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J Virol, March 1998, p. 2398-2405, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A New Internal Ribosomal Entry Site 5' Boundary Is
Required for Poliovirus Translation Initiation in a Mouse
System
Toshihiko
Ishii,1
Kazuko
Shiroki,1,*
Duck-Hee
Hong,1
Takahiro
Aoki,1
Yoshihiro
Ohta,2
Shinobu
Abe,2
So
Hashizume,2 and
Akio
Nomoto1
Department of Microbiology, Institute of
Medical Science, The University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo 108,1 and
Japan
Poliomyelitis Research Institute, Higashimurayama, Tokyo
189,2 Japan
Received 28 July 1997/Accepted 30 November 1997
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ABSTRACT |
Four mutants of the virulent Mahoney strain of poliovirus were
generated by introducing mutations in nucleotides (nt) 128 to 134 of
the genome, a region that contains a part of the stem-loop II (SLII)
structure located within the internal ribosomal entry site (IRES; nt
120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in
human HeLa cells but not in mouse TgSVA cells that had been established
from the kidney of a poliovirus-sensitive transgenic mouse. Their
neurovirulence in mice was also greatly attenuated compared to that of
the parental virus. The poor replication activity of the SLII mutants
in TgSVA cells appeared to be attributable to reduced activity of the
IRES. Two and three naturally occurring revertants that replicated well
in TgSVA cells were isolated from mutants SLII-1 and SLII-5,
respectively. The revertants recovered IRES activity in a cell-free
translation system from TgSVA cells and returned to a neurovirulent
phenotype like that of the Mahoney strain in mice. Two of the revertant
sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the
revertant genomes and exerted a significant effect on the revertant
phenotype. Exhibition of the full revertant phenotype required
mutations in both regions. These results suggested that nt 107 of
poliovirus RNA is involved in structures required for the IRES activity
in mouse cells.
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INTRODUCTION |
The single-stranded genome of
poliovirus has mRNA polarity, is approximately 7,500 nucleotides (nt)
in length, is polyadenylylated (45), and is linked
covalently at its 5' end to a small protein called VPg (30,
41). The RNA itself is infectious; cells transfected with the RNA
produce progeny virions that are infectious. Poliovirus RNA harbors a
long 5' noncoding region of approximately 740 nt that is important for
viral RNA and protein syntheses. A possible cloverleaf-like structure
formed by the 5'-proximal end of the RNA (approximately 90 nt) is a
probable cis element that regulates the synthesis of the
plus-strand RNA (1). nt 120 to 590 of the poliovirus RNA
make up the internal ribosomal entry site (IRES) (32), which
directs the viral translation initiation step in a 5'-end- and
cap-independent manner (17, 25, 29, 44). The IRES is assumed
to carry a number of secondary structures (10, 40), and
multiple host cellular factors are required for its functions.
Translation of poliovirus does not occur in a cell-free wheat germ
translation system, and it occurs only inefficiently and usually
incorrectly in rabbit reticulocyte lysates (RRL) (9). The
poor translation in RRL, however, is markedly improved by the addition
of factors from HeLa cells (5, 9, 33). Other IRESs, such as
the IRESs of encephalomyocarditis virus RNA (18) and
hepatitis C virus RNA (43), are highly functional in the RRL
system. These observations indicate that individual IRESs with
different structures may require quantitatively and/or qualitatively different sets of host factors for their activities.
Determinants for strain-specific neurovirulence (replication ability)
of poliovirus type 1 in the central nervous system (CNS) have been
mapped in the IRES region, particularly at nt 480 of the genome, by
using monkey neurovirulence tests on recombinant viruses between the
virulent Mahoney and attenuated Sabin 1 strains (19, 24).
Similar results were obtained when the recombinants were tested for
their relative neurovirulence levels by using transgenic (Tg) mice
carrying the human gene for the poliovirus receptor (15, 20,
34). Thus, the IRES seems to be an important regulatory element
for strain-specific expression of poliovirus neurovirulence. These two
animal models show no difference in the development of the disease,
even though replication of the virus in vivo must involve a number of
biological interactions between viral and host factors. These results
suggest that host factors of monkeys and mice, including IRES-related
factors, support the expression of poliovirus neurovirulence
(replication) in much the same way. However, it is possible that
species differences between the IRES-related host factors of monkeys
and mice exist.
Several mutants with alterations in the stem-loop II (SLII) region were
constructed from an infectious cDNA clone of the virulent Mahoney
strain of poliovirus type 1 (39). The mutants replicated well in primate cells and in the CNS of monkeys but did poorly in mouse
cells expressing human poliovirus receptor and in the CNS of the Tg
mice carrying the human PVR gene (39). The
replication of the mutant strains in mouse cells was blocked at the
IRES-dependent translation initiation step, indicating that the
function of the SLII as a part of the IRES is deficient in mouse cells
but still active in primate cells. These differences in how the SLII
mutants acted in the two animal models point to an interaction between the SLII and SLII-related host factors that could be a determinant for
host-specific replication of poliovirus.
To gain a deeper insight into the molecular basis of the function of
the SLII region within the IRES, revertants that acquired the ability
to replicate in mouse cells were isolated from the SLII mutants.
Genetic analysis of mutation sites in the revertant genomes revealed
that nt 107 within the 5' noncoding region of poliovirus RNA influenced
the efficiency of the IRES-dependent translation initiation process and
that the remaining mutation sites (nt 120 to 161), in addition to
nt 107, were required for the expression of the full revertant
phenotype.
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MATERIALS AND METHODS |
Cells and viruses.
Suspension-cultured HeLa S3 cells were
grown in RPMI 1640 medium supplemented with 5% newborn calf serum and
used for plaque formation and preparation of poliovirus type 1 Mahoney
strain, PV1(M)OM (38, 39), which is named WT in this paper.
African green monkey kidney (AGMK) cells cultured in Dulbecco modified Eagle medium supplemented with 5% newborn calf serum were used for
plaque formation and transfection with infectious poliovirus cDNAs.
TgSVA cells established from the kidney of a Tg mouse carrying the
human PVR gene (37) were cultured in Dulbecco
modified Eagle medium supplemented with 5% fetal calf serum.
SLII mutants (39) were prepared in AGMK cells at 37°C.
Titers of poliovirus preparations used as inocula were measured by a
plaque assay in AGMK cells.
Mouse neurovirulence test.
At 6 weeks of age, line
IQI-PVRTg21 (heterozygote) (20, 39) Tg mice were inoculated
intracerebrally with 30 µl of poliovirus suspensions at various
titers. The mouse line had been maintained under specific-pathogen-free
conditions before use. The animals were observed every 12 h up to
14 days after inoculation for paralysis or death.
Nucleotide sequence analysis.
The cDNAs corresponding to the
5'-proximal portion of the revertant genomes were prepared by reverse
transcriptase PCR of intracellular viral RNA; total RNAs were isolated
from cells infected with revertants, and oligonucleotides
5'-CTGAGAATTCGTAATACGATCACTATAGGTTAAAACAGCTCTGGGGTTG-3' (nucleotide sequences of EcoRI site, T7 
10
promoter, and nt 1 to 20 of poliovirus RNA) and
3'-CCACCACCTTCAACGGACTA-5' (antisense sequence of nt 1182 to
1201 of poliovirus RNA) were used as sense and antisense primers,
respectively. The reverse transcriptase PCR product was digested with
EcoRI and SphI (nt 1131/1132), and inserted into
the EcoRI and SphI sites of pUC118. These
plasmids were termed pT7-1131-WT, pT7-1131-RSLII-1(I),
pT7-1131-RSLII-1(S), pT7-1131-RSLII-5(H), pT7-1131-RSLII-5(I),
and pT7-1131-RSLII-5(S) (Table 1).
Nucleotide sequences of cDNAs corresponding to approximately the
5'-proximal 500 nt of the genomes were determined by a dideoxy sequencing method with a Pharmacia LKB ALFred DNA sequencer.
Construction of infectious cDNA clones.
cDNAs to the genome
of revertant RSLII-5(H) were prepared with a cDNA Synthesis System Plus
(Amersham) (38). The cDNAs with EcoRI adapters at
both ends were inserted into the EcoRI site of the plasmid
vector pSVA14 (11). Plasmids that carried inserted cDNAs
nearly equivalent in length to full-length poliovirus RNA were chosen.
The cDNAs thus constructed lacked short segments corresponding to the
5'-proximal portion of the genomes.
DNA segments complementary to the 5'-proximal region of nt 1 to 339 (
AgeI site) were prepared from pRSLII-5(H)/WT (Table
1;
see
Fig.
3) (see below). To prepare infectious cDNA clones of
RSLII-5(H),
the DNA fragment was joined to the remaining cDNAs
at the corresponding
AgeI site at nt 339/340. Virus produced in
cells transfected
with the infectious cDNA clone pRSLII-5(H) phenotypically
resembled the
parental revertant RSLII-5(H).
Construction of recombinant cDNAs and viruses.
Recombinant
infectious cDNA clones originating from poliovirus WT (pWT) and
carrying revertant segments from nt 39 to 339 were constructed by
replacing the PmlI (38/39) and AgeI (339/340) fragments of pWT with the corresponding revertant fragments; the resulting clones were designated pRSLII-1/WT, pRSLII-5(H)/WT, pRSLII-5(I)/WT, and pRSLII-5(S)/WT (Table 1). pWT/RSLII-5(H) and
pSLII-5/RSLII-5(H) were constructed similarly.
Modifications of nt 107 in the cDNAs were carried out by PCR. The first
PCR primers were a sense nucleotide sequence from
nt 93 to 115 and an
antisense sequence from nt 389 to 407, and
the second primers were a
sequence from the pSVA14 vector (nt
4639 to 4659) and an antisense
sequence from nt 93 to 115. Plasmids,
pWT, pSLII-1, pSLII-4, pSLII-5,
and pSLII-6 were used as templates.
The sense and antisense primers (nt
93 to 115) were designed to
replace T at nt 107 with A, C, and G. A
cDNA clone in which nt
107 is missing was also constructed by using a
mutant primer.
Two overlapping fragments produced by the first and second PCRs were
used in PCR along with primers of a sense sequence of
pSVA14 vector (nt
4639 to 4659) and an antisense sequence from
nt 389 to 407 to yield a
longer fragment that spanned the full
length of the overlapping
fragments. The
PvuI (nt 4704, vector
sequence)
-
AgeI (nt 339) fragment of plasmid pWT was replaced
by the
corresponding fragments of the modified cDNAs. The infectious
cDNAs
thus constructed were designated p107A-WT, p107G-WT, p107C-WT,
p107del-WT, p107A-SLII-1, p107A-SLII-4, p107A-SLII-5, and p107A-SLII-6.
Plasmids pM-1-WT, pM-5(H)-WT, pM-5(I)-WT, and pM-5(S)-WT were
constructed by the replacement of A at nt 107 with a T in pRSLII-1/WT,
pRSLII-5(H)/WT, pRSLII-5(I)/WT, and pRSLII-5(S)/WT, respectively.
As a
way to confirm the presence of modified sequences, nucleotide
sequences
from nt 1 to 339 of the cDNAs were determined by dideoxy
sequencing
with a Pharmacia LKB ALFred DNA sequencer.
A DEAE-dextran method was used to transfect AGMK cells with cDNAs or
with their RNA transcripts (
11,
38). RNA transcripts
were
synthesized from cDNAs, which were linearized by digestion
with
PvuI, by using the MEGAscript T7 Kit (Ambion).
Cell-free translation.
Mouse TgSVA cell monolayers were
collected and cultured in suspension for 4 to 6 h, and then a
cytoplasmic extract (S10) was prepared as previously described
(16, 39). Conditions for the translation reaction were those
used by Iizuka et al. (16). After incubation at 32°C for
1 h, radioactive products in the translation mixtures were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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RESULTS |
Isolation of revertants from mutants SLII-1 and SLII-5.
Parental WT poliovirus grows well in primate and mouse systems
(20, 21, 27, 39), and although SLII mutants grow well in
primate cells and in the CNS of monkeys, they grow poorly in mouse
TgSVA cells and in the CNS of the Tg mice carrying the human PVR gene (39). Apparently, SLII mutants are
sensitive to species differences in the SLII-related host factors of
primates and mice.
We isolated revertants of SLII-1 and SLII-5 mutants that were able to
grow in TgSVA cells with the intention of learning about
the molecular
basis of SLII function. For possible secondary structures
of the SLII
region of WT and SLII mutants, see Fig.
2. TgSVA cells
were infected
with mutant SLII-1 or SLII-5 at a multiplicity of
infection of
approximately 1 and incubated at 37°C until most
of the cells had
detached. The virus was passaged three times,
after which the virus
preparations started to replicate efficiently
in TgSVA cells, and the
supernatants were checked for plaque formation
on TgSVA cells. Single
plaques formed in TgSVA cells were harvested
and plaque purified again
in TgSVA cells. Of these plaque-purified
revertants, one revertant,
RSLII-1(I), from SLII-1 and three revertants,
RSLII-5(H), RSLII-5(I),
and RSLII-5(S), from SLII-5 were chosen
and used as revertants in this
study (Table
1). Revertant RSLII-1(S)
was obtained in a similar way
from plaque-purified virus recovered
from the spinal cord of a
paralyzed Tg mouse intracerebrally inoculated
with SLII-1
(
39).
The titers of these revertants were measured in HeLa cells and TgSVA
cells, and the logarithmic difference between the titers
was compared
with that of WT to determine the extent to which
the revertants
overcame the limitations of host cell species differences
on their
capacity to replicate (Table
2). The log
difference
for each of the revertants was approximately 1, whereas that
of
WT was 0.2. The results suggested that all the revertants obtained
in this study acquired at least a partial ability to replicate
in mouse
cells.
Mouse neurovirulence of revertants.
SLII mutants are known to
be host range mutants; that is, they appeared to replicate well in the
CNS of monkeys but not in the CNS of the Tg mice (39).
Revertants obtained in this study grew well in TgSVA cells; therefore,
they may have recovered the capacity to replicate in the CNS of the Tg
mice. Accordingly, revertants were tested for their neurovirulence
phenotypes in the Tg mice (Fig. 1). The
Tg mice were inoculated intracerebrally with four revertants,
RSLII-1(S), RSLII-5(H), RSLII-5(I), and RSLII-5(S). As shown in Fig. 1,
these revertants exhibited a neurovirulence phenotype similar to that
of WT whereas SLII-1 and SLII-5 showed much less of the neurovirulence
phenotype than WT, as reported previously (39). These
results suggested that the revertants overcame the host range
restriction observed for SLII-1 and SLII-5 in the CNS of Tg mice.
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FIG. 1.
Neurovirulence test on SLII mutants and their revertants
in Tg mice after intracerebral inoculation. Neurovirulence tests were
performed on SLII mutants and their revertants in Tg mice. Tg mice were
inoculated intracerebrally with the WT, SLII-1, SLII-5, RSLII-1(S),
RSLII-5(H), RSLII-5(I), or RSLII-5(S) and observed for paralysis and
death for up to 14 days after the inoculation.
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Genomic region influencing revertant phenotype.
We attempted
to identify a mutation site(s) that influences the revertant phenotype,
by preparing cDNA clones containing the segment located upstream of the
SphI site (nt 1 to 1131) in all the revertants (Table 1) and
then sequencing nt 1 to about 500. We found that all the mutations that
occurred in this region of the revertant genomes resided between nt 107 and 161. The mutation sites are shown in Fig.
2. The mutations discovered in
pT7-1131-RSLII-1(I) and pT7-1131-RSLII-1(S) were identical; therefore,
revertants RSLII-1(I) and RSLII-1(S) are hereafter both simply referred
to as RSLII-1 (Fig. 2 and 3; Tables 1 and 2). RSLII-5(H), RSLII-5(I), and RSLII-5(S) differed slightly from each other in this genome region
(Fig. 2). Unexpectedly, all the revertants had A at nt 107, unlike WT
and SLII, which had U at nt 107. Other mutation sites were observed in
nt 120 to 161 (Fig. 2).
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FIG. 2.
Secondary structures and sequences of SLII mutants and
their revertants. Possible secondary structures and sequences of SLII
regions of the genomes of WT virus, SLII mutants (SLII-1, SLII-4,
SLII-5, and SLII-6), and their revertants [RSLII-1, RSLII-5(H),
RSLII-5(I), and RSLII-5(H)] are shown. The mutation sites in the
revertants are indicated by shadowed letters or an asterisk that
represents a deleted nucleotide. Nucleotide positions are indicated on
the WT genome.
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To find whether mutations seen in nt 107 to 161 conferred the revertant
phenotype, a segment upstream of
AgeI (nt 399) of
pWT was
replaced by the corresponding segment of pT7-1131-RSLII-1,
pT7-1131-RSLII-5(H), pT7-1131-RSLII-5(I), or pT7-1131-RSLII-5(S);
the
resulting recombinant infectious cDNA clones were designated
pRSLII-1/WT, pRSLII-5(H)/WT, pRSLII-5(I)/WT, and pRSLII-5(S)/WT,
respectively. Recombinant cDNAs pWT/RSLII-5(H) and pSLII-5/RSLII-5(H)
were constructed similarly. The recombinant viruses from these
cDNA
clones were designated RSLII-1/WT, RSLII-5(I)/WT, RSLII-5(S)/WT,
WT/RSLII-5(H), and SLII-5/RSLII-5(H), respectively, and the log
difference between their titers in HeLa cells and TgSVA cells
was
compared to that of WT (Fig.
3). The log
difference between
the titers of RSLII-1/WT in HeLa cells and TgSVA
cells was similar
to that of RSLII-1 (Fig.
3). An allele replacement
experiment
carried out for RSLII-5(H) also gave a very similar result
(Fig.
3). Furthermore, WT/RSLII-5(H) showed a revertant phenotype, and
SLII-5/RSLII-5(H) showed a phenotype like that of SLII-5. These
data
strongly suggested that the revertant phenotypes of RSLII-1
and
RSLII-5(H) are due to mutations in nt 107 to 161.
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FIG. 3.
Genome structures of the SLII mutants and revertants and
logarithmic differences between their virus titers in HeLa cells and
TgSVA cells. The genomes of poliovirus WT, RSLII-1, and RSLII-5(H) are
indicated by  ,  , and  , respectively. Segments of nt 1 to 339 (AgeI fragment) for SLII-1, SLII-5, RSLII-5(I), and
RSLII-5(S) are shown by  ,  ,
 , and
&atyp0220;,
respectively. Virus titers in log10 PFU per milliliters in
HeLa cells and TgSVA cells and the logarithmic differences between the
titers are given on the right of the individual constructs.
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Although all recombinant viruses that carried the 5'-proximal genome
sequences of revertant or WT showed revertant phenotypes,
the log
differences in the viral titers of these viruses were
slightly higher
than that of WT. It should be noted that the reciprocal
recombinants
RSLII-5(H)/WT and WT/RSLII-5(H) had very similar
log differences that
were slightly higher than that of WT. Thus,
all the revertants and the
recombinants, except for SLII5/RSLII-5(H),
constructed here appear to
have a minor defect in their replication
abilities in mouse cells.
These observations may suggest that
a mutation(s) other than in the
5'-proximal 339 nt existed in
the revertant genomes and that such a
mutation(s) influenced the
viral replication efficiency to some extent
in concert with a
mutation(s) in nt 107 to 161.
Mutation sites influencing revertant phenotype.
To narrow the
number of possible sites that might be important for the revertant
phenotype, we generated new recombinants and mutants for investigation
(Fig. 4). First, U at nt 107 was changed
to A in the genomes of SLII-1, SLII-4, SLII-5, and SLII-6, and these
mutants were designated 107A-SLII-1, 107A-SLII-4, 107A-SLII-5, and
107A-SLII-6, respectively (Table 2; Fig. 4). The titers of these
mutants were measured in HeLa and TgSVA cells, and the log differences
were compared with those of the corresponding SLII mutants. The log
differences of 107A-SLII-1, 107A-SLII-4, and 107A-SLII-5 were much
lower than those of the parental SLII mutants (Table 2). Thus, mutation
at nt 107 in the revertant genomes appeared to have a significant
influence on the revertant phenotype, although mutation at nt 107 did
not restore the replication capacity of SLII-6 in the TgSVA cells. We
were unsuccessful in our attempts to isolate a revertant(s) from SLII-6
by the procedure described in Materials and Methods (data not shown).
Only the mutation at nt 107 did not confer the full revertant phenotype
on SLII-1 and SLII-5, suggesting that the remaining mutations observed
in the region from nt 120 to 161 may affect the viral replication
efficiency in TgSVA cells.
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FIG. 4.
Genomic structures of nt 107 to 161 of recombinant
viruses. The recombinant viruses shown were constructed as described in
Materials and Methods. Genomic structures other than nt 107 to 161 were
the same as that of the WT genome.
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We tested the effect of mutations in the region from nt 120 to 161 by
adding a U at nt 107 (as is found in WT and SLII mutants)
into the
background of RSLII-1/WT, RSLII-5(H)/WT, RSLII-5(I)/WT,
and
RSLII-5(S)/WT, thereby creating M-1-WT, M-5(H)-WT, M-5(I)-WT,
and
M-5(S)-WT, respectively. A schematic representation of nt
107 to 161 in
these mutant genomes is shown in Fig.
4. As shown
in Table
2, these
mutants lacked any apparent plaque-forming
activity in TgSVA cells, yet
they manifested cytopathic effects
that were obviously stronger than
those of SLII-1 and SLII-5 when
the cells were infected with the
undiluted virus solutions (data
not shown). These data suggested that
the remaining mutations
also contributed to better revertant
replication in TgSVA cells.
The mutation at nt 107 apparently affected
the expression of the
revertant phenotype more than the region spanning
nt 120 to 161,
although both sites seem to have been involved in the
full revertant
phenotype. Point mutations at nt 107 were introduced
into the
WT genome to yield a series of mutants designated 107A-WT,
107C-WT,
107G-WT, and 107del-WT (Fig.
4). The replication of these
mutants
was changed little from that of the replication phenotype of WT
(Table
2). Thus, the role that A at nt 107 plays in poliovirus
replication was detected by using host range mutants SLII-1, SLII-4,
and SLII-5.
Cell-free translation.
A possible change in the IRES activity
of revertant RNAs was investigated by using a cell-free translation
system. RNAs with free 5' ends were synthesized from cDNA clones of
pWT, pSLII-1, pRSLII-1/WT, pM-1-WT, p107A-SLII-1, pSLII-5,
pRSLII-5(H)/WT, pM-5(H)-WT, pRSLII-5(I)/WT, pM-5(I)-WT,
pRSLII-5(S)/WT, pM-5(S)-WT, and p107A-SLII-5, which had been linearized
by digestion with XbaI (Fig.
5B), and were used as templates in S10
extracts prepared from TgSVA cells. The amounts of RNA templates used
are shown in Fig. 5B. The 66-kDa protein was hardly detected in the
products from RNAs of SLII mutants but was visible in the products of
the WT RNA and revertant RNAs (Fig. 5A). The 66-kDa protein was
detected weakly in the products directed by the recombinant RNAs. These
observations indicated that the IRESs of the SLII mutant RNAs do not
function in the mouse system but that the IRESs of the revertants
recover much of their function. The intensities of the bands observed in Fig. 5A seemed to be compatible with growth phenotypes of individual viruses in TgSVA cells. Enhanced efficiency of translation initiation is therefore highly likely to be responsible for the regained growth
ability of these revertants and recombinants.
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FIG. 5.
Cell-free translation products of RNAs from WT, SLII
mutants, and their recombinants. (B) Template RNAs were transcribed by
T7 RNA polymerase from pWT, pSLII-1, pSLII-5, and recombinant cDNAs of
WT and the revertants that had been cleaved with XbaI. The
length of the template RNA is shown at the bottom. (A) The in vitro
transcripts were incubated at 32°C for 1 h in S10 extract from
TgSVA cells. The position of the 66-kDa product is indicated by an
arrowhead on the right.
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The translation directed by the RNAs from recombinants p107A-SLII-1 and
p107A-SLII-5 was more efficient than that from pSLII-1
and pSLII-5. We
examined the effect of mutation at nt 107 on the
IRES activity by
measuring the levels of mRNA translation from
various RNAs carrying
mutations at nt 107 (Fig.
6).
Translational
efficiency relative to WT RNA appeared not to be changed
much
by any of the mutations at nt 107. The translation from
107A-SLII-4
RNA was more efficient than that from SLII-4 RNA. In the
case
of 107A-SLII-6, the translation efficiency appeared not to be
recovered. The translation efficiencies were compatible with the
plaque-forming ability of the viruses. These experiments indicated
that
nt 107 is involved in the structure required for IRES-dependent
translation initiation, a function that was detectable only by
using
SLII mutants.
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FIG. 6.
Effect of nt 107 on IRES activity in cell-free
translation. (B) Template RNAs were transcribed by T7 RNA polymerase
from pWT, pSLII-4, pSLII-6, and their mutants concerning nt 107 that
had been cleaved with XbaI, as in Fig. 5. (A) The in vitro
transcripts were incubated at 32°C for 1 h in S10 extract from
TgSVA cells. The position of the 66-kDa product is indicated by an
arrowhead on the right.
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DISCUSSION |
Molecular genetic analysis of naturally occurring revertant
viruses from two mutants (SLII-1 and SLII-5), which were able to grow
in TgSVA cells, revealed that all the revertant genomes had a change at
nt 107 from U to A. Mutants 107A-SLII-1 and 107A-SLII-5 showed more
efficient plaque-forming ability in TgSVA cells than did SLII-1 and
SLII-5, respectively. The in vitro translation directed by the RNA from
p107A-SLII-1 and p107A-SLII-5 was also more efficient than that
directed by RNAs from pSLII-1 and pSLII-5. These results suggested that
nt 107 functions in IRES-dependent translation initiation in TgSVA
cells. Trono et al. (42) reported that an insertion of 3, 11, or 15 bases between nt 108 and nt 109 of poliovirus RNA did not
result in any alteration of the replication phenotype. Pelletier et al.
(31) showed that the change of U to A at nt 119 in the
poliovirus type 2 genome was a silent mutation. We also showed here
that mutations at nt 107 of WT had no effect on the wild-type
phenotype. These data suggested that the translational function of nt
107 can be detected only by using the host range mutants and their
revertants.
In addition to nt 107, the remaining mutation sites (nt 120 to 161)
were required for the expression of the full revertant phenotype. This
observation suggested that nt 107 works with the SLII region to
maintain the IRES activity in mouse cells. One or more host-specific
factors may interact with nt 107 and the other mutation sites. At
present, the structural relationship between nt 107 and SLII is not
clear. Le and Zuker (22) reported a predicted consensus
secondary structure that consists of nt 103 to 179 of poliovirus type 3 in which nt 107 and SLII are involved in a stem-loop structure.
Cellular factors may recognize such a structure formed by a longer
nucleotide sequence including nt 107 and the SLII (nt 124 to 162)
region.
The initiation events directed by the IRES probably require many
protein factors including most of the same set of initiation factors
that are used by typical capped cellular mRNAs (36). Cap-binding activity of eIF-4F is not required for translation of the
uncapped poliovirus RNAs, but the RNA helicase activity manifested by
this protein complex may still play an important role in internal
initiation (35). If eIF-4F is required for IRES-dependent
initiation, it may function in a structurally modified form. Within the
5' NCR of poliovirus RNA, eIF-2
associates with nt 97 to 182 and nt
510 to 629 (7); similarly, eukaryotic initiation factor 4B
(eIF-4B) binds to the 5' NCR of foot-and-mouse disease virus RNA
(28). In addition to the standard initiation factors, other
trans-acting protein factors probably mediate IRES-dependent translation initiation of poliovirus. La protein, polypyrimidine tract-binding protein, and poly(rC)-binding protein 2 are reported as
being possible host factors for IRES-dependent translation initiation
(2, 3, 6, 14, 26). Many unidentified host proteins are
reported to bind to defined IRES regions of poliovirus RNA, as
determined by UV cross-linking analysis or a gel shift assay (4,
7, 8, 12). Host cellular proteins that can be UV cross-linked to
the SLII structure are now being investigated.
Although the revertant phenotypes observed for RSLII-1, RSLII-5(H),
RSLII-5(I), and RSLII-5(S) were shown to be due to mutations in nt 107 to 161 of the revertant genomes, these revertants appeared to have a
very minor defect in replication efficiency in TgSVA cells compared
with that of WT as judged by logarithmic differences of virus titers in
HeLa cells and TgSVA cells (Fig. 3). This phenomenon was reproducibly
observed in repeated experiments. However, this discrepancy was very
small and is almost negligible considering the range of error for
plaque assays. Nevertheless, we determined the nucleotide sequence of
the genome region encoding viral protein 2A of the revertant
RSLII-5(H), because protein 2A is known to enhance the efficiency of
poliovirus translation initiation (13, 23). The results
demonstrated that the protein 2A coding sequence was not changed (data
not shown). The reason for this phenomenon is not clear at present.
Expression of IRES function in mouse cells must be influenced by the
efficiency of interactions with mouse host factors; therefore, the
IRESs of SLII mutants may be recognized by such a putative mouse
factor(s) while the revertant IRESs are recognized. Although the SLII
mutants did not show efficient IRES activity in mouse cells and their
S10 extracts (Fig. 4) (39), they did have IRES activities in
HeLa cells and their S10 extracts (39). This difference in
IRES activity may be due to species difference in host factors. If so,
the two different systems may provide powerful tools for identifying a
host factor(s) that restricts IRES activity in murine systems.
| |
ACKNOWLEDGMENTS |
We are grateful to S. Kuge and H. Toyoda for helpful suggestions
and discussions. We thank Y. Sasaki and K. Iwasaki for expert technical
assistance and E. Suzuki and M. Watanabe for help in preparation of the
manuscript.
This work was supported in part by a grant-in-aid from the Ministry of
Education, Science, Sports, and Culture of Japan and the Ministry of
Health and Welfare of Japan and by funds from the Science and
Technology of the Japanese Government.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Phone: 81-3-5449-5501. Fax: 81-3-5449-5408.
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Abstract
Introduction
