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Journal of Virology, April 2000, p. 3709-3714, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel Subgenomic Murine Leukemia Virus RNA
Transcript Results from Alternative Splicing
Jérôme
Déjardin,1
Guillaume
Bompard-Maréchal,1
Muriel
Audit,1
Thomas J.
Hope,2
Marc
Sitbon,1 and
Marylène
Mougel1,*
Institut de Génétique
Moléculaire de Montpellier, IFR 24, CNRS-UMR5535, and
Université Montpellier II, F-34293 Montpellier Cedex 5, France,1 and Salk Institute for
Biological Studies, La Jolla, California 920372
Received 4 October 1999/Accepted 26 January 2000
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ABSTRACT |
Here we show the existence of a novel subgenomic 4.4-kb RNA in
cells infected with the prototypic replication-competent Friend or
Moloney murine leukemia viruses (MuLV). This RNA derives by splicing
from an alternative donor site (SD') within the capsid-coding region to
the canonical envelope splice acceptor site. The position and the
sequence of SD' was highly conserved among mammalian type C and D
oncoviruses. Point mutations used to inactivate SD' without changing
the capsid-coding ability affected viral RNA splicing and reduced viral
replication in infected cells.
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INTRODUCTION |
The retroviral life cycle requires
that significant amounts of RNA remain unspliced and perform several
functions in the cytoplasm. Thus, the full-length RNA serves as the
viral genetic material that will be encapsidated in viral particles and
as the mRNA encoding structural and enzymatic proteins required for
viral replication. Simple retroviruses are defined as those viruses
which produce one single-spliced env RNA from this
full-length precursor RNA, whereas complex retroviruses are
characterized by the production of multiple spliced RNA species
(18). Because cis-acting and coding functions in
the viral genome frequently overlap, most of the studies on RNA
splicing and transport regulation in murine leukemia viruses (MuLV)
have been conducted with extensively reshaped retroviral vectors that
are replication defective (9, 14, 17). Besides these models
with vectors, usage of MuLV canonical or cryptic splice sites has been
reported in the context of insertional mutagenesis occurring in animals
inoculated with the replication-competent wild-type Moloney MuLV
(2, 15, 19, 22, 26). Since such events are accompanied by
the rearrangement of both the target cellular proto-oncogene and the
inserted proviral DNA (20, 26), the mechanisms controlling
the differential usage of the splice sites remain unclear.
In the present work, performed on cells infected ex vivo with the
closely related replication-competent Friend and Moloney MuLVs, we
report for the first time the production of a large subgenomic
transcript in a simple retrovirus. This novel RNA was generated from an
alternative splice donor site, SD', which is conserved among mammalian
simple retroviruses, in combination with the env splice
acceptor site. Mutations of SD' in Friend and Moloney MuLVs affect the
general splicing pattern of viral RNAs and reduce viral replication.
Identification of this novel MuLV subgenomic RNA provides new insights
into simple retrovirus functions.
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MATERIALS AND METHODS |
Mutagenesis and proviral constructs.
The parental prototypic
Friend MuLV strain 57 (16) and Moloney MuLV strain 8.2 (23) were used as permutated molecular clones with a single
copy of the long terminal repeat (24). These clones provided
the wild-type (wt) background sequences for the reconstitution of
replication-competent Friend and Moloney mutants. Mutations were
introduced by PCR with oligonucleotide-directed mutagenesis
(11). The pMSD1/B plasmid carrying the entire Moloney MuLV
sequence with the M1 mutation was previously described (1). The same strategy was used for construction of Friend F1, F2, and FDV
mutants. All mutated fragments introduced in the wt parental MuLV were
sequenced. Details of plasmid construction will be provided on request.
Cell culture, transfection, and infection.
Mus dunni
fibroblasts (Dunni cells) (12) were maintained in
Dulbecco's modified Eagle's medium supplemented with glutamine (2 mM), penicillin, streptomycin, and 10% heat-inactivated fetal calf
serum at 37°C. One microgram of wt or mutant DNA was transfected onto
Dunni cells by the Lipofectamine method according to the instructions
described by the manufacturer (Gibco BRL). Transfections were monitored
by focal immunofluorescence assay (FIA) using anti-env monoclonal antibodies (25). Reverse transcriptase (RT)
activity was measured by using a rapid assay as previously described
(7). For preparation of mutant viral stocks, supernatants
were collected from transfection experiments that led to confluently
positive monolayers in less than 20 days, within the same time period
as wt-transfected cells. Titration of viral stocks on Dunni cells was
also performed by FIA. Average viral titers, expressed as infectious
focus-forming units (FFU) per milliliter, were as follows: Friend,
105; F1, 6 × 102; F2, 7 × 103; FDV, 3 × 104; Moloney,
104; and M1, 2 × 103. To determine and
compare precisely the levels of viral production by cells infected with
wt or mutant viruses, a two-step method was used. First, Dunni cells
were infected with serial dilutions of viral stocks in the presence of
polybrene (8 µg/ml). After 2 or 3 days of infection, we determined by
FIA the exact number of infected foci per dish, which varies linearly
with the viral input in this assay (25). One tissue culture
dish with approximately 600 to 800 FFU was selected. Supernatants
corresponding to monolayers with the same number of foci were filtered
through 0.45-µm-pore-size filters and used in a second round of
infection on Dunni cells. Comparison of viral production was based on
the number of FFU per milliliter determined in the second round of
infection divided by the number of foci obtained in the first round of
infection. In some experiments, RT activity assays were performed in
parallel in order to quantify viral particle contents in supernatants
produced by infected cells.
RNA analysis.
Cells were infected with different wt and
mutant viral strains and cultured in 10-cm-diameter plates for 4 days
at 37°C. Viral RNA was isolated from viral particles essentially as
described previously (14). Cells were washed twice with
phosphate-buffered saline, and total RNA was extracted from cell
pellets with TriReagent (Sigma) according to the manufacturer's
instructions. The samples were treated with RNase-free DNase (RQ1;
Promega) to remove DNA contamination. Cellular RNA concentrations were
quantitated by measuring optical absorption at 260 nm.
For Northern blot analysis, 5 to 10 µg of cellular RNA were
fractionated on denaturing formaldehyde agarose gels (14),
blotted onto Hybond-N nylon, and UV cross-linked to the nylon. Viral
transcripts were detected by hybridization with the
XbaI-KpnI MuLV fragment (nucleotides [nt] 5638 to 8327) radiolabeled by the random primer method (6) and radiography.
Semiquantitative RT-PCR was performed with the Expand reverse
transcriptase and Expand long template PCR system (Boehringer
Mannheim
Biochemicals). Five micrograms of cellular RNA sample
and sequential
threefold dilutions were denatured in the presence
of 220 ng of
oligo(dT)
15 at 65°C for 10 min and chilled for 5
min at
4°C. RNA was reverse transcribed for 1 h at 42°C and shifted
to 52°C for 30 min in a 20-µl reaction volume containing RT buffer,
10 mM deoxynucleoside triphosphate (dNTP) mix, 10 mM dithiothreitol
DTT, 20 U of RNasin (Promega), and 50 U of Expand RT. The mixture
was
then inactivated at 95°C for 2 min and chilled at 4°C. One-fifth
of
each reaction mixture was used as the starting material for
the
different PCRs. Four combinations of oligonucleotide pairs,
where
"s" and "a" indicate the sense and antisense orientations,
respectively, were used to detect specific MuLV transcripts. The
positions of the transcriptional starts were as follows, with
the sizes
of the amplified products indicated in parentheses:
s3350 and a3600
(250 bp) for full-length RNA detection, and s76
and a5620 (259 bp) for
SD, s1450 and a5620 (276 bp) for SD', and
s6153 and a6418 (265 bp) for
total MuLV RNAs. PCRs were carried
out with 10 mM dNTP mix, PCR buffer,
1.5 U of Expand DNA polymerase
mix, and 220 ng of each of the sense and
antisense primers. Denaturation,
annealing, and extension were
performed at 94, 57, and 68°C, respectively.
PCRs were performed on a
RoboCycler Gradient 96 thermocycler (Stratagene)
with 20 cycles for
total, full-length, and
env amplifications
and 24 cycles for
SD' or SD" amplifications. These cycles were
preceded by a 5-min
denaturation at 94°C and terminated by a 10-min
extension at 68°C.
Amplified products were electrophoresed on
agarose gels, and bands were
quantified by FluorImager (Molecular
Dynamics). Experiments conducted
in parallel with addition of
a radiolabeled oligonucleotide were used
to quantitate the amplified
products by PhosphorImager (Molecular
Dynamics), and there was
a good correlation between the two
methods.
Extensive amplifications (30 cycles) were also performed to obtain
sufficient amounts of DNA for sequencing. Amplified DNA
products was
sequenced on an automatic sequencer (ABI PRISM 377;
Perkin-Elmer) with
the dye terminator cycle sequencing ready reaction
kit (ABI PRISM) by
following the recommendation of the manufacturer
and using an
oligonucleotide complementary to nt 5601 to 5620
of
MuLV.
For RNase protection assays, 15 µg of total RNA were generally used.
Riboprobe transcription was performed with linearized
template plasmids
according to standard procedures (
3). The
274-nt Moloney
riboprobe was generated from the SPMLV plasmid
linearized with
EcoRI and includes 5' and 3' non-MuLV vector sequences
(
3,
14). An SPFLV vector was derived after insertion of the
Friend
SacI8273-
BalI213 homologous fragment in
pGEM. The 274-nt
Friend riboprobe was obtained after linearization of
SPFLV with
EcoRI. These probes should both yield a 214-nt
genomic and a 205-nt
spliced fragment after RNase digestion, as
previously described
(
14). RNase protection products were
quantitated by the
PhosphorImager.
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RESULTS |
Identification of a novel MuLV RNA transcript.
Total cellular
RNA extracted from Dunni cells infected with Friend MuLV was examined
by Northern blot hybridization using a probe complementary to the 3'
end of the Friend MuLV sequence. This yielded two expected major bands
corresponding to the full-length genomic transcript (8.3 kb) and the
single-spliced env RNA (3 kb) (Fig.
1). However, we noted an additional faint
band, below the 28S RNA, corresponding to a potential 4- to 5-kb
transcript. This faint band was more readily detected in the FDV mutant
of Friend MuLV, which was obtained after introduction of two synonymous point mutations in the gag region (Fig.
2). Interestingly, the FDV mutations
surround a potential 5' splice site that is implicated in MuLV-driven
rearrangements of the c-myb proto-oncogene (15, 21). In order to determine the precise nature of this new 4- to
5-kb RNA in replication-competent MuLV, extensive RT-PCR amplifications (30 cycles) were performed on two different strains of MuLV. We detected this novel RNA with both Friend and Moloney MuLV (Fig. 3). The sequence of the amplified
fragment containing the splicing junction revealed the use of the
alternative splice donor site mentioned above, designated here as SD'.
SD' is located in the 3' third of the capsid-coding region in the
env intron, at positions 1597 and 1596 of Friend and
Moloney, respectively. The 3' splice site used in concert with SD' was
located at position 5491 for Friend and 5490 for Moloney, corresponding
to the canonical splice acceptor site (SA) that yields the
single-spliced env RNA.
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FIG. 1.
Viral RNA species expressed in MuLV-infected cells.
Cellular RNA from mock-infected cells or cells acutely infected with wt
Friend MuLV or the FDV mutant (see Fig. 2) were analyzed by Northern
blot hybridization with a radiolabeled probe complementary to the 3'
MuLV region (nt 5638 to 8327). This probe reveals the viral genomic RNA
(8.3 kb), the single-spliced env RNA (3 kb), and a third RNA
species (4 to 5 kb). Positions of RNA molecular size markers and 28S
and 18S ribosomal RNAs are indicated.
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FIG. 2.
Sequences of Friend and Moloney MuLV mutants in the SD'
region. Sequences of Friend (FDV, F1, and F2) and Moloney (M1) viruses
containing changes in the SD' region were aligned with the 5' splice
donor site (5' SS) consensus sequence of U1 snRNA. The cleavage site is
indicated by a slash mark. The nucleotide changes maintain the coding
potential of the parental strains and are indicated for each mutant.
For comparison, the sequence of the conserved canonical MuLV 5' splice
donor site (SD) is shown. The number of potential mismatches between
each splice donor site and the U1 snRNA is on the right.
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FIG. 3.
Amplification of a new alternatively spliced MuLV
transcript by RT-PCR. (A) Schematic structures of the unspliced genomic
and alternatively spliced (SD') RNA, including the canonical (SD),
acceptor (SA), and alternative (SD') splice sites. Nucleotides are
numbered starting from the first nucleotide of R according to the
Friend MuLV sequence. Also noted are the gag gene
components, including the matrix (MA), capsid (CA), and nucleocapsid
(NC). Arrows refer to the approximate positions of primers used for RT
with the oligo(dT) primer and for PCR amplification. (B) RT-PCR was
conducted on total RNA samples extracted from mock-infected cells (lane
3) or cells infected with either Friend (lane 4) or Moloney virus (lane
6). Reverse transcriptions were performed with oligo(dT), and PCRs were
performed with oligonucleotides s1450 and a5620 as described in the
Materials and Methods. The alternatively spliced SD' RNA yielded an
amplified product of 276 bp. After 30 cycles, amplified samples were
loaded onto an agarose gel and stained with ethidium bromide. Lanes 1 and 2, 1-kb and 100-bp ladders, respectively.
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Effects of SD' mutations on viral replication.
To evaluate the
influence of the SD' sequence on the MuLV life cycle, we generated
replication-competent MuLV mutants with distinct point mutations in
this sequence. It is important to note that all the mutations
maintained the wt Gag amino acid sequence. In the aforementioned FDV
mutant, the two mutated nucleotides also maintained the potential
parental base-pairing of SD' with the U1 snRNA consensus (Fig. 2). We
generated two other Friend mutants, F1 and F2, and one Moloney mutant,
M1, in which putative mismatches with U1 snRNA were introduced (Fig.
2). The Gag and Env precursor proteins and cleaved products were
detected in all mutants, as assessed by immunoblotting (not shown). The
infectivities of the mutant and wt strains were determined by
monitoring cell surface envelope expression on newly infected cells by
using the quantitative FIA (25) (Fig.
4), as well as by measuring the RT
activity of the various supernatants (not shown). The former technique
is a measure of protein production from the spliced env RNA
while the latter reflects the production of RT by the unspliced
gag-pol RNA. These two tests yielded similar results, indicating a significant reduction (ranging from 7- to 100-fold) in the
infectivity observed with the different SD' mutants (Fig. 4). The F1
and M1 mutants, which displayed the lowest potential base pairing with
U1 snRNA, exhibited the lowest infectious ability. Interestingly, the
replication capacity of the FDV mutant, which maintained the parental
U1 snRNA base pairing, was the least altered.
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FIG. 4.
Replication of wt and mutant viruses. FIA was used to
quantitate infectious virions present in the supernatants harvested
from cell cultures with the same number of wt or mutant MuLV-infected
foci. Infectivities were performed at least three times for each virus
in parallel and each test was performed in triplicate. Bars, the
standard error of the mean of each series.
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Effects of SD' mutations on MuLV splicing.
To evaluate whether
the observed reduction in MuLV titers following mutation of the SD'
sequence was associated with an alteration in the alternative splicing
efficiency, we next examined the splicing patterns of these mutants in
infected cells. Semiquantitative RT-PCR was performed with total RNA
extracted from de novo-infected Dunni cells. Full-length RNA,
single-spliced env RNA, alternatively spliced RNA, and total
MuLV transcripts were monitored with different combinations of
oligonucleotide pairs (Fig. 5A). Although
similar results were obtained when the initial RT reaction was
performed with a specific MuLV or an oligo(dT) primer, we preferred the latter because it allowed amplification of the total cellular RNA
independently of infection efficiency. The RT-PCR band obtained with
the F1, F2, and M1 mutants migrated slightly slower than that detected
with wt MuLV or FDV (Fig. 5A). Sequencing of the amplified product in
F1, F2, and M1 revealed the recruitment of another cryptic donor site,
AAG/GUAAA, designated here as SD". It is present in both Friend and
Moloney and is located 82 nt downstream of SD'. As for SD', SD" was
used in conjunction with the SA canonical acceptor site. Therefore,
impairment of SD'-U1 base pairing in these mutants resulted in the
activation of a cryptic splice donor site (SD").
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FIG. 5.
Effect of SD' mutations on MuLV splicing. The different
RNA species were detected by semiquantitative PCRs (20 to 24 cycles)
performed on threefold dilutions of each sample. The amplified products
were run onto agarose gels containing ethidium bromide. (A) Schematic
MuLV genome map indicating the splice donor (SD, SD', and SD") and
acceptor (SA) sites. The corresponding sequences in Friend (Fr) and
Moloney (Mo) are shown in the upper panel. Approximate positions of the
primers used to amplify the different RNA species are identified by the
numbered arrows. Each viral RNA species detected is indicated on the
left side of the gel, with the corresponding oligonucleotide pairs
identified by numbers on the right. Note that the main products
amplified from the F1, F2, and M1 mutants correspond to usage of the
cryptic SD" and exhibit slower migration relative to products arising
from usage of the alternative SD' in the wt and FDV. (B) Quantification
of the different RNA species was determined by a FluorImager. For each
virus, at least two RNA preparations were isolated and subjected to
three to six semiquantitative RT-PCR analyses. For each RNA species,
the RNA levels are expressed as the ratio of the value obtained after
amplification with the specific oligonucleotide pair divided by the
total RNA value obtained by amplification with primers 6 and 7. Final
RNA levels are represented after normalization to levels of the
corresponding wt strain (± standard error of the mean). Note that both
wt Friend and Moloney, as well as the FDV mutant, produced
alternatively SD' spliced RNA, while F1, F2, and M1 mutants displayed a
spliced RNA resulting from the activation of the SD" cryptic splice
site.
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For comparison purposes, the ratio of the amount of each transcript to
the total level of MuLV RNA was determined and normalized
to the ratio
of the corresponding wt MuLV (Fig.
5B). Differences
were evaluated by
using the two-tailed Student
t test and considered
significant when probability values (
P) were <0.05.
Although semiquantitative
RT-PCR does not allow the precise
quantification of small differences
in RNA expression, this method
revealed dramatic differences in
the relative amounts of alternatively
spliced RNA species between
mutant and wt MuLV. We observed a
significant increase in the
SD' RNA level in cells infected with the
FDV mutant (
P < 0.001),
in agreement with the initial
observation made on Northern blots
(Fig.
1). Also, the F1 and F2 Friend
mutants harbored a much higher
level of SD" RNA than the equivalent M1
Moloney mutant (Fig.
5),
indicating that elements, additional to a
functional alternative
SD' site, influenced SD"
usage.
Since the F1 and F2 mutations appeared to decrease levels of the
canonically spliced
env RNA (Fig.
5B), a more direct
quantification
of
env RNA levels was performed by using the
RNase protection
assay. Total cellular RNA was hybridized to a
riboprobe that spans
the MuLV canonical splice donor site (SD) at
position 205 (Fig.
6A). RNase digestion
yielded a 214-nt protected fragment derived
from the unspliced RNA and
the SD' or SD" alternatively spliced
RNA species and a shorter 205-nt
long fragment corresponding to
the canonical SD-spliced
env
transcript (Fig.
6A). A reproducible
decrease in
env RNA
levels was observed for all mutants, albeit
to different extents, when
compared to the respective wt strains
(
P < 0.04) (Fig.
6B
and C).
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FIG. 6.
Quantification by RNase protection of the canonical
env transcript level in MuLV-infected cells. (A) Friend and
Moloney transcripts were detected by RNase protection with the
uniformly labeled antisense SPFLV or SPMLV probes, respectively (see
Materials and Methods), which overlap the canonical SD site.
Hybridization of the 274-nt probe to viral RNA species that are not
spliced at the canonical SD site (full length, SD', and SD" RNA) yields
a protected 214-nt fragment, while canonically spliced env
RNA yields a protected 205-nt fragment. (B) RNase protection assays
were carried out with 15 µg of total cellular RNA (lanes 2 to 6, 8, 9, and 13) and RNA extracted from 10% total virus pelleted medium from
infected or mock-infected cell monolayers (lanes 10 to 12). The
positions of the probe and the protected fragments corresponding to
canonically spliced env RNA (SD) as well as the
noncanonically spliced RNAs (non SD) are indicated by arrows. The size
markers (lane 14) consist of end-labeled  X174 HaeIII DNA
fragments. (C) Quantification of RNase protection assays. For each
series, the percentage of env-protected fragments versus the
total of protected signals is represented (± standard of the mean).
Each value corresponds to the average of at least three measurements
performed on different RNA preparations.
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Conservation of a potential alternative splice donor site in the
capsid of mammalian simple retroviruses.
gag sequences of
replication-competent MuLV, feline, porcine, and simian C- and D-type
retroviruses were aligned. This showed that a 5' splice site consensus
sequence, corresponding to a conserved putative SD' site, is present
approximately 100 nt upstream of the major homology region (MHR)
(4) in the capsid-coding gene (Fig.
7). Interestingly, the GU dinucleotide
immediately adjacent to the cleavage site, which is present in almost
all mammalian pre-mRNA introns (10), was strictly conserved
among these viral sequences.
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FIG. 7.
Conservation of the SD' site. Sequence alignment of a
putative SD' site in the capsid-coding region of a series of
replication-competent mammalian types C and D retroviruses. The 5'
splice donor site consensus sequence (5' SS) is shown on top, with the
potential splicing cleavage site indicated by a vertical line. All
sequences were located approximately 100 nt upstream of the capsid
major homology region. Numbering is according to the Friend-MuLV 57 sequence. Lower-case letters indicate mismatches between the 5'SS
consensus and the viral sequence. Abbreviations and strains correspond
to the following retroviruses. (i) MuLVs: Friend-MuLV, strain 57;
Moloney-MuLV, strain 8.2; RadLV, radiation leukemia virus; Cas-Br-E,
Lake Casitas brain E neurotropic virus; WNB5, the N- and B-tropic
clones of the WN1802 isolate; and AKV, from clone AKR 623 of endogenous
virus from the AKR mouse strain. All of the above are ecotropic MuLVs.
MCF, clone MCF1233 of the polytropic mink cell focus-inducing viruses.
(ii) Feline leukemia viruses: FeLVA and FeLVB, strains A and B. (iii)
Primate simple retroviruses: simian type C retroviruses include BaEV,
baboon endogenous virus, and GaLV, gibbon ape leukemia virus. Simian
type D retroviruses include MPMV, Mason-Pfizer monkey virus, and
SRV-Pc, a baboon simian retrovirus-like isolate. (iv) Porcine
endogenous virus: a human-tropic C-type porcine endogenous
retrovirus.
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DISCUSSION |
We have identified a new 4.4-kb RNA transcript produced by two
prototypic strains of MuLV. This novel RNA results from splicing between an alternative splice donor site, SD', located in the capsid-coding region, and the canonical env splice acceptor
site. Although in vivo studies of Moloney MuLV-induced promonocytic leukemia have shown that the recruitment of an SD' sequence produces a
rearranged truncated c-myb gene upon insertional mutagenesis (8, 21), this is the first report demonstrating that a
second spliced RNA is produced during the life cycle of a
replication-competent simple retrovirus. Moreover, we found that an SD'
sequence is present at a similar position in the capsid-coding gene of
all the replication-competent MuLV, feline, porcine, and simian C-type retroviruses we examined. This conserved feature also extended to the
more distantly related simian D-type oncoviruses. It will be
interesting to determine whether an additional homologous subgenomic transcript is also present in these retroviruses, as already suggested in a previous report with the gibbon ape leukemia virus (5).
This new 4.4-kb RNA was weakly detectable by Northern blot analysis of
RNA from cells infected with the wt Friend strain, suggesting a low
level of production and/or a potential instability of the mature SD'
transcript. Since the synonymous mutations introduced in the FDV mutant
flanked the SD' site and increased the SD' RNA level, it is likely that
potential local RNA structures also modulate splicing at this site.
Production of two spliced RNAs from a single precursor and maintenance
of the full-length RNA pool require incomplete splicing. According to
our results, production of the alternatively spliced SD' RNA might play
an important role in splicing regulation in MuLV simple retroviruses.
Thus, when SD' splicing was maintained (wt and FDV), cryptic splicing
at SD" was not detected and mutations in Friend-MuLV that activated the
alternative SD' or cryptic SD" were accompanied by a decreased
production of the canonical env spliced RNA (FDV, F1, and
F2). However, a strict competition model between these sites cannot
account for the splicing pattern observed in the Moloney MuLV strains,
since a significant impairment of alternative splicing in the M1 mutant
did not lead to increased levels of the env RNA.
Furthermore, usage of these potentially competing sites did not
strictly correlate to U1 base pairing. For instance, although both SD"
and the canonical SD site harbored two potential mispairings with U1,
an SD" RNA remained undetectable in the wt viral strains. Therefore,
other parameters, such as neighboring (14) or distant RNA
structures (13), likely influence the splicing balance. This
underlines the usefulness of full-length viruses in unraveling
regulatory elements involved in the modulation of RNA expression.
Since decreased levels of infection observed with the mutants resulted
from synonymous mutations in the SD' region, this was the first
demonstration of a cis-acting effect of the capsid region in
the context of a replication-competent retrovirus. The severe drop
observed in replication abilities of the F1, F2, and M1 mutants could
result from a negative effect of the newly produced SD" RNA, loss of
the SD' RNA, or alteration of the splicing balance. Nevertheless,
results obtained with the FDV mutant, which harbors a high level of SD'
RNA and a slightly altered viral titer, suggest that SD' usage is
required for a balanced splicing profile and optimal replication.
In addition, the coding capability of the SD' RNA might be directly
involved in the increased level of infectious retroviral particles. The
alternatively spliced SD' RNA was polyadenylated, as shown by its
presence in the poly(A)+ RNA fraction (not shown), and
could be reverse transcribed with an oligo(dT) primer. Also, the SD'
RNA presents a large coding potential, with multiple translation
initiation codons; these include initiation codons used in the
glycogag, gag, and env open reading
frames, which are active in the full-length or canonically spliced
mRNA, as well as the initiation codon in the capsid that is used in the
chimerical gag-myb transcripts (19, 26). These many putative initiation codons, placed in the context of new gag and env open reading frames, provide the
potential for new MuLV translational products. Identification of this
novel alternatively spliced MuLV RNA at a site that is highly conserved
among types C and D mammalian oncoviruses provides new avenues of
investigation on the influence of new RNA species in replication of
simple retroviruses.
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ACKNOWLEDGMENTS |
We thank laboratory members past and present, including F. Kim,
K. Beemon, J. Richardson, S. Orsoni, B. Hohl, and L. Drumright for help
and advice during the course of this work and N. Taylor, R. Hipskind,
and J. M. Blanchard for critical reading of the manuscript.
This work was supported by grants no. 9521 and 4066 from the
Association pour la Recherche sur le Cancer (to M.M. and M.S.) and by
successive grants from CNRS (ATIPE virology program) and the Fondation
pour la Recherche Médicale and a Philippe Foundation award (to
M.S.). M.M. and M.S. are supported by the Centre National de la
Recherche Scientifique (CNRS) and the Institut National de la
Santé et de la Recherche Médicale (INSERM), respectively. J.D. was supported by a fellowship from the French government (MENRT).
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut de
Génétique Moléculaire de Montpellier II (IGMM), IFR
24, CNRS-UMR5535, Université Montpellier II, 1919, Rte. de Mende,
F-34293 Montpellier Cedex 5, France. Phone: 33 (4) 67 61 36 40. Fax: 33 (4) 67 04 02 31. E-mail: mougel{at}igm.cnrs-mop.fr.
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Journal of Virology, April 2000, p. 3709-3714, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
