their abilities to process the P1 precursor. Comparable cleavage at
either the VP0-VP3 or VP3-VP1 junction requires greater than 1000-fold
more 3Cpro than 3CDpro, and overall processing
of the P1 precursor by 3Cpro is inefficient as indicated by
the presence of the 1ABC polyprotein product (Fig. 2A, lanes
8 and 13). These results illustrate the necessity of
the 3D domain in 3CD for complete and efficient in vitro
processing of the P1 precursor.
The diminished proteolytic cleavage properties of 3Cpro
relative to 3CDpro seen with the P1 structural precursor
are not observed when the non-structural polyprotein precursor 2C-3AB
is used as a substrate (Fig. 3). For this
analysis, reaction conditions using enzyme dilutions were identical to
those detailed above for the P1 precursor. In the case of the 2C-3AB
substrate, all three proteinases appear to have similar activities in
processing the polyprotein into the 2C and 3AB products (Fig. 3A,
lanes 3-17). Results of this experiment are illustrated
graphically in Fig. 3B where production of 2C was calculated
as described in the legend to Fig. 3. We have carried out a kinetic
analysis of cleavage of the 2C-3AB substrate at fixed concentrations of
His-3C or His-3CD and have confirmed that this substrate is cleaved
with equal efficiencies by 3C and 3CD (data not shown). Note also that
since the proteolytic activities of the 3C enzymes (amino-terminally
histidine-tagged 3C and authentic 3C) appear to be nearly equivalent,
the rest of the experiments in this study utilize the histidine-tagged 3C protein as a source of 3Cpro.
Poliovirus polypeptide 3CD is a polyprotein that could potentially
cleave itself through an intramolecular processing event to generate
the mature 3C and 3D protein products (refer to Fig. 1). The relatively
high abundance of the 3CD polyprotein compared with the 3C and 3D
protein products in a poliovirus-infected cell suggests that if
cleavage of the 3C/3D junction does occur in cis, it is a
very inefficient processing event (7). Structural data on the 3C
proteinases of both human rhinovirus 14 (HRV14) (28) and poliovirus
type 1 (16) suggest that the 3C/3D junction is positioned such that it
could not fold into the active site cleft and be accessible to
intramolecular cleavage. These previous data suggest that the
regulation of processing of the 3C/3D junction is mediated through a
trans-cleavage event. To assess the relative efficiencies of
3C and 3CD activities on trans-processing of the 3CD
precursor, enzyme dilution analysis was performed on a purified, radiolabeled 3CD substrate containing an active site cysteine 147 to
alanine mutation (24). This lesion renders the 3C enzyme proteolytically inactive. Fig.
4A shows the results of
incubating radiolabeled 3CD(C147A) substrate in the absence
(lanes 2) or presence of decreasing amounts of purified
His-3CD (lanes 3-7) or His-3C (lanes 8-12). As
seen in lanes 3-7, 3CD is a more effective enzyme at
processing the 3C/3D junction than is 3C. Results of this experiment
are illustrated graphically in Fig. 4B, where percentage of
3D was calculated as described in the legend to Fig. 4. Results of this
analysis indicate that 3CD is a more effective enzyme in
trans-cleavage of 3CD than is 3C with comparable levels of
cleavage at the 3C/3D junction requiring up to 100-fold more 3C than
3CD.
Analysis of Recombinant 3CD Polyproteins Containing Site-specific
Lesions in the 3D Domain--
The experiments presented above
demonstrated that 3D amino acid sequences contribute to the overall
specificity and proteolytic activity of the 3CD proteinase. To define
specific amino acid residues in 3D that contribute to 3CD function, we
sub-cloned site-specific lesions coding for amino acid changes
3D-R136A/D137A (AL10), 3D-E226A/E227A/Y264C (AL14), 3D-K127A, and
3D-N424H into the pET15b-3CDµ10 expression vector. Recombinant 3CD
proteins containing these mutations were expressed in E. coli, purified, and used (along with wild type 3C and 3CD
proteinases) in protein processing assays. For these experiments (shown
in Fig. 5 and Fig. 6), we used a kinetic
analysis at fixed enzyme concentrations (100 nM) since we
anticipated that this more sensitive analysis might be required to
detect less dramatic differences in cleavage activity between wild type
and mutant 3CD polypeptides compared with differences previously
observed between 3C and 3CD. Shown in Fig. 5A are the
results of time course cleavage assays of the P1 polyprotein precursor.
For this experiment, in vitro synthesized P1 substrate was
incubated in the absence (lanes 3 and 4) or
presence of 100 nM 3C (lanes 5-10) or 3CD
(lanes 11-16), and aliquots were removed at the indicated
times. Our data demonstrate that 3CD is able to process the P1
precursor much more rapidly and efficiently than is 3C, confirming the
differences detected in our enzyme dilution assays shown in Fig. 2. P1
cleavage products appeared as early as 15 min when substrate was
incubated with 3CD, whereas comparable levels of processing were not
achieved by 3C until 8 h. Processing of P1 by 3C was also less
efficient than processing of P1 by 3CD as indicated by the accumulation
of the 1ABC intermediate cleavage product in the reactions incubated
with 3C (compare lanes 10, 11, and 16).
Fig. 5B shows the results of a kinetic analysis of P1
processing (identical to that described above) in the presence of 100 nM 3CD-3DK127A (lanes 3-8), 3CD-AL10
(lanes 9-14), 3CD-AL14 (lanes 15-20), or
3CD-3DN424H (lanes 21-26). Three of these mutated 3CD proteinases, K127A, AL10, and N424H, are able to process the P1 precursor at rates similar to that of wild type 3CD (compare
lanes 3-8, 9-14, and 21-26 in Fig.
5B to lanes 11-16 in Fig. 5A). One mutant, AL14, is impaired in its ability to process the P1 precursor (relative to wild type 3CD and the other three mutated proteinases) as
indicated by the lag in the production of VP1 and VP3 (see lanes
15-17 in Fig. 5B) and the increased relative amounts
of 1ABC. These results are represented graphically in Fig.
5C.
Based on our observation that 3CD was able to perform
trans-cleavage of a purified 3CD substrate more effectively
than was 3C on the same substrate, we examined whether this
differential cleavage activity extended to other sites in the P3
non-structural precursor. As seen in Fig. 1, the P3 polyprotein is
thought to be a direct precursor to 3CD as well as a precursor to other
non-structural polypeptides (e.g. 3AB and 3D) involved in
poliovirus RNA replication. We performed a kinetic analysis of
proteolytic cleavage using an in vitro synthesized P3
substrate (containing a proteolytically inactive 3C domain (27)) and
100 nM concentrations of different 3C-containing enzymes.
Results of this analysis show that 3C and 3CD proteinases display
differential proteolytic activity toward multiple cleavage sites in the
P3 non-structural precursor (Fig. 6A). For the primary cleavage
of the P3 precursor, 3C-mediated processing may occur at three sites in
the polyprotein: (i) the 3A/3B junction producing 3A and 3BCD, (ii) the
3B/3C junction producing 3AB and 3CD, and (iii) the 3C/3D junction
producing 3ABC and 3D (refer to Fig. 1). Processing of the primary
cleavage products or multiple processing events within the P3 precursor could also occur, producing any of the mature cleavage products 3A, 3B,
3C, or 3D. The appearance of any of these products would depend on the
efficiency of cleavage at the respective sites within the polyprotein
and on the stability of the cleavage product. Results of the time
course processing assay show that incubation of P3 with either
exogenous His-3C or His-3CD results in the production of 3CD, 3D, and
3AB (Fig. 6A, lanes 7-10 and 11-16,
respectively). The cleavage products 3A (lane 16), 3C
(lanes 14-16), and 3ABC (lanes 13-16) are also
seen at later times when P3 is incubated with 3CD. As documented above
for the data shown in Fig. 4, 3CD appears to be more effective than 3C
at processing the 3C/3D junction with comparable levels of 3D appearing
at 8 h for 3C (lane 10) and between 1 and 2 h for
3CD (lanes 13 and 14). It also appears that 3CD
is more effective at processing the 3B/3C junction than is 3C (compare
the levels of 3CD and 3AB in lanes 7-9 with those in
lanes 12-16), although this difference is not as pronounced as that for the 3C/3D junction. One potential P3 product that was not
observed in this analysis is protein 3BCD (which would have an
electrophoretic mobility between that of P3 and 3CD). Previous studies
using in vitro translation of a P3 precursor containing wild
type 3C sequences indicate that 3BCD is rapidly produced during the
course of translation and that cleavage at the 3A/3B junction may occur
in cis (28).3 These findings, combined with our
results, indicate that cleavage of the P3 precursor at the 3A/3B
junction may only occur in cis. Lack of the 3BCD product
also indicates that the mature 3A protein (Fig. 6A, lane 16)
results from proteolysis of a precursor other than P3 (i.e.
3AB).
Results of time course processing assays (identical to those described
above) with P3 precursor polypeptides as substrates and mutated 3CD
proteinases are shown in Fig. 6, B and C. One of
the mutated 3CD proteins studied, 3CD-N424H (Fig. 6C, lanes 3-8), displays P3 processing activities that are similar to those of the wild type 3CD proteinase (compare lanes 11-16 in
Fig. 6A with lanes 3-8 in Fig. 6C).
Another mutant, 3CD-AL10 (Fig. 6B, lanes 9-14), appears to
process the 3B/3C junction within the P3 precursor with an efficiency
similar to that of wild type 3CD, as indicated by the appearance of 3CD
and 3AB cleavage products at 0.5 and 1 h, respectively. These
times of appearance are similar to the appearance of 3CD and 3AB seen
in wild type 3CD processing of P3 (compare Fig. 6B, lanes
9-14, with Fig. 6A, lanes 11-16). However, the
AL10-mutated proteinase appears to be impaired in cleavage activity at
the 3C/3D junction since both the 3D and 3C cleavage products are
delayed in appearance in the reaction incubated with 3CD-AL10 relative
to the reaction incubated with wild type 3CD proteinase (compare Fig.
6B, lanes 9-14, with Fig. 6A, lanes 12-16).
Both of the other two mutated 3CD proteinases, 3CD-3DK127A (Fig.
6B, lanes 3-8) and 3CD-AL14 (Fig. 6C,
lanes 9-14), showed a decreased cleavage efficiency of the
P3 substrate relative to cleavage of this substrate by wild type 3CD
proteinase. The 3CD-AL14 proteinase processes the P3 precursor with an
efficiency nearly identical to that of the 3C proteinase (compare
lanes 9-14 in Fig. 6C with lanes
5-10 in Fig. 6A). The 3CD-3DK127A proteinase is
severely impaired in its ability to process the P3 precursor relative
to the wild type 3CD proteinase activity on P3 substrates, with only
the 3CD and 3AB cleavage products being produced at late times during
the incubation (Fig. 6B, lanes 3-8). The data from Fig. 6,
A RNA Mobility Shift Assays Using 3C and 3CD Proteinases--
The
ribonucleoprotein (RNP) complex formed by poliovirus 3CD proteinase and
the 5'-terminal RNA sequences of the viral genome (the 5' cloverleaf)
was first reported by Andino et al. (10, 30). These authors
concluded that poliovirus 3CD bound to the first 100 nucleotides of
viral RNA in conjunction with an ~36-kDa cellular protein to form an
RNP complex that is essential for replication of genomic RNA. Further
characterization of the nature of the 3CD-RNP complex has suggested
that although RNA-binding motifs may be contained within the amino acid
sequence of 3C, 3D amino acid domains are necessary for the efficient
formation of the 3CD-RNP complex (10, 15). These data suggested that 3D
residues in 3CD contribute to the RNA binding affinity of the proteinase. To define protein domains in 3D that may contribute to
3CD-RNA interactions, we have used our mutated 3CD polyproteins in RNA
mobility shift analysis with radiolabeled RNA transcripts representing
the first 108 nucleotides of the poliovirus genome. For this study, 0.1 nM radiolabeled RNA probe was preincubated for 10 min at
30 °C with 200 nM purified recombinant poly(rC)-binding protein 2 (PCBP2), a cellular protein that we have previously identified as a binding partner for 3CD in the formation of the ternary
RNP complex (11). Following preincubation, increasing amounts of
purified recombinant 3C or 3CD proteins were added, and incubation was
continued for 5 min at 30 °C. The resulting complexes were resolved
on non-denaturing polyacrylamide gels. Fig.
7 shows the results of incubating
32P-labeled RNA and PCBP2 with increasing amounts of each
of the purified recombinant proteinases. As seen in Fig. 7, both wild type 3C (Fig. 7A, lanes 3-8) and 3CD (Fig. 7A, lanes
9-14) are able to interact with the PCBP2-RNA complex to form a
ternary RNP complex. Formation of the ternary complex appears to
require more 3C than 3CD (compare the relative amounts of free probe
and ternary complex in lanes 3-8 to those in lanes
9-14); however, complex formation by 3C does not appear to be as
inefficient, relative to 3CD, as previously assumed (10, 15).
Incubation of 3CD with PCBP2 and RNA results in the formation of two
RNP complexes with distinguishable electrophoretic mobilities (labeled 3CD/PCBP2/RNA I and II on the right-hand side of
A and B). These two isoforms of the RNP complex
have been observed in previous studies (31); however, the nature of
these two electrophoretically distinguishable isoforms has not been
determined.
Analysis of mutated 3CD polyproteins reveals that the 3CD-3DN424H
mutation has little or no effect on 3CD RNA-binding properties (Fig.
7A, lanes 15-20). All other mutations in 3D sequences
studied show some degree of impaired ternary complex formation relative to that of wild type 3CD. Mutated protein 3CD-AL10 (Fig. 7B,
lanes 9-14) is the least affected in complex formation and is
still able to form the ternary RNP complex at concentrations nearly equal to those of wild type 3CD (compare lanes 9-14 in Fig.
7B to lanes 9-14 in Fig. 7A). Mutated
protein 3CD-AL14 (Fig. 7B, lanes 15-20) is less efficient
in RNP complex formation than is 3CD-AL10; however, 3CD-AL14 is able to
form the 3CD-PCBP2 ternary RNP complex at the highest concentrations of
protein tested (lanes 19 and 20). A single amino
acid substitution of 3D Lys-127 to Ala in 3CD (3CD-3DK127A) results in
a mutated 3CD polyprotein that is severely impaired in its ability to
form the ternary RNP complex (Fig. 7B, lanes 3-8).
In this study, we have used purified recombinant poliovirus 3C and
3CD proteinases in enzyme dilution and time course protein processing
experiments to assess the relative contribution of the 3D domain to the
proteolytic activities of 3CD. Our results with wild type 3C and 3CD
proteinases show that 3CD is able to process the structural polyprotein
precursor more rapidly and efficiently than is 3C. 3CD is also able to
perform trans-cleavage of other 3CD molecules more
efficiently than is 3C, and it processes sites within the P3 precursor
more rapidly. However, 3C and 3CD appear to be equally efficient in the
processing of a non-structural polyprotein precursor, 2C3AB. Moreover,
cleavage of the non-structural precursors 2C3AB and P3 by 3C and 3CD
appears to be less efficient than the processing of the structural
precursor by 3CD.
We examined four mutated 3CD polypeptides containing site-directed
lesions in 3D amino acid sequences in time course processing and RNA
binding assays. Our rationale for examining these particular mutations
for possible defects in specific 3CD functions is based on the
following observations. (i) Analysis of virus and recombinant poliovirus 3D RNA polymerase containing the N424H mutation revealed that this mutation causes a defect in the synthesis of positive strand
RNA unrelated to the RNA elongation activity of 3D polymerase and viral
protein processing (18). This suggested a defect in the initiation of
viral RNA synthesis. We therefore hypothesized that the N424H mutation
may affect the ability of 3CD to interact with 5' poliovirus RNA
sequences. (ii) Mutant virus containing the AL14 mutation in 3D has
been shown to display a severe defect in viral yield and RNA
accumulation; however, this mutation also confers a defect in
polyprotein processing, suggesting that these residues might function
in 3CD proteinase activity (19). (iii) The individual 3D-K127A mutation
had not been previously characterized; however, a basic residue (Lys or
Arg) at 3D amino acid position 127 is conserved throughout the
picornavirus family. This residue is centered in a highly conserved
basic domain (126-KKRDI-130) found within the flexible fingers domain
of all enterovirus and rhinovirus 3D polymerases
(33),4 indicating that it may
be exposed on the surface of the molecule and could play a role in
RNA-protein or protein-protein interactions. Transfections of HeLa cell
monolayers with full-length poliovirus cDNA constructs coding for
3D-K127A in combination with mutations coding 3D-K125A and 3D-K126A, or
3D-K127A in combination with mutations encoding 3D-R128A and 3D-D129A
did not yield virus, suggesting that in the presence of these other
mutations, the K127A mutation is lethal for viral replication (19).
(iv) Mutant virus containing the AL10 mutation in 3D has been shown to
display a severe temperature-sensitive defect in both viral yield and RNA accumulation while displaying wild type protein processing and
translation levels at the non-permissive temperature (19).
One of the mutated proteinases studied, 3CD-3DN424H, displayed
activities similar to those of wild type 3CD in protein processing and
RNA binding. These results indicate that the defect in the initiation
of RNA synthesis previously observed for virus containing this mutation
is not the result of a defect in the RNA binding or protein processing
activities of 3CD. The location of residue Asn-424 in the reported
structure of poliovirus 3D RNA polymerase (33) is at the base of the L
Another mutated proteinase examined in this study, 3CD-AL14, was
deficient in all aspects of 3CD activity, although not as deficient as
3Cpro in processing the P1 capsid precursor. These results
make it difficult to assign a specific function for 3D residues
Glu-226, Glu-227, and Tyr-264 in 3CD proteinase or RNA binding
activities and suggest that mutating these residues may affect 3CD or
3D function through structural disruption of the proteins.
The other two mutated 3CD proteinases, 3CD-3DK127A and 3CD-AL10, showed
differential abilities in protein processing relative to wild type 3CD
proteinase. Both were able to process the P1 precursor at levels
comparable to those of wild type 3CD; however, they displayed decreased
abilities to trans-process the P3 precursor. Whereas it is
possible that the cleavage of the 3B/3C junction in P3 occurs in
cis, the relative half-life of 3CD in the infected cell and
the distal positioning of the 3C amino terminus relative to the
proteinase active site (16) suggest that cleavage of the 3C/3D junction
occurs in trans (7). The impaired ability of these mutated
proteins to process the 3C/3D junction indicates that 3D residues
Lys-127, Glu-226, and Glu-227 may be important determinants for
facilitating the recognition of the P3 precursor substrates by 3CD
proteinase. The AL10 and 3D-Lys-127 mutations involve amino acid
residues contained entirely within a structurally unresolved portion of
the "fingers" domain of 3Dpol. The lack of a defined
structure for this region of 3D suggests that it is disordered in the
3D crystal and may exhibit some degree of flexibility within the
molecule (33). This possibility, along with the nature of the charge of
these residues, makes them likely candidates for facilitating
protein-protein or RNA-protein interactions. RNA binding analysis of
the K127A mutant 3CD protein revealed a severe defect in its ability to
form an RNP complex with viral RNA sequences, since efficient complex
formation was not observed even at the highest concentration of protein
tested. These results suggest that the conserved 3D-KKRDI (amino acid
residues 126-130) may comprise a structurally important RNA binding
domain. Studies on the effects of the K127A mutation on 3D RNA
polymerase function are ongoing.
The data presented in this study demonstrate that amino acid residues
in the 3D portion of poliovirus 3CD form functionally distinguishable
domains that contribute to the differential activities of the 3CD
polyprotein. Wild type 3C proteinase and two of the mutated 3CD
proteinases examined, 3CD-AL10 and 3CD-3DK127A, displayed altered
abilities to process sites within the non-structural P3 precursor and
to form a ternary RNP complex with viral RNA sequences relative to
those of wild type 3CD. These findings show that 3D amino acid
sequences contribute to the proteolytic processing of both the
structural P1 precursor and the non-structural P3 precursor. The
observed ability of the 3CD-AL10 and 3CD-3DK127A proteinases to process
the P1 precursor as efficiently as wild type 3CD also suggests that
substrate recognition determinants in 3D required for processing of P1
may be different from those required for the processing of P3
precursors and for RNP complex formation. Collectively, our results
underscore the multi-functional nature of picornavirus non-structural
proteins involved in proteolytic cleavage and viral RNA replication.
This functional overlap, necessitated by the limited coding capacity of
picornavirus genomes, contributes to the exquisite specificity of
proteins like 3CD for viral polypeptide and RNA substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(5-GGTGTCCTTATCAATATGTG G-3').
(5'-TTGTTCAAGATGTCTCTCGCCTTCTTTCCCATTGC-3').
Mutations in all expression plasmids were verified by
dideoxynucleotide sequencing.
-D-galactopyranoside in
the presence of [35S]methionine overnight at 26 °C.
Induced cells were pelleted, resuspended in 30 ml of buffer A
containing 2 mg/ml lysozyme, and incubated on ice for 1 h.
Histidine-tagged poliovirus [35S]methionine-labeled
3CD(C147A) was purified from the insoluble fraction of the induced
bacterial lysate using nickel affinity chromatography (11).
-D-galactopyranoside-induced BL21(DE3)
cells transformed with pET15b-3CD. Transformed cells were grown at
37 °C until they reached an A600 of ~0.5 at
which time isopropyl-1-thio-
-D-galactopyranoside was
added to a final concentration of 0.5 mM. Incubation was
continued overnight at 26 °C. Cells were then pelleted, resuspended
in 30 ml of I30 buffer (20 mM Tris, pH 7.0, 0.5 M NaCl, 30 mM imidazole, 10% glycerol), lysed
in a French pressure cell, and then subjected to sonication (2 bursts
for 10 s). These bacterial lysates were subjected to centrifugation at 15,000 rpm for 20 min in a JA20 rotor, and the soluble protein fraction was loaded onto a 1-ml Hi-trap metal chelating
column charged with nickel. The column was washed with I60
buffer (20 mM Tris, pH 7.0, 0.5 M NaCl, 60 mM imidazole, 10% glycerol), and recombinant
histidine-tagged 3C (His-3C) was eluted with I200 buffer
(20 mM Tris, pH 7.0, 0.5 M NaCl, 200 mM imidazole, 10% glycerol). To remove the histidine tag,
purified His-3C was dialyzed overnight in the presence of 50 units of
thrombin (Sigma) at 4 °C against 1l of HD buffer (20 mM
HEPES, pH 7.4, 1 mM DTT). Treatment with thrombin resulted
in a molecular weight change consistent with the removal of the
histidine tag and generation of the authentic 3C amino terminus.
Dialyzed 3C was then subjected to anion exchange chromatography on an
Amersham Pharmacia Biotech Mono-Q column using a constant flow of HD
buffer. Purified 3C was recovered in the flow-through fraction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Proteolytic processing map of
poliovirus. Diagrammatic representation of the ~7.5- kilobase
pair poliovirus genome and the viral gene products. The
dark triangles represent 3Cpro- or
3CDpro-mediated processing events at Q-G peptide bonds
within the viral polyprotein. The diamonds represent
2Apro-mediated processing events at two Y-G peptide bonds
within the polyprotein. The star represents a virion
maturation event, which occurs through an undefined mechanism.
View larger version (59K):
[in a new window]
Fig. 2.
In vitro processing of P1
precursor by purified 3CD, His-3C, and 3C proteinases.
A, for cleavage analysis, ~25 fmol (in 1 µl) of P1
precursor protein (translated in vitro in the presence of
[35S]methionine) and 24 µg of protein from a HeLa S10
extract were incubated for 2 h at 30 °C in the absence
(lane 2) or presence of decreasing amounts of each
proteinase (lanes 3-17, enzyme concentrations of each
proteinase (in µM) are indicated above each
lane) in a total reaction volume of 10 µl. Cleavage products were
resolved on an SDS-12.5% polyacrylamide gel. Lanes 3-7,
incubation of P1 with histidine-tagged 3CD; lanes 8-12,
incubation of P1 with histidine-tagged 3C; lanes 13-17,
incubation of P1 with 3C. Lane 1 shows poliovirus marker
proteins. B, graphical representation of P1 processing
results. Note that the enzyme concentrations (x axis) are
shown on a log scale. Percentages of cleavage products were determined
by scanning the autoradiograph of A with a Hewlett-Packard
Desk Scan. Amounts of P1 and VP1 were calculated as the intensity of
the scanned band multiplied by the area of the region scanned (I*A)
using Sigma plot software. The amounts of P1 and VP1 were then
normalized for methionine content by dividing the I*A values by the
total number of methionines in each molecule. These normalized values
were then used to calculate the percentage of VP1 using the formula % VP1 = VP1/(P1 + VP1) × 100%. To calculate the production of VP3
as a function of enzyme concentration normalized I/A values (calculated
as above) for P1, 1ABC, and VP3 were used. Percent of VP3 was then
calculated using the formula % VP3 = VP3/(P1 + 1ABC + VP3) × 100%. 3CD ( ), His-3C (
), 3C (
).
View larger version (33K):
[in a new window]
Fig. 3.
In vitro processing of 2C3AB
precursor by purified 3CD, His-3C, and 3C proteinases.
A, for cleavage analysis, ~20 fmol (in 1 µl) of 2C3AB
precursor protein (translated in vitro in the presence of
[35S]methionine) and 24 µg of protein from a HeLa S10
extract were incubated for 2 h at 30 °C in the absence
(lane 2) or presence of decreasing amounts of each
proteinase (lanes 3-17, enzyme concentrations of each
proteinase (in µM) are indicated above each
lane) in a total reaction volume of 10 µl. Cleavage products were
resolved on an SDS-12.5% polyacrylamide gel. Lanes 3-7,
incubation of 2C3AB with histidine-tagged 3CD; lanes 8-12,
incubation of 2C3AB with histidine-tagged 3C; lanes 13-17,
incubation of 2C3AB with 3C. Lane 1 shows poliovirus marker
proteins. B, graphical representation of 2C3AB processing
results. Note that the enzyme concentrations (x axis) are shown on a
log scale. Percent of 2C was calculated from the normalized I*A values
of 2C3AB and 2C using the formula % 2C = 2C/(2C3AB + 2C) × 100%. 3CD ( ), His-3C (
), 3C (
).
View larger version (27K):
[in a new window]
Fig. 4.
trans-Cleavage of 3CD by purified
3CD and 3C proteinases. A, for
trans-cleavage analysis, 10 pmol of radiolabeled 3CD(C147A)
substrate was incubated for 2 h at 30 °C in the absence
(lane 2) or presence of decreasing amounts of purified
3CDpro (3500 to 35 nM, lanes 3-7)
or His-3Cpro (3500 to 35 nM, lanes
8-12) in a total reaction volume of 10 µl. Following
incubation, Laemmli sample buffer was added to each reaction mixture;
the mixtures were boiled, and the cleavage products were resolved on a
SDS-12.5% polyacrylamide gel. Lane 1 shows poliovirus
marker proteins. Lane 2 shows 3CD(C147A) incubated in buffer
alone. B, graphical representation of the production of 3D
as a function of enzyme concentration. Note that the enzyme
concentrations (x axis) are shown on a log scale. Percentage
of 3D was calculated from normalized I*A values of 3CD and 3D using the
formula 3D/(3CD + 3D) × 100%. 3CD ( ), His-3C (
).
View larger version (43K):
[in a new window]
Fig. 5.
Time course kinetics of P1 processing by
3Cpro, 3CDpro, and mutated 3CD
proteinases. A, in vitro translated P1 was
incubated at 30 °C in the absence (lanes 3 and
4) or presence of 100 nM purified 3C
(lanes 5-10) or 3CD (lanes 11-16). Equal
portions from each reaction were removed at the indicated times,
boiled, and subjected to electrophoresis on a SDS-12.5% polyacrylamide
gel. Lane 1 shows the products resulting from translation of
viral RNA in HeLa cellular extract. Lane 2 shows the
translation mixture incubated in the absence of any exogenous RNA. The
mobilities of the P1 substrate and the resulting proteolytic products,
VP0, 1ABC (uncleaved VP0-VP3), VP1, and VP3 are indicated on the
right. B, in vitro translated P1 was
incubated at 30 °C in the absence (lane 2) or presence of
100 nM purified 3CD-3DK127A (lanes 3-8),
3CD-AL10 (lanes 9-14), 3CD-AL14 (lanes
15-20), or 3CD-3DN424H (lanes 21-26). Equal portions
from each reaction were removed at the indicated times, boiled, and
subjected to electrophoresis on an SDS-12.5% polyacrylamide gel.
Lane 1 shows the products resulting from translation of
viral RNA in HeLa cellular extract. The mobilities of the P1 substrate
and the resulting proteolytic products, VP0, 1ABC (uncleaved VP0-VP3),
VP1, and VP3, are indicated on the sides of the gel. C,
graphical representation of P1 processing results using wild type 3C
( ) and 3CD (
) proteinases and mutated 3CD proteins 3CD-AL10
(
), 3CD-AL14 (
), 3CD-3DN424H (
), and 3CD-3DK127A (
) in
parallel P1 processing reactions. % VP1 and % VP3 were calculated as
described for the enzyme dilution assays of P1 cleavage.
View larger version (53K):
[in a new window]
Fig. 6.
Kinetics of P3 processing by 3C and 3CD.
A, in vitro translated P3 was incubated at 30 °C in the
absence (lanes 3 and 4) or presence of 100 nM purified 3C (lanes 5-10) or 3CD (lanes
11-16). Equal portions from each reaction were removed at the
indicated times, boiled, and subjected to electrophoresis on a
SDS-10 18% polyacrylamide gradient gel. Lane 1 shows translation of viral RNA in HeLa cellular extract. Lane
2 shows HeLa cell extract incubated in the absence of exogenous
RNA. The mobilities of the P3 substrate and the resulting proteolytic
products are indicated on the right-hand side of the figure.
B, P3 processing by mutated 3CD proteins 3CD-3DK127A and
3CD-AL10. In vitro translated P3 was incubated at 30 °C
in the absence (lanes 2) or presence of 100 nM
purified mutated 3CD proteins (lanes 3-20). Equal portions
from each reaction were removed at the indicated times, boiled, and
subjected to electrophoresis on an SDS-10
18%
polyacrylamide gradient gel. Lane 1 shows the products
resulting from translation of viral RNA in HeLa cellular extract. The
mobilities of the P3 substrate and the resulting proteolytic products
are indicated on the right. C, P3 processing by
3CD-3DN424H and 3CD-AL14. In vitro translated P3 was
incubated at 30 °C in the absence (lane 2) or presence of
100 nM purified mutated 3CD proteins (lanes
3-20). Equal portions from each reaction were removed at the
indicated times, boiled, and subjected to electrophoresis on an
SDS-10-18% polyacrylamide gradient gel. Lane 1 shows the
products resulting from translation of viral RNA in HeLa cellular
extract. The mobilities of the P3 substrate and the resulting
proteolytic products are indicated on the right.
D, graphical representation of P3 processing results using
wild type 3C (
) and 3CD (
) proteinases and mutated 3CD proteins
3CD-AL10 (×), 3CD-AL14 (*), 3CD-3DN424H (
), and 3CD-3DK127A (
).
The values for "% P3 products" were calculated from normalized I*A
values of P3, 3CD, 3D, 3ABC, 3C, and 3AB using the formula (3CD + 3D + 3ABC + 3C + 3AB)/(P3 + 3CD + 3D + 3ABC + 3C + 3AB) × 100%.
C, are summarized graphically in Fig. 6D.
View larger version (64K):
[in a new window]
Fig. 7.
RNA mobility shift analysis of 3C, 3CD, and
mutated 3CD proteins. Increasing molar amounts of purified
recombinant 3C and 3CD proteins were incubated with 0.1 nM
radiolabeled RNA probe representing the first 108 nucleotides of the
poliovirus genome in the presence of 200 nM recombinant
PCBP2. A, mobility shift analysis with 50-600
nM 3C (lanes 3-8), 3CD (lanes
9-14), and 3CD-3DN424H (lanes 15-20). B,
mobility shift analysis with 50-600 nM 3CD-3DK127A
(lanes 3-8), 3CD-AL10 (lanes 9-14), and
3CD-AL14 (lanes 15-20). Lane 1 in both panels
shows probe incubated in buffer alone. Lane 2 in both panels
is probe incubated with 200 nM recombinant PCBP2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix in the thumb domain of the molecule, proximal to two other
residues, Met-394 and Val-391, which have been shown to give rise to
conditional polymerase mutants in 3D proteins containing single site
substitutions of M394T and V391L (34, 35). Characterization of the
M394T mutant using an in vitro replication assay revealed
that this mutation confers a temperature-sensitive defect in the
initiation of negative strand RNA synthesis (34). The conditional RNA
synthesis mutant 3D-V391L was initially identified by Hope et
al. (35) based on its inability to interact with poliovirus 3AB
protein in a two-hybrid screen. Poliovirus 3AB contains the amino acid
sequences of a small basic protein, termed VPg (also called 3B), which
is covalently linked to the 5' terminus of all virion RNAs and newly
synthesized viral RNAs in infected cells. It has been hypothesized that
3AB serves as a donor of 3B to the RNA replication complex of
poliovirus and that the 3B portion of the molecule is utilized as a
primer for the initiation of RNA synthesis (9, 32). Based on the location of residues Val-391, Met-394, and Asn-424 in the structure of
3D and the conditional phenotypes exhibited by polymerase molecules harboring single site mutations at these positions, it is possible that
residue Asn-424 functions in 3D-3AB interactions necessary for
initiation of RNA synthesis. This function appears to be independent of
the ability of 3CD to process P1 or P3 structural precursors or to form
a ternary RNP complex with the 5'-terminal poliovirus RNA sequences and
PCBP2.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Yolanda Bell, Stacey Stewart, and Ellie Ehrenfeld for critical review of the manuscript. We are indebted to Karla Kirkegaard for the gift of plasmids AL10 and AL14; Ellie Ehrenfeld and Ollie Richards for the gift of plasmid pEXC-3CD-3DN424H; Louis Leong for the gift of plasmid pTM1-P1; and Jonathan Towner for the gift of plasmid pTM1-2C3AB.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Public Health Service Grant AI 22693 from the National Institutes of Health and by services provided by the IMAGE facility of the School of Biological Sciences, University of California, Irvine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Predoctoral trainee supported by Public Health Service Training
Grant GM 07311. Present address: Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD
20742-3351.
§ To whom correspondence should be sent. Fax: 949-824-8598; E-mail: blsemler{at}uci.edu.
2 L. E.-C. Leong and B. L. Semler, unpublished observations.
3 J. S. Towner and B. L. Semler, unpublished results.
4 Anne Palmenberg, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: 3Cpro, 3C proteinase; RNP, ribonucleoprotein; PCBP2, poly(rC)-binding protein 2; bp, base pair; DTT, dithiothreitol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|