Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH
box protein family, which is defined by six shared sequence motifs. The
contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia
(PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by
alanine scanning. NPH-II-Ala proteins were expressed in
baculovirus-infected Sf9 cells, purified, and characterized with
respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA
binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif
I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in
RNA unwinding that correlated with reduced rates of ATP hydrolysis. In
contrast, alanine mutations at His-299 (motif II) and at Thr-326 and
Thr-328 (motif III) elicited defects in RNA unwinding but spared the
ATPase. None of the mutations analyzed affected the binding of NPH-II
to RNA. These findings, together with previous mutational studies,
indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are
essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif
III and the His moiety of the DExH-box serve to couple the NTPase and
helicase activities. Wild-type and mutant NPH-II-Ala genes were tested
for the ability to rescue temperature-sensitive nph2-ts
viruses. NPH-II mutations that inactivated the phosphohydrolase in
vitro were lethal in vivo, as judged by the failure to recover rescued
viruses containing the Ala substitution. The NTPase activity was
necessary, but not sufficient, to sustain virus replication, insofar as
mutants for which NTPase was uncoupled from unwinding (H299A, T326A,
and T328A) were also lethal. We conclude that the phosphohydrolase and
helicase activities of NPH-II are essential for virus replication.
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INTRODUCTION |
Vaccinia virus NPH-II (nucleoside
triphosphate [NTP] phosphohydrolase II) is an NTP-dependent helicase
that catalyzes unidirectional unwinding of 3'-tailed duplex RNAs
(27). NPH-II is a member of the DExH family of nucleic
acid-dependent NTPases. The DExH proteins are defined by six conserved
motifs arrayed in a collinear fashion (7, 9, 16); five of
these motifs are shown in Fig. 1. The
size of the DExH family is expanding rapidly as the signature elements
are encountered in newly cloned genes and during genome sequencing
efforts. The products of these genes are typically designated putative
helicases, yet relatively few DExH proteins have actually been shown to
unwind duplex DNA or RNA. Vaccinia virus NPH-II was the first
RNA-dependent NTPase to be purified (21, 22), and its
substrate specificity and nucleic unwinding properties have been
thoroughly characterized (10, 22, 27, 28). NPH-II
exemplifies a subgroup of structurally related DExH proteins that
includes human RNA helicase A, the yeast pre-mRNA splicing factors
Prp2, Prp16, and Prp22, Drosophila maleless, and hepatitis C
virus (HCV) RNA helicase NS3 (Fig. 1) (1, 12-14, 18, 19,
26).
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FIG. 1.
Conserved sequence elements define the DExH protein
family. The amino acid sequence of motifs I, Ia, II, III, and VI of
vaccinia virus NPH-II (GenBank accession no. M35027) is aligned with
the sequences (with GenBank accession numbers in parentheses) of the
corresponding motifs of six other DExH-box proteins: human helicase A
(Hel-A) (L13848); Saccharomyces cerevisiae splicing factors
Prp2 (X55936), Prp16 (M31524), and Prp22 (X58681);
Drosophila MLE protein (M7412); and HCV RNA helicase NS3
(M62385). Amino acids that have been shown by mutagenesis to be
important for the ATPase or helicase activities of NPH-II are shown in
shaded boxes. New residues mutated in this study are marked by
asterisks.
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Insights into the function of the conserved motifs have emerged from
structure-function analyses of NPH-II (8, 9). We demonstrated previously that alanine substitutions for Lys-191 in the
GKT element (motif I) or Asp-296 and Glu-297 in the DExH box (motif II)
severely impaired ATP hydrolysis and RNA unwinding without affecting
the binding of the mutated proteins to RNA. Changing the DExH-box His
residue to Ala resulted in a protein that was constitutively active for
ATPase in the absence of a nucleic acid effector; the H299A enzyme did
not unwind duplex RNA, even though RNA binding was unaffected
(8). We have also performed an alanine scan of 10 residues within motif VI of NPH-II (491-QRKGRVGRVNPG-502).
Alanine substitutions at invariant residues Gln-491, Arg-492, Gly-494,
Arg-495, Gly-497, Arg-498, and Gly-502 caused severe defects in RNA
unwinding that correlated with reduced rates of ATP hydrolysis
(9). None of these mutations in motif VI significantly
affected the binding of NPH-II to RNA. The residues shown to be
important for helicase activity of NPH-II are highlighted in shaded
boxes in Fig. 1.
In this study, we extended the mutational analysis to motifs Ia and III
and to additional conserved residues in motifs I and II. The eight new
positions targeted for alanine substitution are marked by asterisks in
Fig. 1. We found that mutations (underlined) of Thr-192 in motif I
(GKT), Arg-229 in motif Ia (PRI), and Glu-300 in motif II (DExHE) coordinately inactivated RNA unwinding
and ATP hydrolysis without affecting RNA binding. In contrast,
mutations of the two threonines in motif III (TAT) abolished helicase
activity with only modest effects on ATP hydrolysis. The
phosphohydrolase activity of the T326A and T328A proteins remained
nucleic acid dependent. These results suggest that motif III acts to
couple NTP hydrolysis to duplex unwinding.
In addition, we exploited our collection of biochemically defined
NPH-II alanine substitution mutants and temperature sensitive (ts) nph2-ts viruses isolated by Fathi and Condit
(4, 5) to examine if the NTPase and helicase functions of
NPH-II are essential for vaccinia virus replication in vivo. This was
done by DNA-mediated marker rescue of the nph2-ts viruses
with wild-type and mutant NPH-II genes. Rescued viruses were genotyped
to determine if the targeted alanine mutation had been incorporated
during recombination to restore the wild-type codon at the
ts lesion. We found that alanine mutations that inactivated
the NTPase of NPH-II were invariably lethal, as judged by the failure
to recover rescued viruses containing the Ala substitution. The NTPase
activity of NPH-II was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which ATPase was uncoupled from
unwinding were also lethal. We conclude that the helicase activity of
NPH-II is essential for vaccinia virus replication. A possible role of
NPH-II-mediated strand displacement during vaccinia virus transcription
is discussed.
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MATERIALS AND METHODS |
Alanine mutagenesis of His-NPH-II.
Phagemid
pTM-His10-NPH-II containing a His-tagged NPH-II gene under
the control of a T7 promoter and a picornavirus translational enhancer
was described previously (8). Uracil-substituted
single-stranded pTM-His10-NPH-II DNA was used as a template
for oligonucleotide-directed mutagenesis. Mutagenic DNA primers were
designed to create alanine substitutions at residues T187, T192, Q194,
P228, R229, E300, T326, and T328. The presence of the desired mutations
was screened initially by the gain or loss of a restriction site and
confirmed by DNA sequencing. The mutated NPH-II genes were exchanged
for the corresponding wild-type gene in the recombinant
His10-NPH-II baculovirus transfer vector pBacPAK-9
(10). The entire NPH-II gene was sequenced in each case to
ensure that no unwanted mutations were present in the expression
vectors. The recombinant baculoviruses expressing the
His10-NPH-II protein were constructed and selected according to protocols supplied by Clontech.
Expression and purification of His10-NPH-II.
Wild-type and alanine-substituted His10-NPH-II proteins
were each expressed by infection of 30 150-cm2 dishes of
Sf9 cell monolayers with recombinant baculovirus at a multiplicity of
10. Infected cells were incubated at 27°C for 72 h. Soluble cell
lysates were prepared as described previously (8, 10). The
His10-NPH-II protein was adsorbed to Ni-nitrilotriacetic acid agarose (10). After extensive washing of the resin with buffer containing 50 mM imidazole, His10-NPH-II was
recovered by elution with 500 mM imidazole. Aliquots (0.3 ml) of each
Ni-agarose fraction were layered onto a 4.7-ml 15 to 30% glycerol
gradient containing 0.3 M NaCl in buffer A (50 mM Tris HCl, pH 8.0, 2 mM dithiothreitol (DTT), 1 mM EDTA, 10% glycerol, 0.1% Triton X-100). The gradients were centrifuged for 20 h at 50,000 rpm in a Beckman SW50 rotor. Fractions (0.17 ml) were collected from the bottoms of the
tubes. The concentrations of NPH-II in the peak glycerol gradient
fractions were determined by quantitative Western blotting as described
previously (9).
Helicase assay.
Reaction mixtures (20 µl) contained 40 mM
Tris HCl (pH 8.0), 2 mM DTT, 2 mM MgCl2, 2 mM ATP, and 25 fmol of [
32P]GMP-labeled tailed double-stranded RNA
(dsRNA) substrate (prepared as described in reference
8). After incubation for 15 min at 37°C, the
reactions were halted by addition of 5 µl of 0.1 M Tris HCl (pH
7.4)-5 mM EDTA-0.5% sodium dodecyl sulfate (SDS)-50%
glycerol-0.1% xylene cyanol-0.1% bromophenol blue. Aliquots (20 µl) were electrophoresed at 15-mA constant current through an 8%
polyacrylamide gel containing 45 mM Tris-borate and 1.2 mM EDTA.
Labeled RNAs were visualized by autoradiography. The extent of
unwinding (displaced RNA/total RNA) was quantitated by scanning the gel
with a Fuji BAS1000 phosphorimager.
RNA binding assay.
Reaction mixtures (20 µl) contained 40 mM Tris HCl (pH 8.0), 2 mM DTT, 1 mM MgCl2, and 25 fmol of
[32P]GMP-labeled 98-mer single-stranded RNA (ssRNA)
(corresponding to the 98-mer strand of the helicase substrate and
labeled to high specific activity). After incubation for 15 min at
37°C, samples were adjusted to 8% glycerol, and 20-µl aliquots
were electrophoresed at 15 mA through a native 8% polyacrylamide gel containing 22 mM Tris-borate and 0.6 mM EDTA. The extent of RNA-protein complex formation (bound RNA/total RNA) was quantitated with a phosphorimager.
Marker rescue.
Confluent BSC40 cell monolayers
(35-mm-diameter dishes) grown at 31°C in Dulbecco modified Eagle
(DME) medium with 5% fetal bovine serum (FBS) were infected with the
ts vaccinia viruses at a multiplicity of 5. The inoculum was
removed after 30 min, and the cells were washed twice and overlaid with
DME-5% FBS. After 30 min of incubation at 31°C, the monolayers were
treated with 1 ml of 0.05% trypsin-0.5 mM EDTA. The cell suspensions
were mixed with 4 ml of DME and then harvested by centrifugation. The cell pellets were washed with 3 ml of HEPES-buffered saline (20 mM
HEPES [pH 7.0], 150 mM NaCl, 0.7 mM Na2HPO4,
5 mM KCl, 6 mM dextrose) and recentrifuged. The pellets were
resuspended in 0.8 ml of cold HEPES-buffered saline by gentle pipetting
and transferred to a chilled tube containing 10 µg of
pTM-His10-NPH-II plasmid DNA. The suspension were mixed by
gentle agitation and chilled on ice for 10 min. The contents were
transferred to chilled Bio-Rad 0.4-cm electrogap cuvettes. The cell
suspensions were electroporated at 200 V (capacitance, 960 µF) in a
Bio-Rad Gene Pulser equipped with a Bio-Rad capacitance extender and
then placed on ice for 10 min. The cells were diluted into 7 ml of
medium at room temperature. An aliquot (3.5 ml) was applied to a
confluent cell monolayer of BSC40 cells (35-mm-diameter well)
maintained at 40°C. After 48 h of incubation at 40°C, cells
were harvested by scraping and centrifugation. The cell pellets were
resuspended in 1 ml of DME. Lysis was achieved by three rounds of
freezing and thawing, followed by brief sonication. The extent of
marker rescue was determined by applying serial 10-fold dilutions of
the lysates onto BSC40 monolayers at 40°C. After 48 h at 40°C,
the cells were stained with 0.1% crystal violet in order to visualize
plaques.
Genotyping of rescued viruses.
Viral revertants were
selected by plating ~10 PFU from each marker rescue onto BSC40
monolayers at 40°C under an agar overlay. Individual plaque isolates
were amplified once by infection of BSC40 cell monolayers at 40°C.
Infected cells were harvested by scraping and centrifugation. The
infected cell pellets were resuspended in 20 mM Tris HCl (pH 8.0)-10
mM NaCl-10 mM EDTA and lysed by addition of 0.75% SDS. The samples
were digested with proteinase K and then extracted twice with
phenol-chloroform (1:1) and once with chloroform. DNA was recovered
from the aqueous phase by ethanol precipitation and resuspended in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA. These DNAs were used as templates for
PCR amplification of the NPH-II gene. PCR was carried out with
Taq polymerase (Boehringer Mannheim) and oligonucleotide
primers flanking the NPH-II coding sequence. The sequence of the
sense-strand primer was 5'-CGTGATAGTTTCTCATTGGCCG-3', and
that of the antisense-strand flanking primer was
5'-GTTAATTCTCCCGTCCTCTC-3'. The PCR products were screened
for the presence of diagnostic restriction endonuclease cleavage sites
linked to the targeted alanine mutations in the transfected NPH-II-Ala
genes.
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RESULTS |
Expression and purification of wild-type and mutated NPH-II
proteins.
Alanine substitutions were introduced at conserved
residues Thr-187, Thr-192, and Gln-194 in motif I, Pro-228 and Arg-229 in motif Ia, Glu-300 in motif II, and Thr-326 and Thr-328 in motif III
(Fig. 1). Wild-type NPH-II, the eight new mutant proteins, and two
other mutants, K191A (motif I) and H299A (motif II), were expressed in
cultured insect cells infected with recombinant baculoviruses carrying
an inserted copy of the NPH-II gene under the control of a polyhedrin
promoter (10). Earlier studies of the K191A and H299A
mutants had been performed with protein expressed in vaccinia
virus-infected mammalian cells (8). Each protein was expressed as a N-terminal His-tagged derivative. We showed previously that recombinant wild-type His-tagged NPH-II is functionally identical to the native enzyme purified from vaccinia virions (8). The recombinant wild-type and mutant NPH-II proteins were purified from
soluble lysates of baculovirus-infected cells by adsorption to
Ni-agarose and elution with 0.5 M imidazole. The proteins were then
purified further by glycerol gradient sedimentation. The peak gradient
fractions from each of the enzyme preparations were used for
biochemical studies of NPH-II activity. The concentration of His-NPH-II
protein was gauged by quantitative Western blotting (10). To
assess overall purity, equivalent amounts of each protein preparation
were analyzed by SDS-polyacrylamide gel electrophoresis, and the gel
was stained with Coomassie brilliant blue. A single 70-kDa polypeptide
corresponding to His-NPH-II was detected in each case (Fig.
2).
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FIG. 2.
NPH-II purification. Aliquots (0.23 µg) of the
glycerol gradient preparations of wild-type (WT) NPH-II and the
indicated NPH-II-Ala mutants were electrophoresed through an 8%
polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized
by staining with Coomassie blue dye. The positions and sizes (in
kilodaltons) of marker proteins are indicated at the left. The
polypeptide corresponding to NPH-II is indicated by the arrow on the
right.
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Helicase activities of mutated His-NPH-II proteins.
Helicase
activity was tested by using a dsRNA substrate formed by annealing a
98-nucleotide RNA strand to a 38-nucleotide radiolabeled RNA strand to
produce a 29-bp duplex with 5' and 3' tails (27). The extent
of RNA unwinding by wild-type NPH-II was proportional to the level of
input protein (Fig. 3). Five of the eight
new mutants displayed severe reductions in helicase activity: T192A
(motif I), R229A (motif Ia), E300A (motif II), and T326A and T328A
(motif III). Their specific activities were <1% of the wild-type
value (Fig. 3). The K191A and H299A proteins were also defective in RNA
unwinding, as noted previously (Fig. 1). In contrast, mutants T187A and
Q194A displayed ~50% of the wild-type specific activity, and P228A
was 20% as active as wild-type NPH-II (Fig. 3). RNA unwinding by
wild-type NPH-II and each of the catalytically active mutants was
completely dependent on added ATP (data not shown).
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FIG. 3.
RNA unwinding by wild-type and mutant NPH-II proteins.
Helicase assays were performed as described in Materials and Methods.
The extent of RNA unwinding by wild-type (WT) and mutated NPH-II
proteins is plotted as a function of the amount of input enzyme. Each
data point represents the average of two independent determinations.
The source of the protein preparation is indicated in the key to
symbols.
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RNA binding by His-NPH-II mutants.
NPH-II binds to
single-stranded nucleic acid in the absence of NTPs. The complex of
NPH-II bound to ssRNA is stable and can be easily resolved from free
RNA by native gel electrophoresis (8, 10, 27, 28). The
radiolabeled RNA ligand used in standard binding assays corresponds to
the 98-mer strand of the helicase substrate. The amount of the shifted
protein-RNA complex formed by wild-type NPH-II and by each of the Ala
mutants varied linearly with input protein; every one of the
Ala-substituted proteins retained the ability to bind RNA (Fig.
4). The electrophoretic mobility of the
RNA-protein complex formed by each of the mutant His-NPH-II proteins
was similar to that of the wild-type enzyme analyzed in parallel (not
shown). All of the NPH-II-Ala proteins displayed an affinity for ssRNA
that was essentially identical to that of the wild-type enzyme (Fig.
4).
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FIG. 4.
Binding of wild-type and mutated His-NPH-II proteins to
ssRNA. Binding of NPH-II to a radiolabeled 98-mer ssRNA was measured in
a gel shift assay as described in Materials and Methods. The extent of
protein-RNA complex formation is plotted as a function of input enzyme.
The symbols denoting identities of the proteins used in the titration
experiments are as shown in Fig. 3.
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To relate the RNA binding properties of the mutant proteins more
directly to the observed effects on helicase activity, we performed
native gel shift assays of the binding of the NPH-II mutants to the
helicase substrate itself (10, 28). All 10 mutated proteins
bound to the helicase substrate with affinity similar to that of
wild-type NPH-II (not shown). Thus, the deleterious effects of the
K191A, T192A, R229A, H299A, E300A, T326A, and T328A mutations on RNA
unwinding could not be attributed to lack of binding to the helicase
substrate.
Mutational effects on nucleic acid-dependent ATPase.
The
extent of ATP hydrolysis by NPH-II in the presence of a ssDNA cofactor
was proportional to input enzyme (Fig.
5). The specific activities of the
mutants, expressed in parentheses as the percentage of the wild-type
value, were as follows: T187A (68%), K191A (<0.2%), T192A (<0.2%)
Q194A (55%), P228A (91%), R229A (2%), H299A (58%), E300A (1%),
T326A (51%), and T328A (52%). The wild-type and mutant proteins were
also assayed for ATP hydrolysis in the presence of a ssRNA cofactor
(not shown). The specific activities of the mutants, expressed in
parentheses as the percentage of the wild-type value, were as follows:
T187A (56%), K191A (<0.2%), T192A (<0.2%), Q194A (67%), P228A
(61%), R229A (4%), E300A (2%), T326A (61%), and T328A (61%). In
light of our previous findings that the H299A mutation rendered the
ATPase activity of NPH-II nucleic acid independent (8), we
assayed each of the mutants for ATPase in the presence and absence of
polynucleotide cofactors. ATP hydrolysis by T187A, Q1941, P228A, R229A,
E300A, T326A, and T328A was stimulated 5- to 30-fold by nucleic acid
(not shown). The K191A and T192A proteins displayed no detectable
activity up to 60 ng of input enzyme in the presence or absence of a
nucleic acid cofactor.
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FIG. 5.
ATP hydrolysis by wild-type (WT) and mutated His-NPH-II
proteins. ATP hydrolysis by NPH-II in the presence of single-stranded
M13mp18 DNA was assayed as described elsewhere (8). ATPase
activity is expressed as nanomoles of 32Pi
released from [ 32P]ATP during a 30-min incubation at
37°C and is plotted as a function of input enzyme. Each data point
represents the average of two independent determinations. The protein
preparations used for each titration experiment are indicated in the
key to symbols.
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The mutational effects on the DNA-dependent ATPase, RNA-dependent
ATPase, and helicase activities of NPH-II are summarized in Tables
1 and 2. We
operationally define as nonessential those residues at which alanine
substitution elicited less than an order of magnitude effect on
helicase or ATP specific activities. By this criterion, conserved
residues Thr-187 in motif I and Pro-228 in motif Ia are nonessential.
The eight other residues analyzed above are defined as important for
NPH-II function, because alanine substitutions reduced ATPase or
helicase activities to less than 10% of the wild-type values. Two
functional classes of important residues are illuminated by our
results: (i) those at which side chain removal coordinately inactivates
the ATPase and helicase functions, e.g., Lys-191 and Thr-192 in motif
I, Arg-229 in motif Ia, and Glu-300 in motif II; and (ii) those at
which side chain removal preserves phosphohydrolase activity but
eliminates the capacity for duplex unwinding, e.g., His-299 in motif II
and Thr-326 and Thr-328 in motif III.
NPH-II RNA unwinding activity is essential for vaccinia virus
replication.
Three ts vaccinia virus mutants isolated
by Fathi and Condit (ts10, ts18, and
ts39) are unable to produce infectious virus at 40°C
because they encode mutated versions of NPH-II (4, 5). The
nph2-ts viruses assemble normal-appearing progeny virions at
the restrictive temperature. These particles contain a wild-type complement of virion polypeptides (including vaccinia virus RNA polymerase and other enzymatic components of the virus core), but they
lack NPH-II (11). We previously correlated the loss of
infectivity of the mutant virions with a defect in the production of
early mRNAs (11). In the present study, we exploited the nph2-ts viruses to test the biological activity of
NPH-II-Ala mutants whose biochemical defects have been determined. This
was done by DNA-mediated marker rescue of the nph2-ts
viruses with wild-type and mutant NPH-II genes.
ts39 encodes a G469S mutation (4); the lesion is
located 22 amino acids upstream of motif VI (QRKGRVGRVNPG). The
biochemical consequences of alanine substitutions at eight amino acids
in this motif are summarized in Table 3.
Marker rescue was performed as follows: NPH-II-Ala DNA was transfected
by electroporation into ts39-infected cells, and the cells
were then plated onto virgin monolayers at the restrictive temperature.
Formation of infectious centers is contingent on reversion of
ts39 to the wild-type Gly at codon 469; this occurs via
recombination between the transfected NPH-II-Ala plasmids and the newly
replicated viral genomes in the transfected cells. The revertants can
then spread to the uninfected cells of the monolayer at 40°C. The
monolayers were harvested 2 days after transfection and tested for
marker rescue by determining the titer of virus capable of plaque
formation at 40°C. It is expected that mutants encoding functional
proteins will rescue like wild-type NPH-II but that those encoding
proteins that are nonfunctional (or ts) in vivo will display
rescue frequencies several orders of magnitude lower than wild-type
NPH-II. For NPH-II alleles containing alanine substitutions in motif
VI, there was a strong correlation between the extents of marker rescue
and the specific ATPase and helicase activities of the encoded gene products. N500A and P501A, which are as active as wild-type NPH-II in
vitro, rescue ts39 as effectively as the wild-type gene,
yielding 5 × 106 to 8 × 106
revertants per transfection. Rescue was absolutely dependent on
introduction of a functional NPH-II gene, insofar as transfection of
the plasmid vector yielded <10 viruses capable of plaque formation at
40°C (Table 3). This finding attests to the low level of spontaneous reversion by ts39. Mutant alleles Q491A, G494A, R495A,
G497A, R498A, and G502A, which encode catalytically impaired
ATPases/helicases (9), yielded only 0.6 × 104 to 1 × 104 revertants per
transfection (Table 3).
A defective allele can give rise to rescued virus by recombination
between the engineered mutation and the ts lesion. Given the
close physical linkage between the ts39 lesion and the Ala substitutions in motif VI, we expect such events to be relatively rare
compared to events in which the Ala mutations are introduced and the
ts lesions are reverted. Because the NPH-II Ala genes were
engineered to gain or lose a diagnostic restriction site at the mutated
codon, we were able to determine if the Ala mutation was incorporated
into the revertant viruses. To do this, we plaque purified rescued
viruses and then screened for the presence of the diagnostic
restriction site in a 1,718-bp NPH-II gene fragment prepared by PCR
amplification of DNA from a monolayer infected with the plaque-purified
virus. If the NTPase or helicase activity of NPH-II is essential for
replication, then we will not be able to recover any rescued viruses
that have taken up the ATPase-inactivating alanine mutations; instead,
all rescued viruses derived from such transfections will be wild type
with respect to the restriction site polymorphism that overlaps the
alanine mutation. On the other hand, if the engineered mutant supports
replication, we will detect the restriction site in the majority of the
rescued virus isolates.
The DNA of plaque-purified revertants from the N500A and P501A
transfections displayed a diagnostic loss of a BstNI site
(Fig. 6B), indicating that the N500A and
P501A mutations were present in viruses that grew at 40°C. Ten of
eleven P501A viruses and two of two N500A viruses that were genotyped
in this way had taken up the Ala mutations (Fig. 6A). We surmise that
Pro-501 and Asn-500 are not important for vaccinia virus replication.
In contrast, none of 24 plaque-purified revertants from cells
transfected with the catalytically defective G502A allele had
incorporated the restriction polymorphism linked to the G502A mutation.
All 24 virus isolates retained the wild-type genotype (Fig. 6). Thus, the low frequency of marker rescue by G502A compared to N500A and P501A
reflected the requirement for a crossover between the Ala-502 and
Gly-496 codons.
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FIG. 6.
Genotyping of rescued viruses. (A) Summary of viral
genotypes determined by restriction analysis of PCR-amplified NPH-II
genes from plaque-purified virus revertants. WT, wild type. (B)
Genotyping by restriction digestion. Representative restriction
endonuclease digests were analyzed by agarose gel electrophoresis. The
NPH-II gene was PCR amplified from DNA isolated from cells infected
with wild-type vaccinia virus or plaque-purified revertants recovered
after transfection with the indicated NPH-II-Ala genes. The 1,718-bp
PCR products were digested with restriction endonuclease
Asp718 (lanes 1 and 2), AvrII (lanes 3, 4, 9, 10, and 11), BstNI (lanes 5, 6, 12, 13, and 14), or
EagI (lanes 15 to 17). For those transfections where the
mutant allele was not incorporated into any rescued viruses (R229A,
G502A, and T328A), a control restriction digest was performed with the
1,718-bp PCR product amplified from the pTM-based plasmid containing
the indicated alanine substitution (lanes 11, 14, and 17). DNA was
visualized by staining the agarose gel with ethidium bromide. The
positions and sizes (in base pairs) of linear DNA markers are indicated
on the left. The 1.7-kbp PCR fragment is depicted as a horizontal bar.
The locations of the restriction sites within the wild-type NPH-II gene
are denoted below the bar. The Q194A mutation eliminates the
Asp718 site. The P228A and R229A mutations destroy the
AvrII site. The N500A, P501A, and G502A mutations eliminate
the downstream BstNI site. The T328A mutation creates a
unique EagI site; digestion at this site (indicated by the
arrow above the bar) generates a doublet of 877- and 841-bp
fragments.
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To analyze the in vivo effects of mutations in motifs II and III, we
performed marker rescue experiments with vaccinia strain ts10. The ts10 lesion (A310E) is situated 10 amino acids downstream of motif II (DEVHE) and 15 amino acids upstream
of motif III (TAT). The seven mutant alleles (D296A, E297A, H299A,
E300A, T326A, and T328A) encoding defective helicases rescued
ts10 with 300- to 1,000-fold-lower efficiency than wild-type
NPH-II (Table 1). The results are instructive insofar as all three
mutations (H299A, T326A, and T328A) that abrogated the helicase
function but spared ATPase activity were all lethal in vivo.
Genotyping of 24 plaque-purified T328A revertants showed that every
isolate was wild type with respect to the EagI restriction
site at the T328 locus (Fig. 6). This finding argues that although the
NTPase activity of NPH-II is essential for vaccinia virus replication,
it is not sufficient if uncoupled from the capacity for duplex
unwinding.
Similar rescue analyses were performed with ts18, which
encodes a G208R mutation (4) located 16 amino acids
downstream of motif I and 20 amino acids upstream of motif Ia. Mutants
T192A in motif I and R229A in motif Ia displayed the lowest extent of ts18 rescue compared to the wild-type NPH-II gene (Table 2); each of these alleles encodes a catalytically defective
ATPase/helicase. Genotyping of 24 plaque-purified R229A revertants
showed that every isolate was wild type with respect to the R229 locus
(Fig. 6). We surmise that the R229A mutation is lethal in vivo. Alleles T187A, Q194A, and P228A, which encode active NTPases/helicases, displayed the highest extents of ts18 rescue (Table 2).
Genotyping of the Q194A revertant showed that 12 of 13 isolates had
taken up the Q194A mutation (Fig. 6). In the case of P228A, 5 of 14 isolates had the mutant genotype. Thus, Gln-194 and Pro-228 are not
critical for virus replication.
| |
DISCUSSION |
In this study, we identified four new amino acid side chains that
are essential for the helicase activity of vaccinia virus NPH-II. This
effectively completes our assessment by alanine scanning of the
functions of conserved helicase motifs I, Ia, II, III, and VI. Of the
22 residues targeted here and in prior studies, 15 are essential for
helicase activity. These are Lys-191 and Thr-292 in motif I, Arg-229 in
motif Ia, Asp-296, Glu-297, His-299, and Glu-300 in motif II, Thr-326A
and Thr-328A in motif III, and Gln-491, Arg-492, Gly-494, Arg-495,
Gly-497, Arg-498, and Gly-502 in motif VI. Individual residues in
motifs I, Ia, II, and VI participate in NTP hydrolysis, whereas motif
III and the His moiety of the DExH box function to couple the energy to
NTP hydrolysis to dsRNA unwinding. None of the mutations that
inactivate the NTPase or helicase functions have a significant effect
on nucleic acid binding.
Functions of the helicase motifs in NTP hydrolysis and duplex
unwinding.
Comparison of our latest mutagenesis results for NPH-II
with those reported for other DEAD- or DExH-box proteins underscores the theme that structure-function relationships at conserved residues in the helicase motifs are context dependent. This is especially so
with respect to the role of motif III in coupling the phosphohydrolase and unwinding steps. For example, changing the SAT motif of translation initiation factor 4A to AAA preserves ATP hydrolysis but abrogates RNA
helicase activity (23, 24). This result agrees with our findings for NPH-II. In contrast, a TAT-to-AAT change in motif III of
HCV RNA helicase, which had no effect on RNA binding, coordinately reduced the phosphohydrolase and RNA unwinding activities (to 21 and
53% of the wild-type values, respectively); i.e., the NTPase and
helicase activities remained coupled (13). The structure of
the HCV RNA helicase NS3 has been solved by X-ray crystallography (33). Motif III is located on a 
-strand that connects a
protein domain containing the GKT and DExH elements to a second domain containing motif VI. Although Yao et al. (33) propose that
the TAT element transmits an NTP-dependent conformational switch, the
mutational findings for HCV RNA helicase do not yet reveal such a role.
It is conceivable that the second threonine of the TAT motif is
involved in energy coupling; the results of mutating that side chain
have not, to our knowledge, been reported.
The histidine moiety of the DExH box of NPH-II functions in coupling
NTP hydrolysis to nucleic acid binding. Changing the histidine to
alanine elicits a gain of function, whereby the enzyme hydrolyzes ATP
in the absence of a nucleic acid cofactor (8). We proposed
that the histidine side chain exerts a negative effect on ATPase
activity of the enzyme in the ground state and that this is relieved
upon nucleic acid binding. The DExA mutation of NPH-II also uncouples
the phosphohydrolase and helicase functions. In the HCV helicase
structure (33), the histidine side chain participates in a
network of hydrogen bond interactions that involves the threonine
(boldface) of the TAT motif (which is critical for
NTPase/helicase coupling in NPH-II) and the glutamate of the DExH box
(which is essential for ATP hydrolysis in NPH-II). A recent study
suggests that the DExH-box histidine couples the ATPase and helicase
activities of HCV NS3. Heilek and Peterson (12) found that
the His
Ala change abolished helicase activity but had no deleterious
effect on nucleic acid-dependent ATPase. In fact, the specific activity
of the DExA mutant was slightly higher than that of wild-type NS3
(12). However, Kim et al. (13) reported that the
same HisAla mutation of NS3 was active as an RNA helicase (with 60%
of the wild-type specific activity). The reasons for the discordant
findings of these two groups regarding the helicase activity of the
DExA protein are unclear. Kim et al. (13) noted that the
DExA mutant of NS3 was constitutively activated for ATP hydrolysis in
the absence of nucleic acid. This feature is reminiscent of our
findings for NPH-II.
Motifs I and II, which are essential for ATP hydrolysis, are in close
proximity in the NS3 crystal structure; the Lys side chain in the
GKT element makes a salt bridge to the Asp of the DExH box
(33). The NS3 structure was solved in the absence of
nucleotide. Structures are available for other helicases (Rep and PcrA)
and NTPases (RecA and p21) with nucleotide bound (17, 20, 30,
31). In p21 bound to GMPPNP, the 
-amino group of lysine in the
GKS element contacts the and phosphates. The adjacent serine
hydroxyl (equivalent to the essential Thr-192 of NPH-II) interacts with
magnesium coordinated to the and phosphates (20).
Similar contacts are proposed for the GKT motif in the RecA-ADP
cocrystal (30). The Lys and Thr side chains of the Rep and
PcrA helicases complexed with ADP are also poised near the phosphate (17, 31). The aspartic acid residue in the B motif
of the p21 structure (equivalent to the Asp in the DExH box) interacts
via a water molecule with the magnesium ion (20). The
aspartate and glutamate residues of the PcrA DExx box are located near
the bound ADP (17, 31). The Glu side chain of the DExx box
is in the same position in space as Glu-96 of RecA (17, 31),
which has been proposed to serve as general base to activate water
during attack on the phosphate (30). The aspartate and
glutamate side chains are both essential for ATP hydrolysis by NPH-II
(8). A second glutamate (boldface) immediately adjacent to
motif II (DEVHE) is also critical for ATP hydrolysis by
NPH-II. This residue is conserved in a subset of DExH proteins that
includes the helicase A and splicing factors Prp2, Prp16, and Prp22,
but it is not conserved in HCV helicase (Fig. 1).
A glutamine side chain (Gln-194) in RecA is proposed to interact with
the phosphate of ATP and thereby elicit a conformational change in
the protein that enhances DNA binding affinity (30). A
glutamine residue is conserved at the corresponding position of the
PcrA structure (31). The analogous residue the DExH family appears to be the glutamine in motif VI (QRxGRxGRxxxG). Mutation of
this glutamine to alanine in NPH-II reduced helicase specific activity
to 2% of wild-type activity, and ATPase specific activity to 9% of
wild-type activity, but had no effect on RNA binding (9).
Note that NPH-II differs from RecA in that NPH-II binds to RNA with
high affinity in the absence of NTPs. A GlnHis mutation in motif VI
of NS3 abolished both ATPase and helicase functions, also without
affecting RNA binding (13). There is complete agreement between our findings (9) and those of Kim et al.
(13) that mutations of the conserved amino acids in motif VI
have no significant effect on RNA binding affinity. In the NS3 crystal
structure, the three conserved arginine side chains of motif VI are
solvent exposed (33). We found that alanine substitution at
each of the three conserved Arg residues of motif VI reduced
phosphohydrolase activity by an order of magnitude (9). Kim
et al. report that mutations of the second and third Arg residues
abolished ATPase activity, but alanine replacement at the first Arg had
no effect on NS3 function (13). This again points up the
context dependence of mutational effects at conserved residues.
The ATPase and helicase activities of NPH-II are essential for
vaccinia virus replication.
Rescue of ts vaccinia
viruses by mutant alleles encoding biochemically characterized proteins
provides a general method to assess whether the catalytic activities of
a given gene product are critical for virus growth. Ellison et al.
(3) have used this approach to demonstrate that the
uracil-DNA glycosylase activity of the vaccinia virus D4 protein is
essential for virus replication, i.e., by showing that mutations at the
active site are incompatible with virus viability. The method is
particularly powerful when the viral gene product has more than one
biochemical activity. This is the case for NPH-II, which catalyzes both
NTP hydrolysis and duplex unwinding. The two activities are
interdependent, insofar as unwinding requires NTP hydrolysis, but not
obligately coupled, as shown by our analyses of mutants that hydrolyze
ATP but do not unwind dsRNA. Plasmid-mediated rescue experiments
described above show that the phosphohydrolase and RNA unwinding
activities of the NPH-II protein are both essential for vaccinia virus
replication. NPH-II-Ala alleles encoding catalytically competent
enzymes (e.g., N500A and P501A) rescued an nph2-ts virus at
high frequency, and progeny revertants containing the Ala mutations
were readily isolated. In contrast, NPH-II alleles encoding
ATPase-defective proteins rescued at low frequency, and none of the
isolated revertants contained the Ala mutation. Mutations that blocked
RNA unwinding without significantly reducing ATPase specific activity
were also lethal. The implication is that duplex unwinding by NPH-II is critical for vaccinia virus replication. The P228A allele, the product
of which is one-fifth as active as wild type NPH-II in RNA unwinding,
is capable of rescuing nph2-ts, whereas mutant alleles
encoding proteins whose helicase activity is 
10% of wild-type activity are lethal. This finding suggests that vaccinia replication is
contingent on a threshold level of duplex unwinding by NPH-II.
It is worth emphasizing that essentiality of the catalytic activities
of DExH proteins cannot be taken for granted. For example, the yeast
DExH-box helicase Rad3 is essential for cell growth (i.e., it cannot be
deleted), but the ATPase activity of Rad3 is not critical for cell
growth, insofar as a rad3 mutant with no ATPase activity is
viable (32). The ATPase and helicase activities of the
Escherichia coli DExH-box helicase PriA are not necessary for PriA to assemble the DNA replication primosome in vitro or for PriA
to function in homologous recombination and double-strand break repair
in vivo (15, 25, 34). Thus, it was not a foregone conclusion
that NPH-II helicase activity would be essential for vaccinia virus
replication.
We showed previously that NPH-II plays an important role during the
transcription of early mRNAs by the vaccinia virus particle (11). In contrast, NPH-II is not required for transcription of early genes in a reconstituted in vitro transcription system. We
suggested that NPH-II facilitates transcription in the virion by
preventing R-loop formation behind the elongating RNA polymerase (11). R-loop formation is favored by the negative
superhelicity generated during transcription of divergently oriented
genes (2), as is the case with early transcription by
vaccinia virions. The encapsidated vaccinia virus DNA topoisomerase
(29) might be expected to relieve some of this superhelical
tension and thereby limit R-loop formation. However, the findings of
Fernandez-Beros and Tse-Dinh (6) that vaccinia virus
topoisomerase preferentially removes positive supercoils, and that its
action on actively transcribed circular plasmids results in the
accumulation of negative supercoils, raises the prospect that the
topoisomerase might actually promote RNA loop formation during mRNA
synthesis by virions. We posit that the RNA-DNA helicase activity of
NPH-II (10) serves to disrupt these R loops. This hypothesis
is consistent with the present findings that the duplex unwinding
activity of NPH-II is essential in vivo. Our findings do not exclude
other functions for the NPH-II helicase activity during vaccinia virus
gene expression.
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Abstract
Introduction
