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Journal of Virology, June 1999, p. 5176-5180, Vol. 73, No. 6
0022-538X/99/$04.00+0
The Major Attenuating Mutations of the Respiratory Syncytial
Virus Vaccine Candidate cpts530/1009 Specify
Temperature-Sensitive Defects in Transcription and Replication and
a Non-Temperature-Sensitive Alteration in mRNA
Termination
Katalin
Juhasz,
Brian R.
Murphy, and
Peter L.
Collins*
Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, Bethesda, Maryland
20892-0720
Received 28 October 1998/Accepted 17 February 1999
 |
ABSTRACT |
The live-attenuated respiratory syncytial virus vaccine candidate
cpts530/1009 was previously shown to contain
two separate amino acid changes in the L protein, mutations 530 and
1009 (Phe-521
Leu and Met-1169Val, respectively, according to
the amino acid sequence of the L protein). Each mutation
independently specifies temperature-sensitive (ts) and
attenuation phenotypes. In this study, we examined the effects of
these mutations on transcription and RNA replication, using
complete infectious recombinant virus as well as a plasmid-based minireplicon system, the latter under conditions in which effects on
replication and transcription are uncoupled. In comparison with
recombinant wild-type virus, the 530 and 1009 viruses were partially restricted at 37°C for RNA replication, mRNA
synthesis, and virus growth. The 1009 virus was partially restricted
for RNA synthesis and virus growth even at 32°C, which suggested that the 1009 mutation has a non-ts component in
addition to the ts component. Interestingly, the synthesis
of polycistronic readthrough mRNAs was elevated 1.6- to 3.8-fold
for the 1009 virus, and this defect was non-ts. Studies
with the minigenome system showed that the 530 and 1009 mutations each directly affect both replication and transcription, that
the effect on replication was marginally greater than on transcription
for the 530 mutation, and that the increase in
readthrough mRNA associated with the 1009 mutation also was
observed with the minigenome system.
| |
TEXT |
Human respiratory syncytial
virus (RSV), a pneumovirus of the family
Paramyxoviridae of the nonsegmented negative-strand RNA viruses, is one of the leading causes of serious viral bronchiolitis and pneumonia in young children (3). Unfortunately, an RSV vaccine is not yet available. To meet this need, we have been developing live-attenuated mutants of subgroup A RSV strain A2 as
vaccine candidates (5-7, 9). Two such vaccine
candidates are the cold-passaged (cp) temperature-sensitive
(ts) viruses cpts530 and
cpts530/1009. The parent for these viruses was
cpRSV, a cp virus made in the 1960s which
contains five amino acid substitutions that confer attenuation in
chimpanzees but do not confer significant growth restriction or
temperature sensitivity in cell culture (11, 12, 16).
The cpts530 virus was derived from cpRSV by
chemical mutagenesis (7). Compared to cpRSV,
cpts530 had acquired the ts phenotype and
exhibited an increased level of attenuation in vivo (7). Sequence analysis showed that it had sustained one additional mutation
in the L protein at amino acid 521, namely, a phenylalanine-to-leucine substitution (15). The cpts530/1009 virus was
derived from cpts530 by a second round of chemical
mutagenesis and was found to be more ts and attenuated than
its cpts530 parent (8).
cpts530/1009 sustained one additional mutation in the L
protein, namely, a methionine-to-valine substitution at amino acid
position 1169 (14). The two mutations were introduced
separately into infectious recombinant RSV (rRSV), yielding
r-530 and r-1009. Genetic and phenotypic analysis confirmed that each
mutation independently conferred the ts and attenuation
phenotypes, although the 1009 mutation was somewhat less
ts than was the 530 mutation. The introduction of both
mutations together with the five cp mutations resulted in
r-cp530/1009, which was phenotypically indistinguishable
from its biologically derived version, cpts530/1009. It
was also shown that these two mutations were additive with regard
to attenuation in the upper respiratory tract of mice, while in
the lower respiratory tract the 1009 mutation was the main contributor
to the attenuation phenotype of cpts530/1009
(14).
Multicycle growth of rRSVs.
The ts phenotype of
rRSV bearing the 530 or 1009 mutation was previously
characterized by determining the efficiency of plaque formation at
various temperatures from 32 to 40°C (14, 15). This
analysis showed that the shutoff temperature (the lowest restrictive
temperature at which there is a 100-fold or greater reduction in the
efficiency of plaque formation compared to 32°C) of recombinant virus
bearing the 1009 or 530 mutation was 39°C. At this temperature,
recombinant virus bearing the 530 mutation was 800-fold more restricted
in plaque formation than one bearing the 1009 mutation, indicating that
the 530 mutation specifies a slightly greater degree of temperature
sensitivity than does the 1009 mutation (14). The shutoff
temperature for r-cp530/1009 was 37°C, at which there
was a 6,000-fold reduction in plaque formation, compared to only 8- to
10-fold for recombinants bearing the 530 or 1009 mutation
(14).
In the present study, the following recombinant viruses were compared:
r-sites, which is a wild-type (wt) recombinant virus identical to that described previously (2) except that six translationally silent restriction enzyme sites have been introduced into the large polymerase (L) gene, which also are present in all of
the other recombinant viruses described here (15, 21); r-530 or r-1009, containing the 530 or 1009 amino acid
substitution; r-cp, which contains the five amino acid
substitutions which specify the attenuation phenotype of
cpRSV; and r-cp530/1009, which contains the
five cp changes as well as the 530 and 1009 amino acid
substitutions (14, 15). The effect of the 530 and 1009 mutations on multicycle virus replication at 32 or 37°C was examined
first since these are the temperatures at which the effects of the
mutations on genome replication and transcription were carried out.
Monolayers of HEp-2 cells were infected at a multiplicity of infection
(MOI) of 0.01 and incubated at 32 or 37°C; medium samples were
harvested at 24-h intervals, and virus was quantitated by plaque assay.
r-
cp was only marginally restricted at both 32 and 37°C
compared to r-sites (0.2 and 0.5 log
10 reductions,
respectively, on
day 7 [Fig.
1]),
consistent with previous results that showed
that it is not a
ts virus (
21). In contrast,
r-
cp530/1009 was
highly restricted in growth at 37°C
compared to r-sites (>6.0
log
10 reduction on day 7), as
expected. r-
cp530/1009 also exhibited
reduced growth at
32°C (1.7 log
10 reduction), which was not previously
appreciated. Compared to r-sites, r-530 was clearly restricted
at
37°C but only marginally restricted at 32°C (1.3 and 0.5 log
10 reductions, respectively). In these comparisons,
differences on
the order of 0.5 log
10 or less, although
reproducible, were considered
to be marginal, and differences on the
order of 1.0 log
10 or more
were considered to be clear. In
contrast to r-530, r-1009 was
clearly restricted at both 32 and 37°C
(1.1 and 1.0 log
10 reductions,
respectively). The finding
that r-1009 was clearly restricted
at 32°C, which was not previously
appreciated, suggested that
its reduced replication involves a
non-
ts component in addition
to the previously described
ts component. The restriction of replication
of r-530 at
37°C was equivalent to, or slightly greater than,
that of r-1009 (1.3 versus 1.0 log
10 reduction). The finding that
the 1009 mutation has a non-
ts component would provide an explanation
for previous biological findings, namely, that a recombinant bearing
the 1009 mutation, although less
ts than one bearing the 530 mutation,
is more attenuated in the lower respiratory tract of mice
(
14).
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FIG. 1.
Kinetics of growth of infectious, recombinant viruses
bearing the cp, 530, and 1009 mutations alone and in
combination. Triplicate monolayers of HEp-2 cells were infected
with the indicated virus at an MOI of 0.01 at 32°C (A) or 37°C (B).
Culture fluids were harvested at 24-h intervals for 7 days. Samples
were analyzed by plaque assay in duplicate, using an immunoperoxidase
staining procedure as previously described (17). Standard
errors (n = 6) from days 0 to 7 at both temperatures
for all viruses ranged from 0.0 to 0.18. Dashed line indicates the
minimum limit of detection (0.7 log10 PFU/ml).
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Levels of transcription and replication of rRSVs.
Since
both the 530 and 1009 mutations are in the L gene, the growth
restriction that they confer probably would be due to effects on
replication and/or transcription. To evaluate this, monolayers of
HEp-2 cells were infected with r-sites, r-530, or r-1009 at an MOI of 4 and incubated at 32 or 37°C. Total RNA was harvested 24 h
postinfection and analyzed by Northern blot hybridization with
double-stranded cDNA probes specific for the NS1, nucleocapsid (N), or
fusion (F) gene of RSV. Hybridization was quantified by phosphorimager analysis (Table
1). At 32°C, RNA replication and transcription by r-530 was equivalent to, or somewhat greater than,
that of the r-sites wt control. In contrast, both processes were reduced for r-1009. This result is consistent with the
contribution of a non-ts component to the attenuation
phenotype of r-1009 but not r-530. Normalized against r-sites, levels
of RNA replication and transcription by r-1009 at 37°C were on
average 48 and 63%, respectively, of those at 32°C, showing that a
contribution by a ts component was evident at the
higher temperature. In comparison, levels of RNA replication and
transcription by r-530 (normalized against r-sites) at 37°C
were 29 and 37%, respectively, of those at 32°C, which is consistent
with the level of temperature sensitivity of r-530 being somewhat
greater than that of r-1009. These data illustrate a significant
restriction of RNA synthesis at this intermediate temperature, which is
fully 2°C below the shutoff temperature of r-530 and r-1009.
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TABLE 1.
Percent RNA replication (synthesis of genome and
antigenome combined) or transcription (synthesis of the indicated
mRNA) at 32 or 37°C by r-530 or r-1009 compared to
wt r-sitesa
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In addition to the effects on the quantity of RNA replication and
transcription by the rRSVs described above, we noticed a
small but
reproducible increase in the level of polycistronic
readthrough
mRNAs produced by r-1009 (Fig.
2). The
ratios between
mono- and polycistronic mRNAs were quantitated
by phosphorimagery
in three independent experiments
performed at 32 and 37°C. Results
for the NS1 and F probes are
illustrated in Fig.
2B and summarized
in Fig.
2C. A 1.6- to 3.8-fold
increase in readthrough mRNAs was
found with r-1009 compared to
wt r-sites, and this increase was
not
ts under
these circumstances (Fig.
2C). The magnitude of the
increase in
readthrough mRNA was dependent on the specific gene
junction
involved and was consistent between experiments.
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FIG. 2.
(A) RSV gene map, showing the relative locations of the
NS1, N, and F probes. (B) Northern blot analysis of intracellular RSV
mRNAs with the NS1 and F probes. HEp-2 cells were infected with
r-sites, r-530, or r-1009 at an MOI of 4 or were mock infected, and the
cultures were incubated at 32 or 37°C. At 24 h postinfection,
total RNA was harvested by using TRIzol (Life Technologies)
and was purified with additional phenol-chloroform extraction and
ethanol precipitation. Total RNA of each preparation was
electrophoresed in 1.5% agarose gel containing 0.44 M formaldehyde,
transferred to nitrocellulose, fixed by UV cross-linking, and
hybridized to a cDNA probe of the NS1 or F gene. For the sake of
brevity, blots are shown for the 37°C samples but not those of 32°C
nor for the N probe. (C) Quantitation of the amount of
readthrough mRNA detected at 32 and 37°C with probes against the
NS1 and F genes. The N probe yielded similar results.
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Effects of the 530 and 1009 mutations on reconstituted RNA
replication and transcription by minigenome C75.
Analysis
of infectious mutant rRSV as described above does not distinguish
clearly between effects of the mutations on RNA replication versus
transcription. This is because for infectious virus, the two processes
are interdependent: any diminution in transcription reduces the supply
of helper proteins, which in turn reduces RNA replication, and any
reduction in RNA replication reduces the amount of template, which in
turn reduces transcription. However, the two processes can be
dissociated in a minigenome system using a mutant
plasmid-supplied negative-sense minigenome, C75 (Fig.
3A; see the Figure legend for a detailed
description). Minigenome C75 encodes two small chloramphenicol
acetyltransferase (CAT) mRNAs and contains a CG (negative-sense)
substitution at the penultimate nucleotide in the trailer region. This
mutation has no apparent effect on encapsidation or on the synthesis of positive-sense RNA, but synthesis of progeny minigenome is
inhibited very strongly, presumably because the antigenome promoter has been inactivated (10, 17a). Therefore, the only
intracellular minigenome would be that synthesized directly
from the plasmid. Because the minigenome template and support
proteins are supplied in trans from transfected plasmids and
are independent of minigenome transcription and replication,
the two processes are no longer interdependent and each process can be
manipulated without indirect effects on the other.
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FIG. 3.
Effects of the 530 and 1009 mutations on reconstituted
RNA replication and transcription by minigenome C75. (A)
Diagram of the cDNA-encoded, negative-sense minigenome C75.
This minigenome, like the previously described C2
minigenome (2) from which it was derived, contains a
negative-sense copy of the 660-nucleotide CAT gene, which is flanked on
the upstream end by the 3'-extragenic leader region and part of the
upstream nontranslated region of the nonstructural protein
(NS)1 gene including the gene start (GS) transcription signal; the
CAT sequence is flanked on the downstream end by the downstream
nontranslated region of the L gene including the transcription GE
signal and the 5' extragenic trailer region. C75 differs from C2 in two
features. First, the CAT gene was broken into two transcriptional units
by the introduction, into a unique NcoI site, of a sequence
cassette containing the junction between the N and P genes of strain
A2. This gene junction consists of the N gene GE signal, the
single-nucleotide N-P intergenic region (N/P IG), and the P gene GS
signal. The resulting minigenome encodes two polyadenylated
mRNAs: an upstream one, mRNA 1, of 577 nucleotides exclusive of
polyadenylate and a downstream one, mRNA 2, of 190 nucleotides. Second,
the penultimate nucleotide position from the 5' end contains a
CG transition (negative sense) which restricts the
minigenome to the synthesis of positive-sense RNA (see the
text). (B) Northern blot analysis of intracellular RNAs encoded by
minigenome C75. Transfections were performed as described
previously (4, 10). HEp-2 cells in six-well dishes
were infected with a vaccinia virus-T7 recombinant at an MOI of 5 and
transfected at the same time with 0.4 µg of C75 plasmid along
with support plasmids pTM1-N (0.4 µg), pTM1-P (0.3 µg),
pTM1-M2(ORF1) (0.1 µg), and empty pTM1 vector (0.1 µg) instead of
pTM1-L as a polymerase-minus control (lanes L-) or the same N, P, and
M2-1 plasmids together with 0.1 µg of the indicated pTM1-L
wt or mutant plasmid. Transfections and incubations were
performed at the indicated temperature; total intracellular RNA was
harvested at 72 h (32°C), 60 h (35°C), or 48 h
(37°C) posttransfection and analyzed by Northern blot hybridization
with negative-sense CAT riboprobe. nd = not detectable.
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RNA replication (synthesis of antigenome) and transcription (synthesis
of mRNA) by the C75 minigenome supported by the N,
phosphoprotein (P), M2-1, and L proteins was examined at 32, 35,
and
37°C (Fig.
3). Total intracellular RNA was analyzed by Northern
blot
hybridization, in this case using strand-specific negative-sense
CAT
riboprobe. The C75-encoded RNAs included the miniantigenome,
mRNAs
1 and 2, and a small amount of mRNA 1-mRNA 2 readthrough
mRNA (Fig.
3B). Unexpectedly, the synthesis of these RNAs by the
reconstituted RSV
system using
wt support plasmids was found to
be
ts, with almost no products detectable at 39°C (data not
shown).
Therefore, 37°C was selected as the highest restrictive
temperature
for analysis, and even at this temperature the level of RNA
synthesized
was at least 10-fold lower than that synthesized at
32°C (data
not shown). A human parainfluenza virus 3 minigenome system which
used the same cell line and vaccinia
virus-T7 recombinant virus
was not
ts, indicating that an
RSV-specific component was responsible
for the unexpected
ts effect (
19). But the
ts effect did
not
appear to be due to a
ts support protein, since these
particular
cDNAs also had been used to reconstitute complete
recombinant
virus which was not
ts. Because of the inherent
ts nature of the
reconstituted RSV system, comparisons were
made between mutants
versus
wt at a given temperature
but not between
temperatures.
The 530 and 1009 mutations were analyzed by their incorporation, singly
and in combination, into the L support plasmid, which
was then examined
in transfections in place of the
wt L support
plasmid.
Total cellular RNA was analyzed by Northern blot
hybridization
with the negative-sense riboprobe (Fig.
3B). The
ts phenotypes
of the 530 and 1009 mutations appeared to be
exaggerated in the
reconstituted system, as had been described above
for the
wt reconstituted
system. For example,
minigenome-templated RNA synthesis supported
by the 530-L
plasmid appeared to be more
ts at each of the three
temperatures compared to that supported by 1009-L and was inhibited
even at 32°C, a temperature which was not restrictive for growth
or
RNA synthesis of r-530 (Fig.
1; Table
1). In addition,
minigenome-templated
RNA synthesis supported by the double
mutant 530/1009-L was strongly
inhibited at 32°C, whereas growth
and RNA synthesis by the r-
cp530/1009
virus were only
moderately inhibited at this temperature. The
exaggerated
temperature sensitivity of the reconstituted system
would account
for the finding that the 530 mutation, which is
the more
ts
of the two mutations, was more inhibitory than the
1009 mutation in the
reconstituted system at 32°C, whereas in
virus the 1009 mutation,
which has the non-
ts component, was more
inhibitory at
32°C. At 35 and 37°C, both RNA replication and transcription
were
affected by the 530 and 1009 mutations alone and in combination.
The
effect seemed to be somewhat greater for replication than
for
transcription, especially for the 530 mutant. For example,
antigenome
synthesis by the 530 and 1009 polymerases was barely
detectable at
35°C and was not detectable at 37°C. However,
phosphorimager
analysis indicated that the magnitude of
the difference between
the effect on transcription and replication was
not great: approximately
twofold for the 530 mutant and less than
twofold for the 1009
mutant. Finally, the increase in levels of
readthrough mRNA observed
with the r-1009 virus also was observed
for the reconstituted
minigenome system (Fig.
3B) and was
independent of
temperature.
In conclusion, the purpose of this work was to provide a further
characterization of two mutations, called 530 and 1009, which
specify
most of the attenuation phenotype of the live-attenuated
cpts530/1009 vaccine candidate and are currently being
combined
with other attenuating mutations to produce new recombinant
live-attenuated
vaccines for RSV subgroup A (
1,
5,
9,
14,
15,
20,
21). The 530 mutation, a Phe-to-Leu substitution at amino
acid
position 521 in the L protein, is a replacement of a nonpolar
aromatic with a nonpolar aliphatic amino acid at a position which
is
well conserved (with >50% identity) among rhabdoviruses and
paramyxoviruses and falls between the first and second conserved
regions of the L protein. The 530 mutation is a
ts
attenuating
mutation which affects both transcription and RNA
replication,
with the effect on replication being somewhat greater. The
use
of the minigenome system confirmed that both processes were
directly
affected. The 1009 mutation, a Met-to-Val substitution at
amino
acid position 1169 in the L protein, is a conservative
substitution
and occurs downstream of the major conserved domains in
the L
protein. The 1009 mutation was found to be somewhat less
ts than
530, as has been observed previously
(
14), and contains a non-
ts component which was
observed in virus infections and the reconstituted
minigenome
system. The
ts and non-
ts components of the 1009 mutation
resulted in decreased RNA replication and transcription. The
non-
ts restriction was associated with a small (1.6- to
3.8-fold) but
reproducible increase in the accumulation of
readthrough transcripts.
An increase in the frequency of
readthrough of the gene end (GE)
signals would be expected to
result in a reduction in the amount
of monocistronic mRNAs and
encoded proteins for upstream genes
and a relative increase for
downstream genes. This latter effect
would be expected
because increased readthrough would be expected
to deliver
more polymerase to downstream genes, making the gradient
of
transcriptional polarity less steep. This effect would not
be large,
given the relative low abundance of the readthrough
mRNAs. It is
possible that this readthrough effect is sufficient
to account for
the reduction in RNA synthesis observed at 32°C
for r-1009.
Alternatively, the non-
ts component of the 1009 mutation
might also involve some other aspect of polymerase
function, and
the increase in transcriptional readthrough might be
an incidental
effect. This possibility is suggested by the finding that
both
RNA replication and transcription by the 1009 polymerase, rather
than transcription alone, were affected at 32°C in the
minigenome
system. However, since
ts effects
appeared to be exaggerated in
the minigenome system and were
prominent even at 32°C, it is difficult
to reliably assess the
non-
ts component of the 1009 polymerase.
Finally, it was
recently shown that the M2-1 protein mediates
antitermination at
GE signals (
9a,
13), and it is interesting
to find in the
present study that antitermination also can be
enhanced by a single
amino acid change in the L
protein.
| |
ACKNOWLEDGMENTS |
We acknowledge Rachel Fearns and Michael N. Teng for their
intellectual input and Jennifer M. Biggs, Myron Hill, and Ena Camargo for technical help.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Infectious Diseases, National Institute of Allergy and Infectious
Diseases, 7 Center Dr. MSC 0720, Bethesda, MD 20892-0720. Phone: (301)
496-3481. Fax: (301) 496-8312. E-mail:
pcollins{at}atlas.niaid.nih.gov.
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