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J. Biol. Chem., Vol. 277, Issue 35, 31551-31562, August 30, 2002
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From the
Received for publication, May 6, 2002, and in revised form, June 13, 2002
The primary oligomerization domain of
poliovirus polymerase, 3Dpol, is stabilized by the interaction of
the back of the thumb subdomain of one molecule with the back of the
palm subdomain of a second molecule, thus permitting the head-to-tail
assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm
is key to the stability of this interface. We show that mutations
predicted to completely disrupt this interface do not produce
equivalent growth phenotypes. Virus encoding a polymerase with changes
of both residues of the thumb to alanine is not viable; however, virus
encoding a polymerase with changes of all three residues of the palm to
alanine is viable. Biochemical analysis of 3Dpol derivatives containing
the thumb or palm substitutions revealed that these derivatives are
both incapable of forming long fibers, suggesting that polymerase
fibers are not essential for virus viability. The RNA binding activity,
polymerase activity, and thermal stability of these derivatives were
equivalent to that of the wild-type enzyme. The two significant
differences observed for the thumb mutant were a modest reduction in
the ability of the altered 3CD proteinase to process the VP0/VP3 capsid
precursor and a substantial reduction in the ability of the altered
3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this
reaction. Because 3C alone can substitute for 3CD in this reaction, we
conclude that the lethal replication phenotype associated with the
thumb mutant is caused, in part, by the disruption of an interaction
between the back of the thumb of 3Dpol and some undefined domain of 3C.
We speculate that this interaction may also be critical for
assembly of other complexes required for poliovirus genome replication.
The RNA-dependent RNA polymerase
(RdRP)1 is the key component
of the replication machinery of RNA viruses. The RdRP from
poliovirus (3Dpol) serves as a paradigm for this class of nucleic acid
polymerases. The crystal structure for 3Dpol (1) revealed that this
polymerase has the typical topology observed for other nucleic acid
polymerases and can be compared with a cupped, right hand with fingers,
palm, and thumb subdomains (Fig. 1A). A unique feature of
this polymerase, however, is the presence of two extensive regions of
polymerase-polymerase interactions, referred to as interface I and
interface II (1). Polymerase molecules interact in a "head-to-tail"
fashion to form long, extended fibers via interface I, and these fibers
interact with each other via interface II. Interface I is formed by an interaction between the back of the thumb of one polymerase molecule and the back of the palm of a second polymerase molecule (Fig. 1B). A few of the critical interactions required for
integrity of interface I are shown in Fig. 1C. Specifically,
Arg-455 and Arg-456 of the thumb subdomain of one polymerase molecule
interact with Asp-339, Ser-341, and Asp-349 of the palm subdomain of
the second polymerase molecule (Fig. 1C).
A variety of biochemical studies have provided additional evidence for
oligomerization of 3Dpol. For example, filter-binding studies,
performed in a manner that separates oligomers greater than 100 molecules from smaller assemblies, demonstrate a
concentration-dependent increase in formation of large
3Dpol oligomers at low pH values (2-4). In addition, glutaraldehyde
cross-linking studies provide evidence for the existence of 3Dpol
multimers (2). Finally, 3Dpol exhibits cooperativity with respect to
polymerase activity (2). However, it should be noted that the observed
cooperative nature of polymerase activity is not observed in all cases
(5) and may either be substrate-dependent (5) or a
reflection of the absence or presence of certain divalent cations, in
particular Zn2+ ions (4). It is worth noting that the
ability of 3Dpol molecules to interact has also been demonstrated by
using the yeast two-hybrid system (6, 7).
Clustered charged-to-alanine mutagenesis of 3Dpol identified residues
Arg-455 and Arg-456 as residues essential for virus viability (8).
Given the role of these residues in the stability of interface I (Fig.
1C) (1), these data provided the first evidence that
oligomerization of 3Dpol might play a significant role in some step of
the virus multiplication cycle. Subsequent studies of additional 3Dpol
derivatives with modifications of residues involved in forming
interface I provided additional support for the importance of this
interface for 3Dpol function and virus viability (4). However, this
study also presented an interesting paradox. Mutation of the residue
that interacts with Arg-455, Asp-349, does not produce a lethal
phenotype (4).
In this study, we test the hypothesis that the lethal phenotype
associated with mutations on the back of the thumb of 3Dpol arises from
a requirement for this subdomain that is independent of
oligomerization. We find that 1) oligomerization via interface I may
not be essential for virus multiplication and 2) the back of the thumb
of 3Dpol may interact with host and viral factors to modulate capsid
protein processing and initiation of protein-primed RNA synthesis,
respectively. The implications of heteromeric interactions between
3Dpol and another viral factor on the mechanism for negative-strand RNA
synthesis will be discussed.
Materials--
DNA oligonucleotides were from Invitrogen and
Integrated DNA Technologies, Inc.; T4 polynucleotide kinase, Deep Vent
DNA polymerase, and restriction enzymes were from New England Biolabs,
Inc.; shrimp alkaline phosphatase was from United States Biochemical
Corp.; T4 DNA ligase and NZCYM were from Invitrogen; QIAEX was from
Qiagen; Sephadex G-25 and RNase A were from Sigma; phosphocellulose
(P-11) and DE-81 filter paper were from Whatman; all nucleotides
(ultrapure solutions) and Q-Sepharose fast flow were from Amersham
Biosciences; RNA oligonucleotides were from Dharmacon Research, Inc.
(Boulder, CO); [ Construction of Expression Plasmids for 3CD and 3C--
Oligos
15 and 16 (Table I lists all
oligonucleotides used in this study) were used to amplify the region
encoding 3CD by using PCR and the viral cDNA (pMoRA, also known as
pXpA-rib+polyAlong (Ref. 9)) as template. The 3CD-coding
region was cloned into pET26Ub-3D plasmid (10) by using the
SacII and AflII sites to give pET26Ub-3CD. To
inactivate the protease activity of 3CD, histidine 40 was changed to a
glycine by using overlap-extension PCR with oligos 15-18 and
pET26Ub-3CD plasmid as template. The product was cloned into
pET26Ub-3CD to yield pET26Ub-3CD-H40G.
Cloning of the 3C gene was achieved by using oligos 15 and 19-21 and
the viral cDNA (pMoRA (Ref. 9)) as template in overlap-extension PCR. The region was cloned into pET26Ub-Chis plasmid by using the
SacII and BamHI sites to yield
pET26Ub-3C-C147G-Chis. "C147G" designates mutation of cysteine 147 to a glycine to inactivate protease activity. The pET26Ub-Chis plasmid
is designed to produce a C-terminal GSSG-His6 tag for any
protein cloned in by using the 3' BamHI site.
The pET26Ub expression plasmid for 3Dpol (pET26Ub-3D-BPKN) was
previously described (10). DNA sequencing at the Pennsylvania State
University Nucleic Acid Facility was used to verify the integrity of
all clones.
Construction of Expression Plasmids for 3Dpol and 3CD
Derivatives--
The thumb mutations of Arg-455 and Arg-456 in 3Dpol
were introduced by using reverse oligonucleotides encoding the Ala
(oligo 8), Ser (oligo 9), or Asp (oligo 10) substitutions and the
forward oligonucleotide (oligo 7). PCR products were cloned into
pET26Ub-3D-BPKN plasmid (10) using the NheI and
EcoRI sites. Palm mutations of Asp-339, Ser-341, and Asp-349
to alanine in 3Dpol were introduced by using overlap extension PCR
using oligos 11-14. The PCR product was cloned into pET26Ub-3D-BPKN
plasmid (10) using the KpnI and EcoRI sites.
The thumb (R455A,R456A) and palm (D339A,S341A,D349A)
mutations were introduced into 3CD by digesting the region containing the mutations in the pET26-Ub-3D-BPKN plasmid and ligating into the
pET26Ub-3CD-H40G plasmid. The BstBI and EcoRI
sites were used to introduce the thumb mutations into 3CD; the
BstBI and MfeI sites were used to introduce the
palm mutations into 3CD. DNA sequencing confirmed the integrity of all clones.
Expression and Purification of 3Dpol, 3CD, and 3C
Derivatives--
All proteins expressed from the pET26Ub-based
plasmids constructed above are N-terminally fused to yeast ubiquitin.
Overexpression of protein in this system is performed in the
BL21(DE3)pCG1 strain of Escherichia coli; this strain
carries the pCG1 plasmid, which constitutively expresses a yeast
ubiquitin protease that processes the ubiquitin fusion protein to
produce the authentic N terminus (10). Expression, lysis,
polyethyleneimine (PEI) precipitation, and ammonium sulfate
precipitation for all 3Dpol and 3CD derivatives were performed as
previously described (10). After suspension of the ammonium sulfate
pellets in buffer A (50 mM Tris, pH 8.0, 20% glycerol, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 60 µM ZnCl2), the samples were dialyzed
overnight against buffer A containing 10 mM NaCl.
Spectra/Por dialysis tubing (Spectrum Laboratories Inc.) with a
molecular mass cut-off of 12,000-14,000 Da was used. All of the steps
of the purification were performed either on ice or at 4 °C. After
dialysis, the conductivity of the protein was adjusted to 50 mM NaCl and the protein was loaded at 1 ml/min onto a
phosphocellulose (P-11) column that was equilibrated with buffer A
containing 50 mM NaCl. Approximately 1 ml of resin was used
per 20 mg of total protein. Protein concentration was measured by using
the Bio-Rad protein assay. The column was washed to baseline with
buffer A containing 50 mM NaCl and protein was eluted by using a six-column volume, linear gradient from 50 to 350 mM NaCl in buffer A. Fractions (0.1 bed volume of the
column) were collected and assayed for purity by SDS-PAGE. Conductivity
of the pooled fractions was adjusted to 50 mM NaCl by using
buffer A and the pooled fractions were loaded at 1 ml/min onto a
Q-Sepharose column equilibrated with buffer A containing 50 mM NaCl. Again, 1 ml of resin was used per 20 mg of total
protein. Washing was the same as for the P-11 column; protein was
eluted using a six-column volume, linear gradient from 50 to 500 mM NaCl in buffer A. Fractions (0.1 bed volume of the
column) were collected and assayed for purity by SDS-PAGE. Conductivity
of the pooled fractions was adjusted to 50 mM NaCl using
buffer A. This pool was loaded at 1 ml/min onto a 0.5-ml Q column
equilibrated as described above. The column was washed to baseline
using buffer B (50 mM HEPES, pH 7.5, 20% glycerol, 10 mM 2-mercaptoethanol, 0.1% Nonidet P-40, and 60 µM ZnCl2) containing 50 mM NaCl.
Protein was eluted from this Q column by using buffer B containing 500 mM NaCl. Fractions (0.5 ml) were collected until the
concentration of the eluted protein fell below the desired value.
Protein concentration was determined by using the following extinction
coefficients: 0.071830 µM
3C was expressed essentially as described for 3Dpol and 3CD (10);
however, a different lysis buffer was employed (50 mM HEPES, pH 7.5, 20% glycerol, 10 mM 2-mercaptoethanol, 5.6 µg/ml pepstatin A, 4 µg/ml leupeptin, 0.1 mM EDTA, and
500 mM NaCl). Phenylmethylsulfonyl fluoride and Nonidet
P-40 were added after lysis to a final concentration of 1 mM and 0.1% (v/v), respectively. PEI was added to a final
concentration of 0.25% (v/v); the lysate was stirred slowly at 4 °C
for 30 min and then centrifuged in a Beckman Ti-60 rotor for 30 min at
30,000 rpm at 4 °C. The conductivity of the PEI supernatant was
adjusted to 50 mM NaCl, and the supernatant was passed
through a Q-Sepharose column followed in tandem by a P-11 column. Both
columns were equilibrated with buffer C (50 mM HEPES,
pH 7.5, 20% glycerol, 10 mM 2-mercaptoethanol, and 0.1% Nonidet P-40) containing 50 mM NaCl prior to loading.
Approximately 1 ml of resin/80 mg of total protein was used for
the Q column, and ~1 ml of resin/500 mg of total protein was used for
the P-11 column. After all of the protein passed through the Q column, the P-11 column was detached and washed with 10 column volumes of
buffer C containing 50 mM NaCl. 3C was eluted from the P-11 column by using buffer C containing 2 M NaCl. Fractions (1 ml) were eluted until the concentration of the eluted protein fell below the desired value. The high NaCl concentration was reduced from
the fractions by dialysis against buffer C containing 250 mM NaCl using Spectra/Por dialysis tubing with a molecular
mass cut-off of 6000-8000 Da. SDS-PAGE was used to assess the purity of the eluted fractions. Protein concentration was determined as
described for 3Dpol and 3CD; the extinction coefficient used was
0.007680 µM Construction of Mutated Viral cDNA Clones and Mutated
Replicons--
Introduction of mutations into the thumb subdomain in
the viral cDNA required overlap extension PCR by using oligos 1-4;
the viral cDNA, pMoRA (9), was used as the template. The PCR
product was digested with MfeI and EcoRI and
subcloned into the intermediate plasmid,
pUC18-BglII-EcoRI-3CD (referred to as pUC-3CD in
Ref. 11). From this subclone, the fragment between BglII and
EcoRI was cloned into the viral cDNA (pMoRA (Ref. 9)) to
yield the thumb mutant viral cDNA (pMoRA-R455A,R456A). For the
introduction of mutations to the palm subdomain, PCR amplification from
the expression vector containing the palm mutations
(pET26Ub-3D-BPKN-D339A,S341A,D349A) was performed using
oligos 5 and 6; the fragment between BstBI and
MfeI was subcloned into the
pUC18-BglII-EcoRI-3CD intermediate and from there
to the viral cDNA using the BglII and EcoRI
sites to yield the palm mutant viral cDNA
(pMoRA-D339A,S341A,D349A). To introduce mutations into the
replicon, the fragment between the BglII and ApaI
sites from the mutated viral cDNAs was cloned into pRLucRA (also
known as pRLuc31-rib+polyAlong (Refs. 9 and 12)) to yield
pRLucRA-R455A,R456A and pRLucRA-D339A,S341A,D349A. DNA sequencing was
used to verify the integrity of all clones.
Infectious Center Assays--
RNA transcripts were generated
according to the instructions of the manufacturer for the T7-MEGAscript
kit (Ambion, Inc.) after linearization with EcoRI. DNase
treatment was used to remove the template; lithium chloride
precipitation was used to remove unincorporated nucleotides. RNA
concentration was calculated by measuring absorbance at 260 nm,
assuming an A260 of 1 was equivalent to 40 µg/ml.
HeLa cells were propagated in DMEM/F-12 (Invitrogen) supplemented with
10% fetal bovine serum (Invitrogen), always keeping the cultures
between 20 and 80% confluence. Subconfluent monolayers of HeLa cells
were detached from the culture flasks by trypsin treatment, washed with
1× phosphate-buffered saline (PBS), and cell number adjusted to 3 × 106 cells/ml in PBS. Cell suspension (400 µl) was
mixed with 10 µg of RNA (wild-type (pMoRA), thumb mutant
(pMoRA-R455A,R456A), or palm mutant (pMoRA-D339A,S341A,D349A) viral
transcripts) in a microcentrifuge tube, transferred to an
electroporation cuvette (0.2-cm gap width; Bio-Rad) and subjected to an
electric pulse at 500 microfarads and 130 V using a Gene Pulser system
(Bio-Rad). Electroporated cells were diluted either 10- or 100-fold in
PBS, and 100 µl of each dilution were plated on 2 × 105 HeLa cells (prepared 1 day in advance) in six-well
dishes; 400 µl of DMEM/F-12 were added to each well. Undiluted
electroporated cells (100 µl) were also plated using the same
procedure. Cells were allowed to adsorb to the plate for 1 h at
37 °C, and then the medium/PBS was aspirated; the cells were covered
with 3 ml of a mixture of 1× DMEM/F-12 plus 10% fetal bovine serum
and 1% low melting point agarose (American Bioanalytical). Plates were then incubated at 37 °C for 3 days. The agarose overlay was removed by using a spatula. Wells were stained with crystal violet, and viral
plaques were counted.
Subgenomic Replicon Assays--
RNA transcripts were generated
as described for the viral genomes from ApaI-linearized
plasmids encoding the wild-type (pRLucRA (Refs. 9 and 12)), the thumb
mutant (pRLucRA-R455A,R456A) or the palm mutant
(pRLucRA-D339A,S341A, D349A) subgenomic replicons. HeLa
cells were propagated as described for the infectious center assays.
HeLa cells were transfected with subgenomic replicons (20 µg) by
using electroporation. Electroporated cells were immediately transferred to prewarmed (37 °C) DMEM/F-12 as follows. The volume of
the electroporated cells added was calculated by multiplying 33 µl by
n + 1 (where n is the number of time points to be
measured). The volume of DMEM/F-12 to which the electroporated cells
were added was calculated by multiplying 500 µl by n + 1 (where n is the number of time points to be measured). After
mixing the appropriate volume of electroporated cells with the
appropriate volume of medium, 500-µl aliquots were prepared in
microcentrifuge tubes for each time point to be measured. These
aliquots were then incubated in a water bath at 37 °C without any
agitation. At fixed time points, cells were pelleted by centrifugation
at 14,000 × g for 2 min in an Eppendorf
microcentrifuge. Lysis was performed by using 100 µl of 1× cell
culture lysis reagent (Promega) and placed on ice for 2 min before
removal of cellular debris and nuclei by centrifugation at 14,000 × g for 1 min. Lysates were left on ice at 4 °C until
all time points were collected. Lysates were assayed for luciferase
activity by mixing 10 µl of lysate with 10 µl of luciferase assay
substrate (Promega) and quantifying light production by using a Lumat
LB 9501 luminometer (Berthold).
HeLa S10 Translation-Replication Reactions (13)--
HeLa cell
S10 extract (S10) and HeLa cell translation initiation factors were
prepared as previously described (14). HeLa S10 translation-replication
reaction mixtures contained 50% by volume S10, 20% by volume
initiation factors, 10% by volume 10× nucleotide reaction mix (10 mM ATP, 2.5 mM GTP, 2.5 mM UTP, 600 mM KCH3CO2, 300 mM
creatine phosphate, 4 mg/ml creatine kinase, and 155 mM
HEPES-KOH, pH 7.4), 2 mM GdnHCl, and viral mRNA at 50 µg/ml.
mRNA Translation (13)--
Poliovirus mRNA translation
was assayed by including [35S]methionine (1.2 mCi/ml;
Amersham Biosciences) in HeLa S10 translation-replication reaction
mixtures. After 3 h of incubation at 34 °C, samples (4 µl) of
the HeLa S10 translation-replication reaction mixtures containing
[35S]methionine were mixed with 100 µl of SDS-PAGE
sample buffer (2% SDS (Sigma), 62.5 mM Tris-HCl, pH 6.8, 0.5% 2-mercaptoethanol, 0.1% bromphenol blue, 20% glycerol). The
samples were heated at 100 °C for 5 min, and 25-µl portions of
each sample were loaded onto a 0.75-mm-thick polyacrylamide gel (29:1,
acrylamide:bisacrylamide) consisting of a 4% stacking gel and a
9-18% gradient separating gel. The gels were fixed and dried;
radiolabeled proteins were detected by phosphorimaging.
Poliovirus RNA Synthesis in Translation-Replication Reactions
(13)--
Poliovirus RNA synthesis was assayed using preinitiation RNA
replication complexes formed in HeLa S10 translation-replication reaction mixtures as previously described (15). Briefly, viral RNAs (50 µg/ml) were incubated in S10 reaction mixtures containing 2 mM GdnHCl for 3 h at 34 °C. Preinitiation
complexes were isolated from the reactions by centrifugation at
13,000 × g for 15 min at 4 °C. Pellets containing
preinitiation complexes were then suspended in 50-µl labeling
reaction mixtures containing [ VPg Uridylylation in Translation-Replication Reactions
(13)--
VPg uridylylation was assayed using preinitiation RNA
replication complexes. Preinitiation RNA replication complexes were isolated from HeLa S10 translation-replication reaction material using
the same procedure as described above for poliovirus RNA synthesis. The
preinitiation RNA replication complexes were suspended in 50 µl of
labeling reaction mixture containing [ Trace Labeling of RNA (rU30)--
The RNA
oligonucleotide was end-labeled by using [ RNA Filter-binding Assays--
Reaction mixtures, typically 50 µl, contained 10 nM trace-labeled rU30 and
wild-type or mutant 3Dpol derivatives of varying concentrations in
reaction buffer (25 mM MES-NaOH, pH 5.5, 60 mM
NaCl, 5 mM MgCl2, 0.1 mM
ZnSO4 5 mM dithiothreitol, 0.25 mM ATP, 20% glycerol). 3Dpol was diluted immediately prior to use in
enzyme dilution buffer (50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 µM ZnCl2
and 20% glycerol). Binding reactions were initiated by the addition of
5 µl of the diluted 3Dpol to the remaining components. Reactions were
incubated on ice for 30 min. This assay employs three filters:
polysulfone membrane, which traps any protein-nucleic acid complexes
larger than 0.45 µm; a nitrocellulose membrane, which traps all other
protein-nucleic acid complexes; and a nylon membrane, which binds all
unbound nucleic acid. Membranes were presoaked in wash buffer (50 mM HEPES, pH 7.5, and 5 mM MgCl2) and assembled, in order from top to bottom, polysulfone,
nitrocellulose, and nylon, in a slot blotter (Invitrogen). After
assembly, 20 µl of wash buffer were applied to each slot and filtered
through all three membranes by applying a vacuum. After washing, 20 µl of each binding reaction were applied to each slot and filtered through the membranes. Membranes were air-dried and visualized by using
a PhosphorImager (Amersham Biosciences) and quantified by using
ImageQuant software (Amersham Biosciences).
Symmetrical Substrate (sym/sub)--
Purification,
5'-32P labeling, and annealing were performed as previously
described (16).
3Dpol Elongation Assays--
Reaction mixtures contained sym/sub
and 3Dpol in reaction buffer (50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 5 mM MgCl2, 60 µM ZnCl2, 500 µM NTP).
Reactions were quenched by addition of EDTA to a final concentration of
50 mM. Specific concentrations of primer/template and
3Dpol, along with any deviations from the above, are indicated in the
appropriate sections below. 3Dpol was diluted immediately prior to use
in enzyme dilution buffer (50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 µM
ZnCl2, and 20% glycerol). The volume of enzyme added to
any reaction was always less than or equal to one-tenth of the total volume.
Product Analysis, Denaturing PAGE--
Reaction products were
analyzed by denaturing PAGE as previously described (16).
Active-site Titration--
Reactions contained 20 µM sym/sub (10 µM duplex), 5 mM
MgCl2, 1 or 2.5 µM 3Dpol, and 500 µM ATP. Reactions were initiated by addition of 3Dpol and
incubated at 30 °C; reactions were quenched at fixed time points by
the addition of EDTA to a final concentration of 50 mM. The
kinetic data best fit to a line. The y intercept represents
the concentration of active 3Dpol.
Dissociation of 3Dpol-sym/sub Complexes--
3Dpol (1 µM) was incubated with stoichiometrically labeled sym/sub
(0.1 µM) for 90 s at 30 °C, at which point trap
(100 µM unlabeled sym/sub) was added to the reaction. At
fixed times after the addition of trap, ATP (500 µM) was
added and the reaction was allowed to proceed for 30 s and then
quenched by addition of EDTA (50 mM). Products were
resolved by electrophoresis on a denaturing, highly cross-linked (1.7%
bisacrylamide), 23% polyacrylamide gel as previously described (16).
The kinetic data best fit to a single exponential.
Inactivation of 3Dpol--
3Dpol (2 µM) was
incubated at 30 °C in 1× reaction buffer (50 mM HEPES,
pH 7.5, 10 mM 2-mercaptoethanol, 60 µM
ZnCl2, and 5 mM MgCl2) and at the
indicated times the reaction was initiated by addition of sym/sub (1 µM) and ATP (500 µM) and allowed to proceed
for an additional 90 s at 30 °C, at which time the reaction was
quenched by addition of EDTA (50 mM). The kinetic data best fit to a single exponential.
Rapid Chemical-quench-flow Experiments--
Rapid
mixing/quenching experiments were performed by using a model RQF-3
chemical-quench-flow apparatus (KinTek Corp., State College, PA).
Experiments were performed at 30 °C by using a circulating water
bath. 3Dpol-sym/sub complex in 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 µM
ZnCl2, and 5 mM MgCl2 was rapidly
mixed with the nucleotide substrate in 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 µM
ZnCl2, and 5 mM MgCl2, and the
reactions were quenched by addition of 0.5 M EDTA to a
final concentration of 0.3 M.
Rate of Nucleotide Incorporation--
3Dpol (2 µM)
was incubated with 2 µM sym/sub (1 µM
duplex) and rapidly mixed with 1000 µM ATP as described
for the rapid chemical-quench-flow experiments. After mixing, reactant
concentrations were reduced by 50%. Reactions were quenched at the
fixed times by addition of 0.5 M EDTA to a final
concentration of 0.3 M. The kinetic data best fit to a
single exponential.
Poly(rU) Polymerase Activity Assays--
Reactions contained
3Dpol (0.5 µM), 1.9 µM dT15 and
0.2 µM poly(rA)400 primer/template in
reaction buffer (50 mM HEPES, pH 7.5, 10 mM
2-mercaptoethanol, 5 mM MgCl2, 60 µM ZnCl2, 500 µM UTP, 0.4 µCi/µl [ Cloning and Transcription of 61-nt oriI--
The region
corresponding to the 61 nucleotides of oriI was amplified from an
expression vector encoding the 2C
gene.3 A BamHI
site and a T7 promoter were included in the forward oligonucleotide (oligo 22) used for the PCR reaction; a BstZ17I site was
included immediately before the EcoRI site in the reverse
oligonucleotide (oligo 23). This PCR product was cloned into a pUC18
vector using BamHI and EcoRI sites; the cloning
was confirmed by sequencing. The pUC18-61nt-oriI plasmid was
linearized using the BstZ17I site and a transcription
reaction was performed according to the instructions of the
manufacturer for the T7-MEGAshortscript kit (Ambion, Inc.). DNase
treatment was used to remove the template; either the QuickClean Enzyme
Removal Resin (CLONTECH) or Micropure-EZ
centrifugal filters (Amicon Bioseparations, Millipore) was used to
remove any protein from the RNA according to the instructions of the
respective manufacturers. A Sephadex G-25 spun column was used to
remove any unincorporated nucleotides. RNA concentration was calculated
by measuring absorbance at 260 nm. The extinction coefficient was
calculated for the 61-nt oriI plus three guanidine residues that are
transcribed at the 5' end of oriI (0.749400 µM VPg Uridylylation Assays--
Reaction mixtures contained 1 µM 3Dpol, 1 µM 3CD, 1 µM
61-nt oriI, and 50 µM VPg in reaction buffer (50 mM HEPES, pH 7.5, 10% glycerol, 5 mM magnesium
acetate, 60 µM ZnCl, 10 mM 2-mercaptoethanol, 10 µM UTP, and 0.004 µM
[ Rationale
If the integrity of interface I (Fig.
1C) is necessary for virus
viability, then disruption of this interface by changing residues on
the back of the thumb subdomain (R455A,R456A) or the palm subdomain
(D349R) should produce virus with equivalent phenotypes. This is not
observed (4). It is possible that changing a single residue on the back
of the palm is not sufficient to disrupt this interface. Therefore, we
engineered a 3Dpol derivative in which aspartic acids 339 and 349 and
serine 341 were all changed to alanines (D339A,S341A,D349A). This
derivative will be referred to as the palm mutant. We have evaluated
this mutant in biological and biochemical assays to determine the
function for interface I in virus multiplication. To compare the data
obtained in this study to that from others, we have reconstructed and
evaluated the R455A,R456A 3Dpol derivative. This derivative will be
referred to as the thumb mutant.
Biological Analysis of Interface I Mutants: Interface I Mutants Do
Not Display Equivalent Phenotypes
To evaluate the viability of virus containing mutations in
interface I, we performed an infectious center assay. HeLa cells were
transfected with wild-type or mutant genomes, and these cells were
plated at various dilutions on a HeLa cell monolayer. After adsorption,
cells were covered with agar media and incubated at 37 °C for 3 days. After removal of the agar, remaining cells were stained with
crystal violet. Without dilution, wild-type and palm mutant virus
spread throughout the entire monolayer (Fig.
2). As expected, thumb mutant virus did
not spread (Fig. 2) (4, 8). By evaluating higher dilutions of the palm
mutant, it becomes clear that this mutant virus is somewhat debilitated
relative to wild-type virus (Fig. 2). The titer of the palm mutant
virus does not appear to be affected because the number of plaques
observed is equivalent to wild-type virus (Fig. 2). However, the
kinetics of multiplication of the palm mutant virus is reduced because the size of the plaques is significantly smaller than observed for
wild-type virus (Fig. 2). These data are consistent with the hypothesis
that the lethal phenotype observed for the thumb mutant virus is
independent of the integrity of interface I.
Structure-Function Relationships of the RNA-dependent
RNA Polymerase from Poliovirus (3Dpol)
A SURFACE OF THE PRIMARY OLIGOMERIZATION DOMAIN FUNCTIONS IN
CAPSID PRECURSOR PROCESSING AND VPg URIDYLYLATION*
§,§,,
,,,
Department of Biochemistry and Molecular
Biology, Pennsylvania State University, University Park, Pennsylvania
16802, the ¶ Department of Microbiology, University of Colorado
Health Sciences Center, Denver, Colorado 80262, and the
** Department of Molecular Genetics and Microbiology,
State University of New York, Stony Brook, New York 11794
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP (6000 Ci/mmol) was from
PerkinElmer Life Sciences; [
-32P]ATP (>7000 Ci/mmol)
was from ICN; synthetic VPg peptide was from Alpha Diagnostic
International (San Antonio, TX); all other reagents were available
through Fisher or VWR.
Oligonucleotides used in this study
1·cm1 (3Dpol) and
0.079510 µM1·cm1 (3CD).
These values were determined by using the protein parameters tool on
the ExPASy site (us.expasy.org/tools/protparam.html). The absorbance
values were measured at 280 nm in 6 M guanidine HCl
(GdnHCl), pH 6.5. The conductivity of the individual fractions was
measured; the fractions were aliquoted and stored at 
80 °C.
1·cm1. The
conductivity of the individual fractions was measured; the fractions
were aliquoted and stored at 80 °C.
-32P]CTP and incubated
at 37 °C for 60 min as previously described (method 4 in Ref. 15).
Under these conditions, radiolabel was incorporated into nascent
negative- and positive-strand RNA as it was synthesized. The reactions
were centrifuged at 13,000 × g to pellet the viral RNA
replication complexes. Radiolabeled viral RNA remained in the
replication complexes.2 The
supernatant, containing unincorporated radiolabel, was discarded. The
pellets were solubilized in SDS sample buffer. The products of the
reaction were phenol/chloroform-extracted, ethanol-precipitated, and
separated by electrophoresis in a 1% agarose MOPS formaldehyde gel.
RNA in the gels was stained with ethidium bromide and visualized by UV
light. 32P-Labeled poliovirus RNA was detected and
quantified by using a PhosphorImager (Bio-Rad).
-32P]UTP rather
than [-32P]CTP. The reaction mixtures were incubated
for 60 min at 37 °C. Following incubation, the reaction mixtures
were centrifuged at 13,000 × g to pellet the viral RNA
replication complexes. Radiolabeled VPgpUpU and radiolabeled viral RNA
remained in the replication complexes and were not released into the
soluble portion of the reaction mixtures.2 The supernatant,
containing unincorporated radiolabel, was discarded. The pellets,
containing radiolabeled viral RNA and uridylylated VPg, were
solubilized in SDS sample buffer. The samples were fractionated by
electrophoresis in a 10% polyacrylamide Tris-Tricine gel, and radiolabeled VPgpUpU was detected by phosphorimaging.
-32P]ATP and
T4 polynucleotide kinase essentially as specified by the manufacturer. Reactions, typically 20 µl, contained 0.1 µM
[-32P]ATP, 10 µM RNA oligonucleotide
(rU30), 1× kinase buffer, and 0.4 units/µl T4
polynucleotide kinase. Reactions were incubated at 37 °C for 60 min
and quenched by incubation at 65 °C for 5 min.
-32P]UTP). Reactions were initiated by
addition of 3Dpol and were carried out in a total volume of 25 µl at
30 °C for 5 min; EDTA (50 mM final) was used to quench
the reactions. The quenched reactions (5 µl) were spotted onto DE81
filter paper discs and dried completely. The discs were washed three
times for 5, 10, and 5 min in 250 ml of 5% dibasic sodium phosphate
and rinsed in absolute ethanol. Bound radioactivity was quantified by
liquid scintillation counting in 5 ml of Ecoscint scintillation fluid
(National Diagnostics).
1·cm1) (17). The three G
nucleotides were included to increase the efficiency of the
transcription reaction; these three G nucleotides do not alter the fold
of oriI in any way as predicted using the mfold RNA folding
server available on the Zuker home page
(bioinfo.math.rpi.edu/~zukerm/).
-32P]UTP (6000 Ci/mmol)). All reactions were adjusted
to a final NaCl concentration of 20 mM. 3Dpol and 3CD were
diluted immediately prior to use. 5 µl of 4 µM 3Dpol
and 5 µl of 4 µM 3CD were mixed together in one tube
immediately prior to the initiation of the reactions and placed on ice;
reactions were initiated by addition of 10 µl of reaction mix
containing the remaining components to the 10-µl 3Dpol/3CD mix.
Reaction mixtures were incubated at the indicated temperatures for 30 min and quenched with an equal volume (20 µl) of 100 mM
EDTA in 90% formamide containing 0.05% bromphenol blue and xylene
cyanol dyes. Quenched reactions (5 µl) were analyzed by using
Tris-Tricine SDS-polyacrylamide gel electrophoresis. Gels contained
15% acrylamide and 0.4% bisacrylamide. The cathode buffer (upper
chamber) contained 0.1 M Tris, 0.1 M Tricine,
and 0.1% (w/v) SDS; the anode buffer contained 0.2 M
Tris·Cl, pH 8.9. Gels were run at 80 watts (for a 33 × 39-cm
gel) for 2.5 h. Products were visualized by using a PhosphorImager
and quantified by using ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
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Fig. 1.
The primary oligomerization domain (interface
I) of 3Dpol. A, 3Dpol has the "right-hand" topology
typical of all nucleic acid polymerases. B, 3Dpol molecules
form head-to-tail fibers via interface I. Residues on the back of the
thumb of one molecule interact with residues on the back of the palm of
a second molecule. C, shown are a few of the critical
interactions observed crystallographically for interface I. Arginines
455 and 456 of the thumb subdomain interact with aspartic acids 339 and
349 and serine 341 of the palm subdomain.
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Fig. 2.
Mutations in interface I do not produce
equivalent phenotypes. HeLa cells were transfected with wild-type
poliovirus RNA, RNA containing the indicated mutations in 3Dpol, or no
RNA (mock transfection). Serial dilutions were then plated onto a
monolayer of untransfected HeLa cells. Plates were covered with agar
media and incubated at 37 °C for 3 days. Shown here are the plates
after removal of the agar overlay and staining with crystal violet.
Although virus containing mutations to the thumb subdomain
(R455,456A) has a lethal growth phenotype, virus containing
mutations to the palm subdomain (D339,S341,D349A) is viable
with a slow-growth phenotype.
To determine whether these phenotypes reflected defects to RNA
synthesis, we evaluated the mutants by using a subgenomic replicon assay (9). The replicon contains a luciferase gene in place of the
capsid-coding sequence, permitting RNA synthesis to be monitored
indirectly by measuring luciferase activity (Fig.
3A) (9). Because the initial
production of luciferase is dependent upon translation of transfected
RNA, this assay can also provide an indication of whether RNA stability
or translational efficiency is affected by the introduced mutations.
The thumb mutant was incapable of RNA synthesis, and the palm mutant
showed a decreased rate of RNA synthesis at 37 °C (Fig.
3B). By lowering the culture temperature to 30 °C, we
were able to completely reverse the phenotype observed for the palm
mutant relative to the wild-type control (Fig. 3C). Although
we were not able to plaque the thumb mutant virus under these
conditions, the replicon assay showed a very slow, steady accumulation
of RNA for this mutant (data not shown and Fig. 3C). RNA
stability and translation did not appear to be affected for any of the
mutant replicons tested in this assay, as wild-type and mutant
replicons displayed the same level of luciferase activity at the 1-h
time point (Fig. 3, B and C).
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) or wild-type sequence in the
absence of GdnHCl (
). GdnHCl is a reversible inhibitor of poliovirus
RNA replication; therefore, the luciferase activity measured in the
presence of GdnHCl is the result of translation of the input RNA. In
the absence of GdnHCl, transfections were also performed using
replicons containing mutations in either the thumb subdomain
(R455A,R456A) (
) or palm subdomain (D339A,S341A,D349A) (
).
Transfection reactions were incubated at 37 °C. At various times
after transfection, cells were processed (see "Experimental
Procedures") and luciferase activity evaluated. The thumb mutant
replicon is incapable of RNA synthesis, whereas the palm mutant
replicon has a decreased rate of RNA synthesis compared with the
wild-type replicon in the absence of GdnHCl. C, analysis at
30 °C. The difference between the palm mutant and the wild-type
replicon was completely eliminated by reducing the temperature.
Interestingly, the thumb mutant appears to be replicating at a very
slow, but observable, rate at the reduced temperature.
Biochemical Analysis of Interface I Mutants
The Thumb Mutant Is Impaired for Capsid Precursor
Processing--
Although the overall stability and translatability of
genomes encoding mutations in interface I were equivalent to wild-type RNA, it was possible that these mutations caused problems with processing of the viral proteins required for replication. To test this
possibility, a cell-free translation/replication system was employed
(13). The system was programmed with wild-type or mutant genomes, and
reactions were performed for 3 h at 34 °C in presence of
[35S]methionine. Labeled proteins were visualized by
phosphorimaging after separation by SDS-PAGE (Fig.
4A). Consistent with the
replicon data, both mutants produced levels of protein equivalent to
wild-type control (Fig. 4A). All of the expected precursor
and processed forms of the capsid (P1, VP0, VP1, and VP3) and
nonstructural (P3, P2, 3BCD, 3CD, 3D, 3C, 3AB, 2BC, 2C, and 2A)
proteins were observed (Fig. 4A). Processed and precursor
proteins containing the palm mutant 3D subunit showed altered mobility
owing to the change in charge of this protein caused by removal of the
two carboxylate side chains (Fig. 4A). Interestingly, the
amount of VP0/VP3 precursor protein produced by the thumb mutant was
elevated relative to the wild-type control (Fig. 4A,
lane 3), suggesting a defect to processing of
this precursor. Consistent with this observation, the amount of VP3
protein was reduced (Fig. 4A, lane 3).
A corresponding reduction in VP0 was not as evident for the thumb
mutant owing to the comigration of this protein with the 2C protein
(Fig. 4A, lane 3). We conclude that
the reduced accumulation of RNA observed for interface I mutants is not
a reflection of reduced, altered, or aberrant processing of the viral
nonstructural proteins.
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The Thumb Mutant Is Defective, and the Palm Mutant Is Impaired for
RNA Synthesis--
The cell-free translation/replication system
facilitates direct evaluation of RNA synthesis (13). Translation is
performed in the presence of 2 mM guanidine, an inhibitor
of RNA synthesis. Initiation complexes are isolated by centrifugation,
RNA synthesis can then be evaluated after suspension of the pelleted
complexes in buffer containing nucleoside triphosphates. Use of
[-32P]CTP permits visualization of the RNA product
after agarose gel electrophoresis by using a PhosphorImager. The
interface I mutants were evaluated by using this system; the results
are shown in Fig. 4B. Although the palm mutant was capable
of producing genomic RNA, the thumb mutant was not. The palm mutant
exhibited a 4-fold reduction in the quantity of genomic RNA produced
relative to the wild-type control. This observation is consistent with
the reduction in the rate of RNA accumulation inferred from the
subgenomic replicon data (Fig. 3B).
Both Thumb and Palm Mutants Exhibit Equivalent Defects in
Oligomerization Capacity--
The disparity in phenotypes observed for
the thumb and palm mutants suggested the possibility that the
interaction domain observed crystallographically (1) might not be
responsible for the oligomerization of polymerase observed
biochemically. To exclude this possibility, we expressed the thumb and
palm polymerase derivatives in E. coli, purified the
enzymes, and evaluated their ability to oligomerize in solution by
using the filter-binding assay established to monitor RNA binding by
and oligomerization of 3Dpol (2). For this assay, equilibrated mixtures
of protein and labeled nucleic acid are pulled by vacuum through a
stack of three membranes: polysulfone, nitrocellulose, and nylon.
Complexes retained on the polysulfone membrane are selected based upon
size (0.45-µm cut-off), as neither protein nor nucleic acid bind to this membrane. In the case of 3Dpol, these complexes should contain on
the order of 100 molecules given the dimensions of the enzyme. All
soluble protein-nucleic acid complexes will bind to the nitrocellulose. Any unbound nucleic acid will bind to the nylon, thus increasing the
accuracy of quantification. Each polymerase was evaluated at varying
concentrations (0.1-5 µM) with an end-labeled
rU30 oligonucleotide (10 nM). Complexes bound
to each membrane were visualized by phosphorimaging (Fig.
5A), and those bound to the polysulfone, the nitrocellulose, or both were quantified by using the
ImageQuant software from Amersham Biosciences (Fig. 5,
B-D). As the concentration of wild-type 3Dpol was
increased, an accumulation of very large complexes was observed (Fig.
5B). These large complexes formed at the expense of the
smaller complexes (Fig. 5C). In contrast, over the same
concentration range, neither interface mutant formed very large
complexes (Fig. 5B); essentially all of these complexes were
bound to the nitrocellulose membrane (Fig. 5C). The overall RNA-binding affinity was evaluated by plotting the sum of all bound
species as a function of 3Dpol concentration and shown to be the same
for all derivatives (Fig. 5D). In addition, in contrast to
previous reports (2-4), cooperative RNA binding was not observed (Fig.
5, B-D). We conclude that oligomerization observed
biochemically is the result, at least in part, of interface I observed
crystallographically. The finding that neither mutant forms large
polymerase fibers suggests that the RNA synthesis phenotype is not
related to polymerase fiber formation.
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0.45 µm (~100 3Dpol molecules), the nitrocellulose membrane binds
protein-RNA complexes <0.45 µm and any free protein, and the nylon
membrane binds all of the free RNA. WT, wild-type 3Dpol;
R455,456A, 3Dpol-R455A,R456A; D339,S341,D349A,
3Dpol-D339A,S341A,D349A. B, quantification of the
protein-RNA complexes on the polysulfone membrane shows that mutations
to the thumb and palm subdomains preclude formation of long 3Dpol
fibers. C, quantification of the protein-RNA complexes bound
to the nitrocellulose membrane. D, evaluation of the total
RNA bound by each 3Dpol derivative reveals that both mutants bind RNA.
It is evident that disruption of interface I does not perturb the
affinity for RNA relative to the wild-type enzyme. Interface I Is Dispensable for Polymerase Functions Required for Nucleotide Incorporation-- To begin to define the function of the back of the thumb, we constructed, expressed and purified two additional thumb mutants (R455S,R456S and R455D,R456D). Serine was introduced to test the possibility that exposure of even a small aliphatic moiety would alter the conformation of the thumb. Aspartic acid was introduced to completely disrupt any residual interaction between the thumb and palm that might remain, with the hope of making any biochemical differences more obvious. The active fraction of each purified enzyme preparation was similar and ranged from 96.5 to 98.5% (see "Active site titration" in Table II). Consistent with the results obtained by using the filter-binding assay, the stability of each polymerase derivative with a primer/template substrate that mimics an elongation intermediate varied less than 2-fold from the wild-type value (see "koff 3Dpol-sym/sub" in Table II). Each derivative was more resistant to thermal inactivation than the wild-type enzyme (see "Thermal inactivation" in Table II). This observation may suggest that the observed inactivation in vitro is related to polymerase multimerization. The rate of nucleotide incorporation of each derivative was within 80% of the value measured for wild-type 3Dpol (see "Rate of AMP incorporation" in Table II). Finally, consistent with previous findings (4), interface I mutants display differences in poly(rU) polymerase activity relative to the wild-type enzyme. The thumb derivatives exhibit a 1.5-3-fold reduction in poly(rU) polymerase activity (see "Poly(rU) polymerase" in Table II); the palm derivative exhibits a 1.7-fold increase in poly(rU) polymerase activity (see "Poly(rU) polymerase" in Table II). We have shown that the rate-limiting step measured by using this assay is template switching (5). Therefore, it is difficult to reach any conclusions from these data when subtle changes are observed. Taken together, these data suggest that all of the functions required for processive elongation of RNA are retained by polymerases that are incapable of forming fibers.
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Mutations on the Back of the Thumb of 3Dpol Interfere
with VPg Uridylylation--
Because elongation does not appear to be
affected by interface I mutations, we focused our attention on the
initiation reaction. Initiation of poliovirus negative- and
positive-strand RNA synthesis requires, minimally, the polymerase, the
22-amino acid peptide primer (VPg), and a template (18). For
negative-strand RNA synthesis, it has been suggested that the template
may either be poly(rA) tail at the 3' end of viral RNA (18) or a 61-nt
stem loop RNA located in 2C-coding sequence (Fig.
6A) (19). We will refer to
this 61-nt cis-acting replication element (cre) (20) as oriI (internal
origin of replication). By using VPg, oriI, and UTP, 3Dpol can activate
a VPg tyrosine for nucleophilic attack on the -phosphorus of UTP to
form VPg-pU, which, in turn, is used as the source of the nucleophile
for addition of a second uridylate residue to form VPg-pUpU (Fig.
6B) (19). Uridylylation is templated by a single adenylate
residue located in the loop of oriI (see boldface
A in Fig. 6A) by using a slide-back mechanism
(21) similar to that described by Salas and colleagues (22) for
bacteriophage 
29. This reaction is stimulated by more than 20-fold
by adding viral protein 3CD to the reaction mixture (19). The
mechanistic basis for this observation is not known. Protein 3CD is a
precursor form of the 3C protease and 3D polymerase. This protein has
protease activity and RNA binding activity but lacks polymerase
activity (23, 24). Although it is currently not clear that oriI serves as the site for initiation in vivo, the specificity of this
reaction is consistent with this role (19, 21, 25).
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We evaluated the ability of each derivative to catalyze
oriI-dependent, 3CD-stimulated uridylylation of VPg. In
these reactions, the incorporation of [-32P]UTP into
VPg-pUpU is monitored by SDS-PAGE using a Tris-Tricine buffer system.
The phosphorimage of a typical gel is shown in Fig. 6C. Both
VPg-pU and VPg-pUpU can be resolved from the labeled UTP, which can be
retained on the gel to permit accurate quantification. Interestingly,
all of the thumb mutants are significantly impaired for uridylylation
(Fig. 6C, lanes 2-4) relative to
wild-type 3Dpol (Fig. 6C, lane 1). The
palm mutant does not exhibit a significant difference relative to
wild-type 3Dpol (Fig. 6C, lane 5).
Quantification of the data revealed that there was an 8-fold reduction
in uridylylation for the R455A,R456A and R455S,R456S derivatives and a
108-fold reduction in uridylylation for the R455D,R456D derivative
relative to wild-type 3Dpol, and the palm mutant was essentially
unaffected (Fig. 6D). At 30 °C, the observed difference
for the R455A,R456A and R455S,R456S derivatives diminished to only
3-fold without any impact on the extent of the defect observed for the
R455D,R456D derivative (Fig. 6E). The diminution in the
magnitude of the defect to uridylylation at 30 °C for the
R455A,R456A derivative may explain the ability of the R455A,R456A
replicon to produce RNA at 30 °C (Fig. 3C). By omitting
3CD from the reaction, it became clear that the primary effect of the
thumb mutations was on the capacity of these derivatives to be
stimulated by 3CD (Fig. 6F). In the absence of 3CD, the
thumb derivatives catalyzed uridylylation at a rate that was within
2-3-fold of the value for the wild-type enzyme, suggesting that the
intrinsic ability of these enzymes to catalyze uridylylation is not
affected. Comparison of the data presented in Fig. 6D with
that presented in Fig. 6F showed that 3CD stimulated the
wild-type enzyme 46-fold, the R455A,R456A and R455S,R456S enzymes
13-fold, and the R455D,R456D derivative not at all.
We also performed experiments to rule out other trivial explanations
for the observed effects on the uridylylation reaction. The linearity
of the reaction was unchanged (data not shown). In addition, the
observed reduction was not because of significant changes in the
K0.5 values for UTP (data not shown). However, there was a subtle difference between the VPg
K0.5 values for the thumb derivative (12 µM) relative to wild-type 3Dpol (6 µM) (Fig. 7). This difference cannot account
for the effects observed here, as VPg was employed at a concentration
of 50 µM. This concentration of VPg is sufficient to
saturate the thumb mutant.
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Finally, to corroborate the data obtained by using the reconstituted
system, we evaluated the accumulation of VPg-pU(pU) in the cell-free
translation/replication system. As expected, the thumb mutant was
incapable of uridylylating VPg in this system as well (Fig.
8). It should be noted that the
radiolabeled products that appear in the presence of guanidine reflect
the labeling of cellular components in the reactions.
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Evidence for an Interaction between the Back of the Thumb of 3Dpol
and an Undefined Domain of Protein 3C--
The simplest interpretation
of the observation that the thumb mutants cannot be stimulated by 3CD
is that a physical interaction exists between 3Dpol and 3CD that
permits uridylylation-competent complexes to form more efficiently
and/or product to form faster. The extent of the effect of
uridylylation observed with the thumb derivatives was consistent with
predictions made by evaluating the structure of interface I (Fig.
1C). In particular, the R455S,R456S derivative was
stimulated to a greater extent than the R455D,R456D derivative.
Therefore, we hypothesized that the interaction of 3Dpol with 3CD was
mediated by an interaction with the back of the palm of the 3D domain
located on the 3CD protein. To test this hypothesis, we subcloned the
original thumb and palm mutations into our 3CD expression vector,
expressed, purified, and characterized these proteins in the
uridylylation assay. Both the thumb and palm mutant 3CD derivatives
were active in this reaction (Fig. 9). To
determine whether the stimulatory activity of 3CD resided in the 3Dpol
domain of this protein, we constructed a 3C expression vector,
expressed, purified, and characterized this protein in the
uridylylation assay. To our surprise, 3C was capable of stimulating uridylylation of VPg within 2-fold of that observed for 3CD (Fig. 9,
bar 4). By increasing the concentration of 3C, we
could increase the stimulation to the level observed for 3CD (Fig. 9,
bar 5). These data suggest that 3C contains all
of the determinants for stimulation, i.e. formation of an
activated uridylylation complex. However, the 3Dpol domain of 3CD
appears to contribute to the overall stability of this complex.
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Consistent with the possibility for an interaction between the back of
the thumb of 3Dpol and some region of 3C is the observation that the
3CD-R455A,R456A derivative is more active than others (Fig. 9,
bar 2). This derivative should not have 3CD-3CD
interactions competing with formation of 3Dpol-3CD interactions
required to form the activated uridylylation complex.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The crystal structure for 3Dpol was the first available for an RNA-dependent RNA polymerase (1). This structure confirmed predictions that the overall fold of all nucleic acid polymerases is similar but also identified several unique features for this enzyme. In particular, a polymerase lattice is formed by head-to-tail interaction of polymerase subunits (interface I) and interaction between the resulting polymerase fibers (interface II). Molecular genetic evidence supported a biological function for these fibers (8). Biochemical studies suggested that the ability to form fibers facilitated complete coating of template RNA, possibly a prerequisite for activity, and explained the cooperativity observed with respect to polymerase activity (2) and RNA binding (2-4). These observations led to the prediction that all RdRPs may function similarly to 3Dpol (26). Indeed, evidence for oligomerization of the RdRP from hepatitis C virus, NS5B, has been pursued rather aggressively (27, 28). Given that DNA-dependent DNA and RNA polymerases do not need to oligomerize for activity or nucleic acid binding (29-31) and a single subunit of an RdRP has all of the elements present in these enzymes (1), it has been difficult to reconcile the observation that RdRP oligomerization is required for activity and/or RNA binding.
Although disruption of interface I by altering residues on the back of the thumb of 3Dpol is lethal to the virus (4, 8), disruption of interface I by altering residues on the back of the palm is not (4). Our palm mutant (D339A,S341A,D349A) was predicted to be as disruptive as the thumb mutant (R455A,R456A) reported previously (Fig. 1C). Interestingly, this palm mutant also supported virus viability (Fig. 2), in addition to translation (Figs. 3 and 4A) and genome replication (Figs. 3 and 4B). Moreover, the finding that both the palm and thumb mutants are incapable of forming long fibers (Fig. 5) suggests that the capacity to form fibers is not essential for virus multiplication. In addition, this observation strengthens the argument that the lethal phenotype associated with the thumb mutant is unrelated to formation of polymerase fibers.
The thumb mutant was not capable of processing the VP0/VP3 junction as well as the wild type (Fig. 4A). Clearly, this phenotype must be a reflection of an alteration in 3CD protease activity. It is known that 3CD is more active at capsid precursor processing in vitro than 3C (32, 33). Interestingly, processing of the VP0/VP3 junction by 3CD requires a cellular cofactor (34). It is possible that one function for the back of the thumb may be to interact with this cellular cofactor. Although this processing defect will limit or even preclude virus spread, this defect should not have any impact on genome replication. Processing of the nonstructural proteins in the thumb mutant was not affected (Fig. 4A). However, the thumb mutant replicon failed to produce RNA (Fig. 3), and the thumb mutant RNA failed to support replication in the cell-free translation/replication system (Fig. 4B). We concluded that the residues on the back of the thumb play a unique, undefined role in viral RNA synthesis.
None of the thumb mutants exhibited any significant alteration in RNA binding properties (Fig. 5 and Table II), and cooperative binding to RNA was not observed by using the RNA oligonucleotide employed in this study (Fig. 5D). The fraction of enzyme active in each preparation, the stability of each polymerase elongation complex, the thermal stability of each polymerase derivative, and the observed rate of nucleotide incorporation for each derivative were essentially unchanged relative to wild-type 3Dpol (Table II). The nucleic acid substrate employed in this study has been shown to predict biological phenotypes and is thought to recapitulate the elongation phase of viral RNA synthesis (16). A previous study of interface I mutants showed minimal to substantial changes in RNA binding and/or elongation for derivatives analogous to those presented here (4). The magnitude of the effect was dependent upon the RNA ligand/substrate employed. Because these two studies were performed under substantially different conditions with different RNA ligands/substrates, it is not possible to explain the differences in outcome. Taken together, our data support the conclusion that changes in biochemical properties required for RNA synthesis do not explain the lethal phenotype of the thumb mutant.
Consistent with previous studies (4), a modest reduction in the poly(rU) polymerase activity was observed for the thumb mutants (Table II). For the R455A,R456A derivative, a 1.5-fold reduction was noted (Table II). We have shown that the rate-limiting step measured by using the poly(rU) polymerase assay is template switching (5). Template switching is thought to be the primary mechanism for homologous recombination in poliovirus (35, 36). Because the recombination frequency for poliovirus ranges from 0.01 to 0.1/genome (35, 36), a 50% reduction in this value is likely too small to cause the lethal phenotype observed for the R455A,R456A mutant virus.
The elongation phase of viral genome replication appeared fine, so we turned our attention to the initiation phase. It has been suggested that the cis-acting replication element located in the 2C-coding region of the poliovirus genome (20) acts as an internal origin of replication (oriI) (19). The thought is that 3Dpol would initiate VPg-primed RNA synthesis at this internal site to produce VPg-pUpU, which, in turn, would be transferred to the 3' end of viral RNA and extended by 3Dpol to produce full-length, VPg-linked RNA. An in vitro system recapitulating the VPg uridylylation step was recently reported (19). The basal reaction is stimulated substantially by the presence of the viral 3CD protein; however, the molecular basis for this stimulation is not known. Interestingly, when the thumb mutants were evaluated in this assay, we observed a substantial reduction (8-100-fold at 37 °C) in the efficiency of VPg uridylylation (Fig. 6). The affinity for VPg was not changed (Fig. 7). The basal activity of each derivative was within ~2-fold of the wild-type value (Fig. 6). In addition, a defect to VPg uridylylation was also observed in the cell-free translation/replication system (Fig. 8). These data provided an intellectually satisfying explanation for the phenotype associated with the thumb mutant virus.
By mutating the back of the thumb of 3Dpol, the VPg
uridylylation reaction could not be stimulated by 3CD (Fig. 8). The
simplest explanation was that a physical interaction between 3Dpol and 3CD was required to simulate VPg uridylylation and that this
interaction was lost by altering the back of the thumb. Surprisingly,
this interaction did not occur between the back of the thumb of 3Dpol and the back of the palm of the 3Dpol domain of 3CD as the 3Dpol palm
domain of 3CD could be modified without consequence to the stimulatory
capacity of 3CD (Fig. 9). In fact, the entire 3Dpol domain of 3CD could
be deleted and the stimulatory activity retained (Fig. 9). Our working
hypothesis is shown in Fig.
10A. The activated uridylylation complex forms sequentially; 3CD binds to oriI and recruits 3D to the site on oriI that serves as a template for uridylylation. Recruitment is mediated by an interaction between the
back of the thumb of 3Dpol and an undefined domain of 3C. Although an
interaction between 3D and 3CD has been shown by using the yeast
two-hybrid system, these studies did not identify an interaction
between 3D and 3C (7). It is possible that the 3D-3C interaction
requires RNA for maximal stability.
|
The R455A,R456A derivative exhibited an 8-fold reduction in uridylylation efficiency relative to wild-type 3Dpol in the reconstituted system (Fig. 6, D and E). In contrast, this mutant fails to produce uridylylated VPg in the cell-free extract (Fig. 8). This difference may reflect the inability to detect the first round of VPg uridylylation and may indicate that genomes produced within replication complexes serve as the primary templates for VPg uridylylation observed in extracts. This scenario would also explain the observation that the palm mutant shows no defect to VPg uridylylation in the reconstituted system but a subtle defect in the cell-free extract (Fig. 8) on par with the defect observed for RNA synthesis (Fig. 3B).
Is the magnitude of the defect to VPg uridylylation observed for the thumb mutant in the reconstituted system sufficient to account for the complete inability of this mutant to produce viral RNA? It is possible that the 3D-3C interaction is required for other aspects of the genome-replication process. For example, it has been suggested that poliovirus RNA must "circularize" via protein-protein interactions for negative-strand RNA synthesis (37, 38). Protein 3CD and cellular factors have been implicated in this protein bridge. It is known that 3CD binds to the cis-acting elements at the 5' (oriL) and 3' (oriR) ends of the viral genome (24, 39). As shown in Fig. 10B, it is possible that the ability of 3CD to function in genome circularization requires the 3D-3C interaction. This possibility would explain the severity of the defect associated with the thumb mutant. Additional studies will be required to investigate this possibility.
Although the palm mutant virus was viable, this virus did
not grow as well as wild-type virus (Fig. 2). The defect to this virus
was attributed to a modest reduction in the kinetics of RNA synthesis
(Figs. 3A and 4B). Interestingly, the defect to RNA synthesis observed for the palm mutant relative to wild type could
be reversed completely by lowering the temperature (Fig. 3B), consistent with changes in the stability of
protein-protein interaction. It is difficult to imagine that such an
intricate interaction domain like interface I would arise fortuitously. It is possible that the phenotype observed for the palm mutant is a
consequence of the inability of this mutant to form polymerase fibers.
Additional studies of this mutant may reveal the true function for
polymerase fibers.
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ACKNOWLEDGEMENTS |
|---|
We thank Brandon Bilohlavek for construction of the thumb mutant expression plasmids, Chong Ha for construction of 3CD expression plasmids and optimization of the purification protocol, and Mehul Suthar for construction of the pET26Ub-Chis plasmid.
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FOOTNOTES |
|---|
* This work was supported in part by the National Institutes of Health through Howard Temin Award CA75118 (to C. E. C.) from NCI and Grants AI45818 (to C. E. C.) and AI42189 (to D. J. B.) from NIAID.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.
§ These authors contributed equally to this work.
Current address: Dept. of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115.
To whom correspondence should be addressed. Tel.:
814-863-8705; Fax: 814-865-7927; E-mail: cec9@psu.edu.
Published, JBC Papers in Press, June 19, 2002, DOI 10.1074/jbc.M204408200
2 D. J. Barton, unpublished results.
3 C. E. Cameron, unpublished results.
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ABBREVIATIONS |
|---|
The abbreviations used are: RdRP, RNA-dependent RNA polymerase; DMEM, Dulbecco's modified Eagle's medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; oligo, oligonucleotide; sym/sub, symmetrical substrate; PEI, polyethyleneimine; PBS, phosphate-buffered saline; nt, nucleotide(s); GdnHCl, guanidine HCl.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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