From the
The poliovirus polypeptide 3AB, the precursor of the
genome-bound VPg protein, stimulates in vitro the synthesis of
poly(U) directed by the viral polymerase 3D
Poliovirus, a member of the Picornaviridae family, possesses a
single-stranded genome of positive polarity(1) . The mechanism
of replication of poliovirus RNA is understood only in general terms.
Upon infection of permissive cells, the viral RNA gains access into the
cytoplasm, where the replication of the genome takes
place(2, 3) . First, the viral RNA, after its
translation to produce virus-specific polypeptides, serves as a
template to synthesize minus strand RNA. The minus strand is used to
synthesize more plus strand RNAs, which can be used as templates for
new minus strand RNAs, as messenger RNAs, or as genomic virion
RNAs(4) . All nascent viral RNA strands synthesized in the cell
are covalently linked to the VPg protein (VPg =
3B)(5, 6) . The mechanism by which this peptide is bound
to the 5`-end of the viral genome constitutes one of the puzzling
unanswered questions in the poliovirus replication field. The viral
polymerase (3D
The proposed function of 3AB in
poliovirus RNA replication mainly arose from genetic studies. The first
genetic evidence was reported by Berstein and Baltimore(21) .
These authors described a cold-sensitive poliovirus mutant encoding for
a 3A protein with a single amino acid insertion. This mutant showed a
small plaque phenotype and severe replication defects at the
restrictive temperature. Since then, multiple poliovirus 3A mutants
with defects in RNA replication have been
isolated(22, 23) . Many of these mutations were mapped
to the hydrophobic domain of 3A, which presumably promotes the binding
of the protein to the cellular membranes. Amino acid changes in the
hydrophobic domain commonly result in death or mutant viruses with
drastic impairments in the replication of the RNA(23) . If RNAs
obtained by transcription from these mutant cDNAs are used to program
HeLa cell extracts for in vitro translation, in some cases
aberrant polyprotein-processing patterns are obtained. Thus, the
hydrophobic region of 3A may serve to anchor the polypeptide, or part
of it, to the membranous replication complex, a process that can
modulate the proteolytic cleavage of the polypeptide. Recently, a key
finding to understanding the function of 3AB in RNA replication has
been reported(24) . 3AB protein purified to almost homogeneity
possesses an intrinsic stimulatory activity of the viral polymerase
3D
To confirm the data on RNA binding, a gel retardation
assay was used in some instances to detect RNA-protein complexes. The
purified proteins were incubated with 10 ng of a 51-nucleotide-long
riboprobe of positive polarity, which contains a region of the 2B
poliovirus gene (nucleotides 3925-3952) in addition to some
plasmid-derived nucleotides. The analysis of the free and bound probe
forms was carried out as described previously(26) . To quantify
the amount of 3AB protein present in crude extracts, we used an anti-3A
antiserum that recognizes an epitope located between amino acids
30-44 of the 3A protein.
In order to ascertain the function of 3AB during the
poliovirus replication cycle, a biochemical and molecular genetic
analysis of this gene has been carried out. Recently, a novel procedure
for the study of protein RNA interactions has been
developed(26) . This device allowed us to demonstrate an RNA
binding activity for 3AB protein. The RNA binding capability of 3AB was
studied by assaying a number of mutants. Among these, five out of the
nine positively charged lysine residues in the 3AB molecule were
tested, leading us to the identification of amino acid residue 107 as
an important part of the RNA binding domain. Interestingly, the
hydrophobic domain of 3AB was not shown to be relevant to eliciting an
RNA binding activity, despite the fact that this region of the protein
has been involved in the replication of the genome. Probably, 3AB is
endowed with different and independent domains implicated in RNA
synthesis. In addition, Lama and Carrasco
Under our experimental conditions,
the binding of 3AB seems to be nonspecific. The purified protein bound
equally well to three different biotinylated riboprobes representing
both extremes of the genome and an internal fragment of the
complementary viral RNA (minus strand RNA). Another report has shown
the binding of 3AB to
The RNA binding-defective 3AB
proteins showed reduced stimulatory activities of the poly(U) synthesis
directed by the viral polymerase. Thus, the single amino acid
substitution at position 107 showed 10-20% of the wild type
stimulatory activity whereas no transactivation was observed with
either the double mutant K9E/K107E or the 3A protein, lacking the
complete VPg sequence. A reduction was observed with the single mutant
K9E-3AB, although this reduction was partially overcome at greater 3AB
concentrations. This defect does not seem to be important since the
K9E-3AB poliovirus mutant replicated at wild type levels. Introduction
of a positively charged residue at position 9 may drastically change
the electrostatic and/or structural requirements for RNA binding
function. Glutamic acid at position 9, but not at position 107,
drastically changed the electrophoretic behavior of 3AB in
SDS-polyacrylamide gels (see for instance Fig. 3A).
However, the key findings of this work were two: 1) the purified 3A
protein does not show either RNA binding or transactivating activity,
and 2) mutations in the 3A and VPg region cause a synergistic effect if
placed together in the same 3AB molecule. This synergistic effect was
also observed in the plaque size phenotype and in the time of
appearance of CPE after transfection of HeLa cells with the mutant
RNAs. Taken together, these results suggest that 3AB, but not 3A, plays
an important role in the synthesis of virus-specific RNAs. This
function must probably rely on its RNA binding and transactivating
activities. Additional mutants with defects in both RNA binding and
transactivation of 3D
The level of accumulation of both
plus and minus viral RNA strands was inhibited by more than 90% at 4 h
postinfection, and the virus yield was reduced more than 100-fold in
HeLa cells infected with the K107-3AB poliovirus mutants. Therefore,
these alterations seem to take place in the replication of both strands
of the genome, probably at an early step in the infection when the
amount of viral RNA is low. The replication impairment may be due to a
defect in the elongation step or in a common step in the synthesis of
both kinds of strands. Otherwise, we cannot rule out that the
uridylylation of VPg is being affected in these mutant viruses.
Nevertheless, the strong correlation between the in vitro stimulation of poly(U) synthesis produced by 3AB and the
replication rate of the mutant viruses makes it likely that the RNA
synthesis defect takes place in a step that requires the stimulatory
effect of 3AB to be carried out. Unfortunately, we are not able to
contrast both hypotheses. In vitro experiments would be
necessary to demonstrate a reduced capability of the mutant 3ABs to
function as substrates in the uridylylating reaction. However, the
responsible enzyme has not been identified yet. On the other hand, VPg
uridylylation has been studied in vitro with polio-infected
HeLa cell extracts. In order to prepare these extracts, cells are
infected with as much as 500 pfu/cell(19) . These experiments
are incompatible with the replication-defective mutants described
herein.
The function of 3AB does not seem to be essential for the
virus. A null 3D
Important considerations must be kept in mind to understand how 3AB
works in vivo. We have demonstrated that 3AB seems to bind RNA
probes in a nonspecific way, but then how is the specificity for polio
RNA obtained? Our attempts to demonstrate such preference by, for
instance, the 3`-end of the genome have failed to date. In any case,
this selectivity should be extraordinarily high to ensure that the
replication complexes can trap viral RNAs in a cytoplasm full of
millions of molecules of cellular RNAs. Another intriguing possibility
comes up if 3AB binds much faster to the messenger RNA from where it is
being translated. In this way, the chance to bind to cellular RNAs
would be reduced to a minimum without the requirement for any specific
signal in the poliovirus genome. This ``trapping'' activity
might not be so important at late steps of infection, when thousands of
virus-specific RNA molecules are found in the cytoplasm. This could be
an explanation for the overcoming of the growth defects observed with
the K107E mutants at late steps of the infection. Otherwise, we cannot
rule out the existence of factors that in conjunction with 3AB direct
the viral polymerase to specific regions of the genome.
Another
puzzling question arising from our results is, ``Why does
3D
The above model
implies that in vivo the stimulatory activity of 3AB is
partially due to its interaction with either 3D
The expert help of Noem Sevilla is acknowledged. We
thank Luis Blanco, Margarita Salas, and Isabel Najera for critical
reading of the manuscript. The invaluable help of Luis Carrasco is also
acknowledged.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(Lama, J., Paul, A., Harris, K., and Wimmer, E. (1994) J. Biol. Chem. 269, 66-70), suggesting that 3AB could be
modulating the activity of the viral polymerase in poliovirus-infected
cells. To address the exact function of 3AB in the viral replication
cycle, a biochemical and molecular genetic analysis of 3AB has been
carried out. 3AB protein bound RNA probes in two different assays, and
amino acid positions implicated in the RNA binding activity of 3AB were
determined. Mutant proteins with reduced RNA binding activity were
unable to stimulate 3D
polymerase activity.
Purified protein 3A showed no RNA binding or 3D
stimulatory activity, but 3A and VPg mutations conferred a
synergistic effect on the 3AB functions. Polioviruses encoding for
these mutant 3ABs were constructed. These mutant viruses translated
their RNA genomes in vitro and processed their polyproteins as
wild type virus did. Cells infected with 3AB mutant viruses showed over
90% inhibition in the accumulation of plus and minus viral RNA strands
and more than 100-fold reduction of virus yield at 4 h postinfection.
Our results suggest that 3AB protein functions in vivo as a
co-factor of the viral polymerase and that the activity of 3AB may be
regulated by proteolytic processing.
) is the only polypeptide isolated
from infected cells with RNA-dependent RNA polymerase
activity(7, 8) . Thus, it has been considered the
protein responsible for the elongation of the RNA strands. In addition,
most if not all of the poliovirus nonstructural proteins have been
implicated in the replication of the
genome(9, 10, 11, 12, 13, 14, 15) .
Special interest has been focused on the 3AB protein. 3AB is the most
abundant precursor of the VPg protein(16) . Protein 3AB
localizes to the membranous replication complexes, where active
replication takes place(17, 18) . It has been postulated
that an uridylylated form of VPg or, more likely, an uridylylated form
of 3AB might serve as a primer to initiate polymerase activity by
3D
(19, 20) . However, the
nucleotidylyl transfer reaction has never been reproduced in vitro using purified components.
. Thus, in vitro poly(U) synthesis by
3D
is stimulated more than 50-fold in the
presence of recombinant purified 3AB. The transactivation activity
occurs in the absence of any detected uridylylation and was completely
dependent on the presence of a nucleic acid primer. These results
suggested that 3AB could function in vivo as a co-factor of
3D
in viral transcription. However, genetic
evidence supporting this hypothesis is still lacking. We now report the
characterization of mutant polioviruses with severe defects in viral
RNA replication. These viruses encode for 3AB proteins unable to
transactivate the viral polymerase.
Cells and Viruses
Virus propagation and virus
plaque assays were performed on HeLa R19 cell monolayers grown in
Dulbecco's modified Eagle's medium supplemented with 10%
calf serum. HeLa cell lysates for in vitro translation were
made from HeLa S3 cells grown in minimum essential medium modified for
suspension cells supplemented with 5% calf serum. The viruses used in
this work were obtained after transfection of HeLa cell monolayers with
infectious RNAs derived from the genomic cDNA cloned in the pT7XLD
vector(25) . Wild type and K9E-3AB viruses were plaque-purified
and amplified in HeLa cells up to titers of 0.5-1
10
pfu/ml.
(
)Stocks of the viruses
K107-3AB and K9E/K107 3AB were obtained after 3 days of transfection
with the corresponding RNAs. These stocks have titers of 0.5-1
10
pfu/ml and were directly used in the experiments
described. Additional plaque purification and amplification of these
viruses gave place to the generation of revertant viruses with wild
type plaque phenotype. The complete 3AB gene of the viral RNA of all
the stock viruses was sequenced. Virus RNA purification, cDNA
synthesis, and PCR from the cDNA obtained were performed to ensure that
the mutations introduced had been retained in the viral genome. To
create mutant cDNAs with the desired mutations the 3AB gene was
PCR-amplified from the corresponding pT7lac3AB plasmid. To do so, the
following primers were used: 5`-3A (5`-CCC GGG CAT ATG GGA CCA CTC CAG
TAT AAA GAC-3`) and 3`-3BC (5`-GCG TAA TCG AAC CCT GGT CCT TGT ACC TTT
GCT GTC CGA AT-3`). To amplify mutant DNAs K107-3AB and K9E/K107 3AB,
the PCR reaction was carried out with primers 5`-3A and 3`-3BC K107E
(5`-GCG TAA TCG AAC CCT GGT CCT TGT ACC TCT GCT GTC CGA AT-3`). After
PCR amplification, the fragments were digested with AvaII, and
the 3AB gene was exchanged with the equivalent AvaII DNA
fragment of the pT7XLD plasmid. The 3AB regions of the wild type and
mutant cDNAs were completely sequenced by the dideoxy method
(Sequenase, U.S. Biochemical Corp.). Transfection of HeLa cells was
carried out with Lipofectin reagent. Ten µg of in vitro transcribed RNA, without further purification, were mixed with 15
µg of Lipofectin and incubated at 37 °C for 2 h with HeLa cells
that had been previously washed with prewarmed phosphate-buffered
saline to remove calf serum. After this time, the cells were kept in 2%
calf serum for 3 days at 37 °C, and the viruses were recovered by
three cycles of freeze-thawing. To quantify the cytopathic effect, the
percentage of cells showing clear symptoms of morphological rounding
was estimated de visu.
RNA Binding Assays
To determine the RNA binding
capabilities of different 3AB proteins, we routinely used a method
recently described(26) . This procedure is based on the binding
of biotinylated RNA probes (riboprobes) to renatured proteins bound to
nitrocellulose sheets (Northwestern). Briefly, the proteins (purified
or crude extracts) were separated in 20% SDS-polyacrylamide gels and
transferred to nitrocellulose. After washing the nitrocellulose sheets
in a renaturation buffer, the nitrocellulose was first incubated with a
biotinylated riboprobe (40 ng/ml). Then unbound RNA was washed with
phosphate-buffered saline-Tween 20, and the membranes were incubated
with streptavidin-conjugated peroxidase. After washing off the
peroxidase, the nitrocellulose was soaked in luminol-luciferin solution
and exposed to x-ray films. The biotinylated riboprobes were obtained
by in vitro transcription of the appropriate DNAs in the
presence of 0.5 mM 21-UTP. The riboprobe
``5-leader'' consists of the genomic RNA sequence (plus
strand) between nucleotides 1 and 745. The riboprobe 3`-end contains
nucleotides 6516-7440 of the 3`-end of the genomic RNA, whereas
the riboprobe ``2.5 BamHI'', of negative polarity,
encompasses the complementary region to nucleotides 2099-4600 of
the genome.
Expression and Purification of Recombinant
Proteins
Expression in E. coli of 3A and 3AB proteins
was carried out from the plasmids pT7lac3A and pT7lac3AB, respectively,
as described previously(27) . The mutant 3AB proteins used in
this work were obtained by PCR-based random mutagenesis. To purify wild
type and K9E-3AB proteins, we followed a previously described
procedure(24) , but in this case, during the elution of proteins
from the S-Sepharose resin, the 100 mM salt wash was omitted.
The protein K107E-3AB behaved as the wild type through the DE-52
column; however, at the S-Sepharose binding step the pH of the column
had to be changed from MOPS (pH 7.2) to MES (pH 6.5). The posterior
elution was carried out in 200 mM NaCl, 25 mM MES (pH
6.5). As control, wild type 3AB protein was also purified in an
identical way, that is at pH 6.5 during the binding and elution of the
S-Sepharose column. K9E/K107E-3AB was purified as described above, but
after the DE-52 column the protein was concentrated with
ultrafiltration membranes (Amicon). Since this protein did not bind to
the S-Sepharose column under any of the conditions tested, this
fraction was no longer purified. Purification of 3A was stopped after
concentration of DE-52 fractions. However, this protein was not
recovered from the unbound proteins originally passed through the DE-52
resin, since under equilibration conditions the protein bound to the
column and was later recovered with a 50 mM NaCl wash.
Enzymatic Assay for Poliovirus RNA
Polymerase
Incorporation of [H]UMP was
measured using a poly(A)-dependent, oligo(dT)
-primed
poly(U) polymerase assay in the presence of purified
3D
. Recombinant 3D
, in a
poly(U)-free form, completely dependent on the presence of a nucleic
acid primer for its activity, was kindly provided by Dr. S. Plotch.
Reaction conditions for the polymerase assay have been described
before(24) . Standard transactivation reactions were carried out
in the presence of 20 ng/µl of 3A or 3AB proteins. Poliovirus
polymerase was used at 0.2 ng/µl.
Quantification of Plus and Minus Viral RNA
To
quantify the levels of viral specific RNA, HeLa cells were infected at
a multiplicity of infection of 0.1 pfu/ml. After 4 h of infection at 37
°C the cells were lysed, and the total cytoplasmic RNA was isolated
by the Nonidet P-40 lysis method (28) and used to synthesize a
cDNA strand with avian mieloblastosis virus retrotranscriptase
(Promega). Retrotranscriptase reactions were carried out in a 40-µl
mixture containing 10 mM Tris-HCl (pH8.3), 50 mM KCl,
1.5 mM MgCl, 0.01% gelatin, 10 mM
dithiothreitol, 0.1 mM dNTPs, 3 units of avian mieloblastosis
virus retrotranscriptase, 15 units of RNAsin inhibitor, 0.25 µM of the first primer, and 250 ng of total cytoplasmic RNA. After
incubation at 42 °C for 30 min, the retrotransciptase was destroyed
by boiling, and 0.25 µM of the second primer and 1 unit of Taq DNA polymerase were added (Perkin-Elmer). PCR
amplification took place during either 30 cycles (minus RNA) or 25
cycles (plus RNA) under the following conditions: 1 min at 93 °C, 1
min at 37 °C, and 5 min at 72 °C (last cycle, 10 min at 72
°C). The amount of amplified fragment was quantified in 1.0%
agarose gels stained with ethidium bromide. To detect plus strand viral
RNA, the retrotranscriptase reaction was carried out with the primer
3`-2B (GGC CCG GAT CCT TAT TAT TGC TTG ATG ACA TAA GGTA). The primer
5`-2A (GGC CGG CCC GGG ATT CGG ACA CCA AAAC) was later added in order
to proceed with the PCR reaction. To detect minus strand polio RNA, the
order of addition of the primers was reversed. As an internal standard
for the reaction the
-actin mRNA was amplified by RT-PCR in the
same test tubes. To this end, the primer 3`-
-actin (GGA AGC TTC
TAG AAG CAT TTG CGG TGG ACG ATG GAG GGG CC) was included into the
retrotranscriptase reaction, and the primer 5`-
-actin (GGG AAT TCA
TGG ATG ATG ATA TCG CCG CG) was later added during the PCR reaction.
-actin primers were used at 1 µM. Amplification of
polio and
-actin cDNAs results in fragments of 727 and 1170
nucleotides in length, respectively.
In Vitro Translation of Transcripts Derived from Mutant
cDNAs
Translations were carried out in HeLa S3 cell extracts
treated with micrococcal nuclease and programmed with infectious RNAs
synthesized by in vitro transcription of wild type or mutant
cDNAs. The preparation of extracts was essentially as described by
Molla et al.(29) . Reactions were incubated at 30
°C for 15 h with 100 ng of transcript RNA in the presence of 0.7
µCi/µl of [S]methionine. The synthesized
proteins were analyzed by autoradiography of SDS-polyacrylamide gels.
3AB Binds RNA in a Nonspecific Manner
Previous
experiments have shown a stimulating activity of poly(U) synthesis
directed by the viral polymerase 3D in the
presence of purified 3AB protein (24). These results suggested that 3AB
could be a co-factor of 3D
in viral
transcription. This stimulating activity was completely dependent on
the presence of the 3AB polypeptide but was not due to a
nucleotidylylation reaction on the 3AB protein, since the polymerase
activity of 3D
was dependent on the presence of
a nucleic acid primer in the reaction mixture. An alternative
hypothesis to explain the stimulatory effect of 3AB might rely on the
ability of the protein to increase the affinity or the binding of
3D
to either the template or the primer RNA.
Paul et al.(30) have shown that under conditions where
3D
and poly(A) template were reduced to a
minimum, the degree of transactivation was maximal, promoting more than
100-fold stimulation. These results suggest that 3AB may enhance the
interaction between the template RNA and the viral polymerase. A
prediction of this model is the interaction of 3AB with RNA and 3AB
with 3D
. As a first step in the elucidation of
the mechanism of action of 3AB, the RNA binding activity of purified
3AB was assayed (Fig. 1). Wild type purified 3AB was used to
analyze the binding activity to different biotinylated RNA probes. As
shown in Fig. 1A, purified 3AB bound three different
heteroligomeric RNA probes. The signal given by the binding reactions
increased linearly with the amount of 3AB protein used. The 5`-end of
the genome has been involved in the translation and replication of the
genome, and different independent domains have been identified for each
of these functions(31, 32) . A pseudo-knot structure has
been identified in the 3`-end of the genome(33) . To test the
binding of 3AB to these structures, riboprobes spanning both ends of
the genome were synthesized. The 3AB protein bound both probes,
suggesting that either 3AB binds specifically to both ends of the
genome or 3AB binds nonspecifically to polyribonucleic acid molecules.
3AB also bound to a riboprobe of negative polarity encompassing the
complementary region between nucleotides 2099 and 4600 of the viral
genome (Fig. 1A). Indeed, this binding reaction took
place to a greater degree than in the other cases, although these
differences may be due to differences in the level of incorporation of
biotinylated UTP in the larger, minus strand RNA probe. Thus, purified
3AB binds to different RNA molecules and does not seem to show any
sequence specificity for binding to the poliovirus genome. Further
corroborating these results, homopolymeric RNAs, either poly(A) or
poly(U), efficiently compete with the binding of the 2.5-BamHI
biotinylated riboprobe to 3AB (Fig. 1B). In fact, we
have tested other riboprobes, finding similar RNA binding capabilities
in all cases (data not shown). In order to confirm the RNA binding
activity found with the Northwestern assay, we carried out a gel
retardation assay to check the effect of 3AB on a
P-labeled riboprobe. As shown in Fig. 1C,
this procedure allow us to detect a retardation effect on the mobility
of a 51-nucleotide-long riboprobe. This binding was also avoided in the
presence of competitor cold poly(A) RNA.
Figure 1:
RNA binding activity
of purified 3AB protein. A, poliovirus 3AB protein was
purified from a recombinant E. coli clone, and different
amounts of protein were electrophoresed in SDS-polyacrylamide gels.
After transferring to nitrocellulose sheets, the proteins were
incubated with biotinylated RNA probes, and the bound RNA was detected
by the streptavidin-peroxidase-luciferin method after exposure of the
gels to x-ray films. RNA binding activity was assayed with a minus
strand RNA probe (2.5-BamHI) or with two different plus strand
probes encompassing both extremes of the genome (5`-leader and 3`-end). B, 5 µg of purified 3AB were used to estimate the RNA
binding capability in the presence of competitive amounts of nonlabeled
poly(A) or poly(U) RNA, following the procedure described above. C, different amounts of purified 3AB were incubated with a
51-nucleotide-long P-labeled riboprobe in the absence or
in the presence of competitive cold poly(A) RNA. The positions of the
free riboprobe and the 3AB-RNA complex were determined after
autoradiography of a 6% acrylamide gel.
Identification of Amino Acid Residues in 3AB Involved in
the Binding of RNA
The Northwestern procedure makes use of
proteins separated on polyacrylamide gels and transferred to
nitrocellulose sheets. Given sufficient quantities of the recombinant
protein, this assay permits the analysis of mutated proteins in total
lysates without further purification. Fig. 2shows the RNA
binding activity of a set of 14 different 3AB variants, including the
3A protein, which lacks the VPg sequence. A new binding protein is
observed when wild type 3AB protein is expressed in Escherichia
coli cells (Fig. 2, lane3). This band
does not appear in the control extract without the recombinant protein
( Fig. 2, lane2). Amino acid substitutions at
positions 9, 13, 54, 62, 63, 67, 75, 77, 79, 81, and 97 did not affect
the binding of the biotinylated riboprobe. Neither the introduction of
positively charged residues nor -helix-breaking proline residues
into the hydrophobic domain seems to affect the interaction of 3AB with
RNA. However, amino acid substitutions in this region produce mutant
viruses with defects in RNA replication(22) . Amino acid
substitution at position 107 (lysine 107 to glutamic acid) seems to
abolish the RNA binding capability of 3AB. Even though similar amounts
of K107E-3AB and, for instance, K97R-3AB, were contained in the E.
coli extracts, no new band was found in the position expected for
the recombinant K107E-3AB protein (Fig. 2, lane6), whereas a clear band was revealed in the position of
the K97E-3AB protein. No RNA binding activity was detected in the
K9E/K107E-3AB protein, which contains a double amino acid change in the
3A and VPg regions of the polypeptide. No binding was detected with the
3A protein.
Figure 2:
RNA binding activity of mutated 3AB
proteins in crude E. coli extracts. In the upperpanel (Northwestern) E. coli BL21(DE3)
cells were induced to synthesize either wild type or 3AB mutated
proteins. Fifty micrograms of total protein from crude extracts were
electrophoresed in SDS-polyacrylamide gels and transferred to
nitrocellulose to test the RNA binding capability, as shown in Fig.
1A. As a negative control, total proteins from bacteria
without the expression plasmid were run in lane2. Lane1 contains 2.5 µg of purified 3AB. In the lowerpanel, after processing to measure the binding
of the biotinylated riboprobe, the same nitrocellulose sheets were
washed and incubated with an anti-3A antibody to determine the position
(labeled with an arrow in the upperpanel)
and the amount of the recombinant proteins.
The level of expression of different 3AB proteins in E. coli was quite variable, making it difficult to compare the
binding activities of different mutated proteins. Thus, we decided to
purify the wild type 3AB and 3A proteins and the mutants K9E, K107E,
and K9E/K107E-3AB proteins (Fig. 3). Wild type and K9E-3AB
proteins were purified following a previously published
protocol(24) , although the mutated 3AB proteins changed their
chromatographic behavior to different degrees and small modifications
in the procedure were necessary. In the case of the proteins K107E and
K9E/K107E-3AB, only 70 and 40% respectively, of the recombinant protein
remains in the unbound fraction after the DEAE-cellulose purification
step (data not shown). This result suggests that both residues at
positions 9 and 107 are in the outer surface of the molecule, since
both proteins seem to interact more strongly with the positively
charged DEAE resin, as compared with either the wild type or the single
mutant K9E. As expected, the 3A protein, which does not contain the
highly basic VPg moiety, bound to the DEAE resin under conditions where
no wild type 3AB protein remained bound to the column (data not shown).
Proteins K107E and wild type 3AB were further purified through
S-Sepharose resin. However, to allow efficient binding of the former,
the pH of the column was reduced to 6.5. To assure that this change did
not alter the RNA binding and transactivating activities of the
protein, the wild type protein was purified at pH 6.5 also. Fig. 3A shows the degree of purity of the recombinant
proteins. The proteins purified through DE-52 and S-Sepharose resins
were more than 95% pure, whereas the proteins 3A and K9E-K107E-3AB were
obtained at 90 and 70% of purity, respectively, as estimated by
densitometric analysis of SDS-polyacrylamide gels. In Fig. 3B equal amounts of the purified proteins were used to estimate their
binding to the biotinylated RNA probe. In the upperpanel the proteins purified through S-Sepharose resin were analyzed.
Wild type protein and K9E-3AB mutant bound RNA to a similar degree.
However, the amino acid substitution at position 107 (K107E) greatly
reduced the binding activity, as compared with the wild type protein
purified in an identical way (pH 6.5). In the lowerpanel, the effect of the double substitution (K9E/K107E)
was assayed. To compare with accuracy the RNA binding activity of all
the variants, the proteins recovered after the DE-52 elution step were
assayed in Northwestern assays. As can be seen, the binding of the
biotinylated probe produced an intense signal with wild type 3AB,
whereas the K107E-3AB protein showed a drastic decrease in the RNA
binding capability. No binding was detected either with the double
mutant K9E/K107E-3AB, or with the wild type 3A protein. These results
demonstrate that amino acid position 107 in 3AB is important for RNA
binding activity. The results shown in Fig. 3B also
suggest that, even though the amino acid substitution at position 9
(K9E) does not produce any effect by itself, this change seems to cause
a synergistic effect on amino acid substitutions more than 100 residues
apart in the 3AB molecule. Introduction of a positively charged residue
at position 9 may alter the structure of the K107E-3AB protein, making
it less active. This structural alteration may not take place with the
natural residue (lysine) at position 107. Otherwise, a lysine at
position 9 may form part of the RNA binding domain but play a minor
role, only detectable if other important positions (e.g. amino
acid 107) are altered. To assure that the differences observed with the
biotinylated probes were reliable, the RNA binding activity of wild
type and K107-3AB proteins were tested by a gel retardation assay with
a P-labeled riboprobe (Fig. 3C). Incubation
of the RNA probe with identical amounts of either wild type or
K107E-3AB showed a different pattern for the RNA-protein complex. Thus,
incubation with 1 µg of K107E-3AB gave place to an RNA-protein
complex migrating slightly farther than the one observed with 1 µg
of wild type protein. Higher concentrations of wild type protein
produced a strong band corresponding to the retarded probe, whereas
incubation with the mutant protein only elicited a weak retardation.
These results corroborate the data presented in Fig. 3B demonstrating that the RNA binding activity is not completely
abolished in the K107E-3AB mutant. No retardation was found either with
3A or with the K9E/K107E-3AB (data not shown).
Figure 3:
Purification and RNA binding activities
of 3A and 3AB mutant proteins. A, five micrograms of the
purified 3AB proteins or 3.5 µg of 3A protein were submitted to
SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue.
Recombinant 3AB proteins with single amino acid substitutions at
positions 9 (K9E) and 107 (K107E) were purified from E.
coli-expressing cells through DEAE- and S-Sepharose
chromatographic steps. The recombinant 3AB protein with a double amino
acid substitution at positions 9 and 107 (K9E/K107E) and the wild type
3A protein were purified only through DEAE-cellulose (DE-52). The pH of
the buffers used to elute the S-Sepharose columns is indicated in the top of the panel. B, five micrograms of
purified 3AB and 3A proteins were assayed for RNA binding activity as
shown in Fig. 1A. The upperpanel shows the
activity of the fraction eluted from the S-Sepharose column at either
pH 7.2 or 6.5 (S-200 fractions). The lowerpanel shows the activity of the proteins eluted from the DE-52 column.
This gel has been overexposed to detect minimal amounts of bound
riboprobe. C, different amounts of wild type and K107E-3AB
proteins purified through DEAE- and S-Sepharose resins (the latter at
pH 6.5) were used to carry out a gel retardation analysis with a P-labeled probe, as indicated in Fig. 1C. The
positions of free and protein-bound probes are shown. Competitor
poly(A) RNA was included in some reactions.
Binding of RNA by 3AB Is Required for the Stimulation of
3D
The following step was to ascertain
how the RNA binding defects of 3AB affect the stimulatory activity on
the viral polymerase. Fig. 4shows the poly(U) synthesis mediated
by 3D in the absence or presence of different 3A
and 3AB proteins. As described previously, the wild type 3AB protein
induces a potent stimulation in the synthesis of poly(U)(24) .
Thus, addition of 10 ng/µl of wild type 3AB induced a 30-fold
stimulation on 3D polymerase activity. A small reduction of the
stimulating activity was found with the K9E-3AB protein. This effect
became clear at low concentrations of K9E-3AB. Nevertheless, when high
concentrations were assayed (16 ng/µl) the mutated protein produced
more than a 25-fold activation of 3D
(Fig. 4). On the other hand, amino acid substitution
at position 107 induced an important decrease in the 3D
stimulatory activity. Thus, while 8 ng/µl of the wild
type protein gave place to a 30-fold stimulation of
3D
, the poly(U) synthesis in the presence of the
same amount of K107E-3AB was only 3-fold stimulated, whereas no
transactivation occurred either with the double K9E/K107E-3AB mutant or
with the 3A protein. The results show a clear correlation between the
RNA binding and 3D
stimulatory activities of 3A
and 3AB proteins and, therefore, suggest that the binding of RNA by 3AB
is an essential step to exert its stimulatory effect on the viral
polymerase.
Figure 4:
Effect of RNA binding-defective 3AB
proteins on the polymerase activity of 3D. Poly(A)-dependent,
oligo(dT)-primed poly(U) synthesis catalyzed by purified poliovirus RNA
polymerase was assayed in the presence of different amounts of wild
type 3AB, K9E, K107, K9E/K107-3AB, or wild type 3A proteins. The
mixture was incubated for 80 min as indicated under ``Materials
and Methods.'' The data are plotted as the -fold stimulation
compared with the synthesis of poly(U) in the absence of 3AB or 3A
proteins.
Characterization of 3AB Poliovirus Mutants with Severe
Defects in RNA Replication
In order to understand the role of
3AB in viral replication, the 3AB mutations were introduced into the
viral cDNA genome. In vitro transcription to produce
infectious RNAs was carried out with T7 RNA polymerase. The transcript
RNAs were mixed with Lipofectin and used to transfect HeLa cell
monolayers. Then the appearance of the cytopathic effect was followed.
Wild type RNA produced around 50% CPE by 2 days after transfection (). The mutant K9E-3AB showed a small delay in the
appearance of the CPE. However, the mutant RNA encoding for the 3AB
protein with the lysine to glutamic acid change at position 107 showed
almost 2 days of delay in the appearance of the cytopathic effect, and
this was still greater in the case of the double K9E/K107E-3AB mutant.
The titer of the viruses recovered after 72 h of transfection was
estimated. Transfection with either wild type or K9E-3AB mutant gave
place to virus titers over 1 10
pfu/ml. However,
the viruses recovered after transfection with K107E-3AB, and
K9E/K107E-3AB showed titer reductions of 3 log units, suggesting that
the defect of the encoded 3AB proteins severely impaired the
replication of these viruses. As expected for replication defects, the
viruses recovered showed a drastic plaque size phenotype reduction (Fig. 5). Thus, viruses recovered after 3 days of transfection
with K107-3AB RNA produced plaques with an average diameter of 2.4 mm
(small-plaque phenotype), in contrast to the wild type and the K9E-3AB
counterparts, with an average plaque diameter of 4.9 mm after 72 h of
incubation at 37 °C (data not shown). Interestingly, the double
mutant showed a minute plaque size phenotype (0.8 mm in diameter).
These results suggest that, as in the RNA binding and
3D
stimulatory functions, mutations at positions
9 and 107 of 3AB have a synergistic effect on the replication of the
virus. The RNA sequence of each of these viruses was verified by RT-PCR
sequencing of the virion RNA. The same plaque phenotype was observed
when the amplified 3AB regions of each mutant were reintroduced in the
wild type cDNA clone and used to produce new infectious RNAs (data not
shown). Attempts to amplify and grow the K107E-3AB mutants repeatedly
failed. When these viruses were further grown in HeLa cell monolayers,
the obtained viruses rapidly reverted to the normal plaque size
phenotype. As an example, the plaque sizes of the viruses recovered 3
or 6 days post-transfection can be compared in Fig. 5. This was
not surprising, since although the RNA sequence of the pooled virion
RNA was shown to be correct for any of the viruses recovered after 3
days of transfection, the analysis of the plaque phenotype showed the
co-existence of large plaque revertant viruses among the small plaque
mutant ones. The proportion of revertants was as much as 1% of the
total population in the case of the double mutant after 3 days of
transfection (data not shown). None of the mutant viruses was
thermosensitive, and identical plaque size phenotypes were observed in
different cell lines (data not shown). The failure to grow these
viruses above 1
10
pfu/ml was a great handicap in
subsequent work. The growth pattern of the isolated mutants was studied
in one-step growth curves. HeLa cells were infected at the maximal
multiplicity of infection (0.1 pfu/cell) permitted by the low titer
stocks. The intracellular virus yield was monitored at different times
postinfection (Fig. 6). The mutants K107E and K9E/K107-3AB showed
different kinetics as compared with the wild type virus. This effect
was only patent at early times of infection. Thus, at 4 h
postinfection, the production of viruses was 100-1000-fold greater in
wild type virus than in any of the K107E mutants. This effect
disappeared later in the infection, and the rate of virus production
between 4 and 8 h was identical for all the tested viruses. Despite
this partial recovery, the total viral production at 8 h postinfection
was 10 times lower in both K107E-3AB mutant polioviruses. These results
suggest that the viruses that encode 3AB proteins defective in RNA
binding and 3D
transactivating activities suffer
an impairment in virus growth during an early step of the infection. No
deviation from the wild type behavior was found with the K9E-3AB
mutant, suggesting that the stimulatory function of 3D
provided by the mutated 3AB was enough to permit the
replication of the virus at wild type levels.
Figure 5:
Plaque phenotype of poliovirus mutants
encoding for 3AB proteins with defects in the in vitro stimulation of poly(U) synthesis catalyzed by 3D. Infectious RNAs
from wild type, K9E, K107E, and K9E/K107E-3AB pT7XLD constructs were
obtained by invitro transcription of full-length
cDNA clones. HeLa cells were transfected with infectious RNAs by the
Lipofectin procedure. At 3 or 6 days post-transfection the cells were
scraped and lysed to recover viruses. The plaque phenotype of these
viruses was determined after infection of HeLa cells overlaid with 0.7%
agar. Two different dilutions are shown for each
virus.
Figure 6:
One-step growth curves of wild type and
3AB poliovirus mutants. HeLa cell monolayers were infected with a
multiplicity of infection of 0.1 pfu/cell and incubated at 37 °C.
At different times postinfection the cells were scraped, and the
intracellular virus production was assayed. Virus yield is given as the
log pfu/ml.
In light of these
results we decided to measure the viral RNA synthesis early in
infection. An RT-PCR procedure was used to estimate the accumulation of
plus and minus viral RNA strands. The cells were infected with 0.1
pfu/cell, and after 4 h the viral specific RNA levels were measured (Fig. 7). As an internal control for the retrotranscriptase and
PCR reactions, the -actin mRNAs were amplified in the same test
tubes. The PCR signal for both plus and minus polio RNA obtained with
any of the K107E mutants was equal or less than the signal observed
with a 10-fold dilution of the RNA isolated from cells infected with
the wild type virus. However, the amount of
-actin mRNA was not
significantly altered after 4 h of infection with any of the viruses
tested. These estimations mean over 90% inhibition in the accumulation
of viral RNAs of K107E and K9E/K107E-3AB polioviruses. No significant
difference in the amount of polio-specific RNAs was found with the
K9E-3AB mutant virus. The defect observed in the K107E viruses seems to
occur at a step affecting the synthesis of both plus and minus strands.
In the case of the K107E mutant polioviruses, the impossibility of
obtaining high titer viral stocks impeded the detection of the viral
proteins synthesized in infected cells. No difference in the pattern of
viral proteins was found in HeLa cells infected with either K9E-3AB or
wild type poliovirus (data not shown). To test whether the amino acid
substitution at position 107 affects the synthesis and processing of
the polyprotein, we decided to analyze the proteins synthesized in
vitro with nuclease-treated HeLa cell extracts. These lysates
permit the faithful translation of poliovirus RNA. Indeed, under
optimized conditions, translation of poliovirus RNA leads to de
novo viral RNA synthesis and the formation of infectious viral
particles(29) . Therefore, this procedure perfectly mimicked the
biochemical processes that take place in vivo. Translation of
mutant and wild type RNAs was performed in uninfected HeLa cell
extracts programmed with viral RNAs (Fig. 8). The proteins were
labeled with [
S]methionine and separated in
SDS-polyacrylamide gels. No difference was observed in the general
pattern of synthesized proteins. Indeed, 3AB was clearly observed in
all cases, suggesting that the 3AB-3C processing is not being affected
by the lysine to glutamic acid change at position 107. Note that 3ABs
containing a glutamic acid at position 9 migrated slightly faster than
the other 3AB proteins. Taken together, these results suggest that the
defects found in the K107E-3AB poliovirus mutants cannot be due to
abnormal processing of the polyprotein but rather are due to defects in
intrinsic activities of the 3AB protein.
Figure 7:
Detection of plus and minus strand polio
RNA in HeLa cells infected with 3AB poliovirus mutants. HeLa cells were
infected with either wild type poliovirus or with the K9E, K107E, or
K9E/K107E-3AB mutant viruses at a multiplicity of infection of 0.1
pfu/cell. After 4 h of infection the cells were lysed, and the total
cytoplasmic RNA was isolated. Identical amounts of these RNAs were used
as templates for an RT-PCR reaction to determine the amount of plus and
minus polio RNA and -actin mRNA. The upperportion shows the 737-nucleotide-long product amplified by RT-PCR, which
corresponds to the 2AB encoding region of the genome and the
1170-nucleotide fragment corresponding to the
-actin mRNA. To
estimate with accuracy the amount of RNA, the RT-PCR analysis was also
carried out with serial dilutions of the RNA recovered after 4 h
postinfection with wild type virus. RT-PCR reactions with RNA isolated
from mock-infected cells were carried out in parallel. The lowerpart of the figure represents the densitometric analysis
of the
-actin and 2AB amplified fragments. Details are given under
``Materials and Methoeds.''
Figure 8:
In vitro synthesis of poliovirus
proteins in HeLa cell extracts programmed with wild type or 3AB mutant
RNAs. The RNAs obtained by in vitro transcription of cDNA
clones were used to program HeLa S3 cell extracts treated with
micrococcal nuclease. The synthesized proteins were labeled with
[S]methionine and electrophoresed in
polyacrylamide gels. Control reaction was programmed with no
exogenously added RNA. The positions of some viral proteins are
denoted.
(
)have
shown evidence for a membrane-permeabilizing activity of 3AB protein
and proposed that this protein may be involved in the generation of the
cytopathic effect observed in infected cells. Thus, 3AB could perform
multiple functions during poliovirus infection, perhaps at different
steps of the replication cycle.
P-labeled poly(A) probes that were
retarded in gel shift assays(30) . The authors suggested that
the binding of 3AB may be specifically directed to the 3`-end-poly(A)
region of the genome, but no experimental evidence was shown to support
this hypothesis. Although we cannot rule out a specific binding of 3AB
to any part of the genome, the available data supports the possibility
that 3AB protein by itself binds to any heteropolymeric or
homopolymeric ribonucleic acid.
will be required to
further confirm the generality of this conclusion. A more extensive
analysis might identify positions in 3AB apart from the VPg domain but
still involved in these functions.
transactivating 3AB protein
(K9E/K107E-3AB) was able to maintain the replication of the virus,
although to greatly reduced levels. This does not mean at all that the
proposed function is not important for the virus. On the contrary, its
importance is supported by the high phenotypic reversion rate showed by
the K107E-3AB mutant viruses. Different reports have identified amino
acid changes in 3AB that exert a severe impairment in the replication
of the RNAs(21, 22, 23) . Despite this, the
exact role of 3AB in the replication of the genome has never been
understood(1, 34) . At least in one case, the altered
function of 3AB was shown to be necessary only at an early step in the
infection cycle(21) . Surprisingly, the synthesis of both
positive and negative strands was severely depressed, as occurs with
the replication mutants shown here. It would be of interest to
determine whether that 3AB protein is able to stimulate poly(U)
synthesis by 3D
. Kuhn et al. described
a poliovirus mutant (VPg26) with a double amino acid substitution at
positions 107 (K107E) and 97 (K97R) of 3AB protein(35) . The
authors showed a deviation from the kinetics of virus production
similar to our K107E-3AB mutants, but the nature of this alteration was
not studied. Other amino acid substitutions at position 107 showed
virus-yield defects early in the infection, corroborating the idea that
this position plays an important role in the replication of the virus.
need a transactivator to reach full
activity?'' This modular system must contribute to the poliovirus
cycle with some advantages. The replication of viral RNAs can be
divided into two separate reactions, initiation and elongation, which
may be carried out by different enzymes(2, 36) . In some
way, these reactions share some similarities with the synthesis of RNA
directed by DNA-dependent RNA polymerases. In both cases initiation
relies on the recognition of specific signals in the RNA or DNA
template molecules. For transcription to occur, first the polymerase
recognizes and stably binds to specific promoter regions forming a
``closed complex.'' Later, the closed promoter complex will
isomerize to an ``open state,'' allowing the release of the
polymerase to carry out the elongation of the RNA chain. The OmpR
transcriptional activator protein enhances the binding of E. coli RNA polymerase to synthetic promoters. This interaction leads to
the activation of transcription on weak promoters, whereas the same
interaction produces a negative effect on strong promoters(37) .
These findings suggest that an increase in the affinity of the
polymerase to the promoter can lead to an activation of the initial
step but may cause a delay in subsequent steps, probably by
``freezing'' the polymerase in the closed complex state.
During RNA-dependent RNA synthesis a similar process may be taking
place. Thus, after binding of the polymerase (or a precursor thereof)
to the viral genome (initiation), the system must switch to the
elongation step. We propose that 3AB may be involved in this switching
step. Initially, 3AB may be directly implicated in the recognition of
these signals, increasing the affinity of the polymerase to these
regions. Then, the proteolytic processing of 3AB to VPg and 3A (without
affinity to RNA) could allow the release of the viral polymerase and
the commitment of the process into the elongation step. Therefore, 3AB
could work as OmpR protein does, but in this case the function may be
modulated by proteolytic processing. Evidence supporting this model
comes from the fact that 3AB but not 3A binds RNA and that this
activity seems to play an important role in the in vitro transactivation of 3D
.
or a precursor thereof and that this interaction increases
the chance of the polymerase to take part in the initiation process.
Interestingly, Molla et al.(38) have shown that 3AB
can co-immunoprecipitate with either 3D
or
3CD
, suggesting a stable interaction of 3AB with
these proteins. The identification of 3AB and 3D
mutants unable to interact with each other will help to
elucidate the interactions that modulate the poliovirus replication
complex. Alternatively, a prediction of this model implies that if the
cleavage of 3AB to 3A and VPg is avoided, the synthesis of RNA chains
would be stopped or delayed in the initiation step, just after addition
of a short oligonucleotide to the primer protein. Also, if 3AB or a
precursor of this protein is priming the initiation reaction, this
model would explain the impossibility of detecting elongating RNA
chains with the 3AB primer protein covalently bound to the 5`-end of
the genome. Nevertheless, the proofs for this model will have to await
the development of in vitro systems where the different steps
involved in the synthesis of poliovirus RNA can be dissociated.
Table: 0p4in
Plaque
phenotype was determined for those viruses obtained after 72 h of
transfection.(119)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.