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Journal of Virology, October 1998, p. 8073-8082, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vivo Selection of Rous Sarcoma Virus Mutants
with Randomized Sequences in the Packaging Signal
Nicole A.
Doria-Rose and
Volker M.
Vogt*
Section of Biochemistry, Molecular and Cell
Biology, Cornell University, Ithaca, New York 14853
Received 9 April 1998/Accepted 15 June 1998
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ABSTRACT |
Retrovirus genomes contain a sequence at the 5' end which directs
their packaging into virions. In Rous sarcoma virus, previous studies
have identified important segments of the packaging signal, 
, and
support elements of a secondary-structure prediction. To further
characterize this sequence, we used an in vivo selection strategy to
test large collections of mutants. We generated pools of full-length
viral DNA molecules with short stretches of random sequence in and
transfected each pool into avian cells. Resulting infectious virus was
allowed to spread by multiple passages, so that sequences could compete
and the best could be selected. This method provides information on the
kinds of sequences allowed, as well as those that are most fit. Several
predicted stem-loop structures in were tested. A stem at the base
of element O3 was highly favored; only sequences which maintained base
pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the
randomized region in all selected L3 stem mutants; we interpret this to
mean that it compensates for the defects in L3. Randomized mutations
adjacent to G213 maintained the wild-type base composition but not its
sequence. The kissing-loop sequence at end of L3, postulated to
function in genome dimerization, was not required for infectivity but
was selected for over time. Finally, a deletion of L3 was constructed
and found to be poorly infectious.
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INTRODUCTION |
The packaging of the RNA genome into
retrovirus particles is a specific and efficient process. The nature of
packaging is only partially understood, but cis- and
trans-acting factors have been identified. Expression of the
Gag protein alone is sufficient for virus assembly and is also
sufficient for encapsidation of the genome (29). An RNA
packaging signal, called or E, has been found in the 5' end of the
genome of many retroviruses (reviewed in references
5 and 9). The core of is
generally located between the primer binding site and the start of
gag, although other sequences can contribute to packaging
efficiency. The segment of RNA encompassing is predicted to be
highly structured. RNA stem-loops have been implicated in packaging for
a number of retroviruses (7, 20, 23, 28, 42).
Two of the best-studied packaging signals are in murine leukemia virus
(MLV) and human immunodeficiency virus type 1 (HIV-1). MLV has been
particularly well studied because of its utility as a vector in gene
therapy. The MLV packaging signal has been mapped to a 350-nucleotide
(nt) sequence in the 5' untranslated region (UTR), downstream of the
splice donor (22). In vitro and in vivo studies show that
four stem-loop structures in the RNA are necessary and sufficient for
packaging, although sequences downstream improve the efficiency
(27, 28). In HIV-1, is less well defined. As in MLV, a
sequence in the 5' UTR is important for packaging. Several studies
indicate a role for four stem-loops located between the 5' splice donor
and the beginning of gag (reviewed in reference
5). A sequence of 46 nt which includes the third stem-loop was shown to be sufficient for packaging of a reporter gene
into virus-like particles in a vaccinia virus expression system
(16). Deletion and mutation of the other stem-loops cause packaging defects in a variety of in vivo expression and in vitro assay
systems (4, 6, 8, 25, 30), indicating their importance.
However, other sequences that have been reported to affect packaging
include (i) the transactivation-responsive element (TAR) and other 5'
sequences and (ii) the Rev-responsive element (25).
Furthermore, the function of the stem-loops appears to be context
dependent: the addition of certain reporter genes inhibits packaging
(5, 24); and the position of the 46-nt packaging sequence
was found to be critical for its function, presumably because
surrounding sequences affected its ability to fold into a stem-loop
(16). The complete packaging signal may be composed of
several widely separated sequences, and its secondary and tertiary structure are almost certainly important for its recognition by the
assembling virus.
In Rous sarcoma virus (RSV), the RNA packaging signal has been mapped
to the 5' UTR (19, 21). A 160-nt sequence, located between
U5 and the start of gag, is sufficient to direct packaging of a reporter gene (2). RNA secondary-structure prediction (14) yielded a model of the 5' UTR consisting of several
stem-loop elements. One element, O3, was shown to be essential:
mutations which disrupted base pairing of the stem at the base of this
element reduced packaging of a reporter gene 100-fold, while
compensatory mutations which restored base pairing rescued packaging to
nearly half of wild-type levels (2, 20).
In RSV, overlaps sequences that are predicted to have other
functions in virus replication. Wild-type viruses have dimeric genomes.
The packaging signal overlaps a putative dimerization initiation
sequence in RSV (13); a similar signal is found in one of
the stem-loops of the HIV-1 (4). The relationship of
packaging and dimerization is unclear (10, 31, 33). The 5'
UTR of RSV also contains three small open reading frames (ORFs) which
serve to regulate Gag protein translation (10, 11, 15, 18,
33). Therefore, in studying the effect of mutations in , one
must consider the effects on dimerization and translation also.
Our approach to the study of packaging was inspired by the RNA in vitro
selection procedure (12, 17, 37). This method, which takes
advantage of the fact that RNA has both informational and functional
capacity, begins with a pool of RNA molecules of random sequence. A
selection pressure, such as requiring the RNA to bind to a protein, is
applied. The bound molecules are isolated, reverse transcribed, and
amplified. This creates a new pool, which can be transcribed back into
RNA and selected again. After multiple rounds, the best sequences are
highly enriched in the pool. Our method, in vivo selection, uses the
natural replication cycle of the virus in place of the in vitro steps
of selection, reverse transcription, and amplification. By building a
randomized stretch into a proviral clone and transfecting the resulting
mixture of DNA into cells, one generates a population of viruses which
can evolve over time. This method was first used by Berkhout and Klaver to examine the bulge and loop of the HIV transactivation-responsive region sequence (3); they randomized three nucleotides at a time. The study presented here extends the method, using much longer
random stretches and multiple random stretches to test larger
secondary-structure elements.
The in vivo selection technique has allowed us to map more of the RSV
packaging signal. One predicted stem was shown to be essential, while
two other stems were disrupted with no reduction in virus infectivity.
A predicted dimerization initiation signal was shown to be favored but
not strictly required.
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MATERIALS AND METHODS |
Plasmids.
All nucleotide sequences and numbers in this
report refer to the RNA sequence of the Schmidt-Ruppin A (SR-A) strain
of RSV (36) (Genbank accession no. L29198). Plasmid
RCAS.bp
A/B was the generous gift of Stephen Hughes. This plasmid, a
member of the RCAS series, contains an infectious RSV DNA sequence in a
pBR322 vector, without src or selectable genes
(40). Except for part of the pol gene,
RCAS.bpA/B has the SR-A sequence. We constructed a derivative of
this plasmid, called RCAS.SE, which contains a unique SpeI
site corresponding to nt 205 in the viral RNA and an EagI
site at nt 325. A second RCAS derivative was made in which nt 205 to
325 are replaced with a nonviral sequence. This plasmid, called
RCAS.stuffer, was made by replacing the small SpeI-EagI fragment of RCAS.SE with a 2.1-kb
SpeI-EagI fragment from YEP24 (New England
BioLabs).
Construction of randomized pools.
The wild-type RSV sequence
from nt 205 to 325 was replaced by mutagenic cassettes. The sequences
of the cassettes are shown in Fig. 2. For L3 Base, L3 Mid, L3 KL, and
O3 Mid, oligonucleotides corresponding to sense-strand nt 193 to 331 in
the viral RNA (RCAS.SE sequence) were synthesized by the Cornell
BioResource Facility. Within each oligonucleotide, one or two stretches
of sequence, 6 to 14 nt in length, were replaced with random sequence
(25% each base at each position). The oligonucleotides were made
double stranded by PCR to facilitate cloning; the sequences of the
primers are presented in Table 1. PCR was
performed with one of the upstream primers SpeI top, SpeI long, and
SpeI G/T and downstream primer EagI bottom or EagI bottom2. For O3
Base, a pair of oligonucleotides was synthesized by Genosys, Inc.; one
corresponded to sense-strand nt 193 to 263 and contained random
nucleotides at positions 228 to 237; the other corresponded to the
antisense-strand nt 331 to 247. This pair was mixed for PCR with
primers SpeI top and EagI bottom. In all cases, the PCR mixtures
contained 100 ng of each long oligonucleotide, 375 ng of each primer, 1 µl of Taq polymerase (Boehringer Mannheim Corporation
[BMB]), 1.5 mM MgCl2, 10 mM Tris (pH 8.3), 50 mM KCl, and
0.2 mM each deoxynucleoside triphosphate (dNTP). PCR products were
purified by phenol extraction and ethanol precipitation, or by using a
High-Pure kit (BMB), and then digested with SpeI and
EagI. Cut DNA was purified by phenol extraction and ethanol
precipitation prior to ligation.
To generate vector for mass cloning without contamination by wild-type
sequences, plasmid RCAS.stuffer was used. Ten micrograms
of DNA was
digested with
SpeI and
EagI. Due to the large
size
of the plasmid backbone (11 kb), we were unable to recover
sufficient
DNA for cloning by the standard procedures of separation on
an
agarose gel and extraction of the relevant band. Therefore, the
fragments were separated on a 5 to 20% sucrose gradient in a buffer
consisting of 10 mM Tris (pH 8), 1 mM EDTA, and 50 mM NaCl.
Centrifugation
was at 50,000 rpm for 5 h in an SW60 rotor
(256,000 ×
g). Fractions
were collected; those
containing the 11-kb backbone and no insert
were recovered, and the DNA
was concentrated by isopropanol precipitation.
For the ligation, 400 ng
of backbone and 1 ng of insert were mixed
with 500 U of T4 DNA ligase
(New England Biolabs or BMB). Forty
nanograms of the ligation mix was
used to transform
Escherichia coli DH5
by electroporation
(BioRad Gene Pulser II). After electroporation,
1 ml of SOC medium
(
32) was added, and the cells were incubated
for 30 min at
37°C with shaking at 225 rpm. Ten microliters of
cells was plated on
ampicillin-containing agar; the resulting
colonies were counted to
estimate the number of transformants.
The remaining cells were added to
100 ml of Luria broth containing
100 µg of ampicillin per ml and
grown overnight at 37°C with shaking
at 225 rpm. Plasmid DNA was
purified by standard techniques (
32,
39).
Cells and virus.
Turkey embryo fibroblasts (TEFs) were the
generous gift of Rebecca Craven. These cells do not contain endogenous
sequences capable of recombining with RSV (41).
Early-passage cells (from between 6 and 15 passages after the initial
preparation) were used in all experiments. TEFs were grown in
low-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with
1% vitamins, 20 mM L-glutamine, 100 U of penicillin G and
100 µg of streptomycin sulfate per ml, 2% heat-inactivated chick
serum (Gibco BRL), and 10% fetal calf serum (Gemini). Cells were kept
in 5% CO2 at 39°C. The chick serum was found to contain
avian retrovirus particles, which could be detected in nested PCRs;
therefore, the chick serum was omitted from the medium when virus was
to be collected for RNA analysis.
Infections were carried out by overnight incubation of TEFs with virus
in complete medium. Transfections were performed by
the DEAE-dextran
method. For a 100-mm-diameter plate, 10 µg of
plasmid was added to
1.5 ml of Tris-buffered saline containing
100 µg of DEAE-dextran.
Cells were washed twice with phosphate-buffered
saline and twice with
Tris-buffered saline, and then the DNA was
added. The plates were
incubated at 39°C and rocked every 15 min
for 60 to 90 min. The DNA
was aspirated, and the cells were shocked
with 4 ml of dimethyl
sulfoxide 27% in serum-free DMEM. After
4 min, the cells were washed
with medium and fed with complete
DMEM. To estimate the transfection
efficiency, one plate was transfected
with the reporter plasmid
pHBAPr-1-
gal, which contains the
lacZ gene driven by the
-actin promoter (
26), in parallel with each
experimental
transfection; histochemical staining was performed
to allow counting of
the transfected cells (
1). We found that
1 × 10
5 to 5 × 10
5 cells were stained per
100-mm-diameter plate.
The presence of virus was monitored by an exogenous reverse
transcriptase (RT) assay as described previously (
34).
Medium
from transfected or infected plates was filtered through a
0.45-µm-pore-size
filter. Virus was collected from 2.5 ml of medium
by centrifugation
through a 15% sucrose cushion at 200,000 ×
g for 18 min at 4°C.
The pellet was resuspended in 30 µl
of 1× VSB (25 mM Tris [pH
7.5], 50 mM NaCl, 1 mM EDTA). Of this, 5 µl of virus was added
to 20 µl of a reaction cocktail for final
concentrations of 50
mM Tris (pH 8), 20 mM dithiothreitol, 0.05%
Nonidet P-40, 6 mM
MgCl
2, 60 mM NaCl, 5 µg of oligo(dT)
and 10 µg of poly(A) per
ml, 10 µM cold dTTP, and 8 µCi of
[
-
32P]dTTP (3,000 Ci/mmol; Amersham). The reaction
mixtures were incubated
for 2 h at 37°C. Five microliters of
each reaction mixture was
spotted on DE81 paper (Whatman), washed three
times in 2× SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate),
rinsed in
H
2O and then in 95% ethanol, dried, and
quantitated by Cerenkov
counting in a scintillation counter.
Analysis of viral RNA.
Virus was collected as described
above. The pellets were resuspended and lysed in 100 µl of VLB (100 mM NaCl, 50 mM Tris [pH 7.5], 10 mM EDTA, 0.5% sodium dodecyl
sulfate) with 10 µg of yeast tRNA and 10 µg of proteinase K. RNA
was purified by phenol extraction and ethanol precipitation.
Alternatively, pelleted virus was resuspended in 10 mM Tris (pH 8)-20
mM NaCl, and the RNA was purified by using a QIamp kit (Qiagen). To
generate cDNA for sequencing, RT-PCR was performed. cDNA synthesis
reactions were performed in a 20-µl volume containing the RNA
recovered from 0.8 ml of medium, 1 µl of avian myeloblastosis virus
RT (BMB), 800 ng of primer 844, 0.8 mM each dNTP, 0.5 µl of RNase
inhibitor (PRIME), 50 mM Tris (pH 8.5), 8 mM MgCl2, 30 mM
KCl, and 1 mM dithiothreitol. RNA and primer 844 were heated to 80°C
for 5 min and chilled on ice to remove secondary structure; all
components except the enzyme were mixed and incubated for 10 min to
allow annealing of the primer to the RNA. Reactions were warmed to
42°C, and enzyme was added. The reaction mixtures were incubated at
42°C for 90 min. This was followed by PCR using all of the cDNA
synthesis reaction mixture, to which was added 375 ng each of primers
844 and 5'EcoRI and 1.3 µl of Taq polymerase (BMB), in a
total volume of 100 µl with final conditions of 18 mM Tris (pH 8.3),
46 mM KCl, and 2.8 mM MgCl2. No additional dNTPs were
added. When insufficient product resulted, nested PCR was performed
with the product of the first reaction as a template, with primers 52 and 619. PCR products were purified by using a SpinBind column (FMC)
and sequenced with the Gibco BRL double-stranded DNA cycle sequencing
system or by the BioResource Center, Cornell University, on an Applied
Biosystems ABI Prism 377. Alternatively, the RT-PCR products were
subcloned into pBluescript KSII+ (Stratagene), using the
EcoRI site introduced at nt 10 by primer 5'EcoRI and an
XhoI site at nt 644, and individual constructs were
sequenced.
Sequence analysis.
Secondary-structure analysis was
performed by using mfold (43, 38) on the server located at
http://www.ibc.wustl.edu/~zuker/rna/form1.cgi. An alignment of the
first 400 nt of viral RNA sequence from different avian sarcoma and
leukosis virus strains was performed with MegAlign. The strains and, in
parentheses, their GenBank accession numbers are CT10 (Y00302), FSV
(J02194), HBI (M11784), HPRS-103 (Z46390), MC29 (J02247), PR2257T
(X51863), Prague B[LA23] (J02339), Prague B[LA23 mutant A]
(M31387), Prague C (J02342), Prague C [duck adapted] (X51860),
Schmidt-Ruppin A (L29198), SRA-V (U41731), Schmidt-Ruppin D
(D10652), UR2 (M10455), and Y73 (J02027).
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RESULTS |
In vivo selection.
In vivo selection is based on the
production of a large pool of viral genomes containing one or
more stretches of randomized sequence. When such a pool of
randomized genomes is transfected into cells, any genome
capable of Gag expression will direct virion production. However,
many of the genomes will have defective packaging signals, and thus
many of the virions will not contain viral RNA. Only those which do
carry genomes are infectious; therefore, when cell-free virus is
used to infect fresh cells, only viruses with functional
packaging signals will spread. After many passages, those with
better packaging signals will come to dominate the population.
Generating the pool of mutant genomes is a multistep process (Fig.
1B). We began with a full-length,
infectious clone of RSV,
plasmid RCAS.bp
A/B (
40), which
contains the complete SR-A provirus,
with some
pol sequence
from strain BH, on a pBR322 backbone. We
modified this construct by
introducing unique
SpeI and
EagI restriction
sites flanking the core of
. The resulting plasmid, RCAS.SE,
was as
infectious as the parental virus (data not shown). A further
construct,
RCAS.stuffer, was made by replacing the
SpeI-
EagI fragment
in RCAS.SE with an
irrelevant 2.1-kb fragment from a yeast plasmid.
RCAS.stuffer,
rather than RCAS.SE, was digested and used for all
of the cloning
steps, in order to avoid contaminating the pools
with wild-type
sequence arising from incompletely digested plasmid.
As expected, this
construct was not infectious (data not shown).
To purify large
quantities of the RCAS backbone, we sedimented
the digested DNA through
a 5 to 20% sucrose gradient, collected
fractions that contained the
backbone but not the stuffer insert,
and after concentration used this
DNA in ligation reactions. For
each experiment, the 120-nt
SpeI-
EagI fragment corresponding to
was
replaced with a mutagenic cassette containing the random
stretches of 6 to 14 nt (25% of each nucleotide [Fig.
2 and
3]).
The cassettes were created with long oligonucleotides that were
amplified to make them double stranded (Fig.
1A). After digestion
with
the two restriction enzymes, the cassettes were ligated into
the RCAS
backbone.
E. coli cells were transformed with the ligation
mixture by electroporation and then grown overnight in batch culture
in
presence of ampicillin. Plasmid DNA was purified from the cultures
and
then transfected into TEFs to initiate an infection.
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FIG. 1.
In vivo selection: experimental procedures and cloning.
(A) Production of mutagenic cassette. A long oligonucleotide (white
box) is synthesized with a viral sequence and a stretch of randomized
sequence (striped segment). PCR is performed to first make the DNA
double stranded and then amplify it. The resulting cassette is digested
with restriction enzymes and purified. (B) Cloning of plasmid pool. The
cassette produced is ligated into an infectious clone of the virus, in
this case an RCAS plasmid. E. coli is transformed with the
ligation reaction and grown as batch culture; a small aliquot is used
to quantitate the number of transformants. Plasmids are purified from
the batch culture. (C) In vivo selection experiment. The pool of
plasmids is transfected into cultured cells. The resulting virus is
passaged many times. After one or more passages, the virus is collected
and its genome is recovered, amplified, and sequenced.
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FIG. 2.
Sequences of viral RNA from the SR-A strain of RSV and
from RCAS.SE-derived virus from nt 194 to 332 (top two lines) and
sequences of the five mutagenic cassettes used in the randomization
experiments. N, randomized position; ., base that matches the SR-A
sequence.
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FIG. 3.
Secondary-structure prediction for RNA. The computer
program mfold was used to predict the structure of nt 138 to 335, a
sequence which includes the minimal packaging region M
(2). The elements L2, O3, L3, and L4 are shown
(14). Thick gray lines indicate the sequences that were
randomized in the different experiments of this study.
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The virus produced by the transfected cells was used to infect fresh
TEFs, and after a period of cell growth the virus in
the medium again
was collected and passaged to new cells for up
to seven rounds of
infection, representing up to 56 days of culture
(Fig.
1C). Virus shed
into the medium was monitored by standard
exogenous RT assays
(
34). After one or more rounds of infection,
the virus was
collected and its RNA purified and subjected to
RT-PCR to
generate cDNA. cDNAs were subcloned, and the individual
clones
were sequenced; alternatively, if only a single sequence
was used for
transfection, then the cDNA was sequenced directly.
To assess the number of sequences in each random pool, we performed two
types of controls. The number of
E. coli transformants,
i.e., the pool size, was estimated by plating 1% of the cells
after
electroporation onto selective medium and counting the resulting
colonies. Pool sizes typically ranged from 1 × 10
5 to
5 × 10
5. Up to ca. 5 × 10
5 TEFs
could be transfected on one plate, and thus most or all
of the plasmid
pool could be sampled in one transfection. For
the L3 KL pool with
seven randomized nucleotides (see below),
that is enough to ensure that
all possible sequences were sampled
(4
7 = 1.6 × 10
4 possible sequences). Other pools had more randomized
positions,
such that the total number of possible sequences was much
larger
than the pool size; the largest was 17 nt, for a total of
1.7
× 10
10 possible sequences. In such experiments,
only a small fraction
of the possible sequences could be sampled.
In addition to pool size, the extent of randomness in each pool was
investigated by sequencing of the relevant stretch of
DNA in plasmid
preps of four to eight randomly picked
E. coli transformants. We calculated the frequency of each base averaged
over
all positions in the randomized stretch. For most experiments,
this was
reasonably close to 25% for each nucleotide. However,
as noted below,
the L3 Base and L3 Mid pools showed a distinct
nucleotide bias, with
many more purines than pyrimidines at all
positions. This bias can be
attributed to an error in the oligonucleotide
synthesis.
O3 Base.
To test the method, we first examined a sequence
whose importance was known. Knight et al. (20) showed in a
packaging assay that a base-paired stem, here called O3 Base, was
critical. Point mutations on one side of the stem abolished packaging,
while mutants with compensatory changes which restored base pairing had
40% of the wild-type activity in the assay. We tested this stem in the
in vivo selection system by randomizing 10 nucleotides (nt 227 to 236),
of which the first 8 are one side of the stem and 2 are predicted to be
unpaired (Fig. 4A and B). We constructed a pool containing 5 × 105 transformants, slightly
less than the theoretical complexity for 10 random nt, 106,
and hence not all sequences would be expected to be represented. In two
independent experiments after one cycle of infection, a single sequence
was recovered (Fig. 4C). It contains only two changes relative to the
wild-type sequence: G232A and A236U. The G-to-A change preserves base
pairing in the predicted stem, and A236 is predicted to be unpaired. In
a third experiment, a different mutant, containing only the A236U
mutation, was recovered (Fig. 4D). The lack of recovery of the
wild-type sequence presumably is due to its absence in the pool. We
interpret these results to strongly support the hypothesis that base
pairing in O3 Base is critical for infectivity and to validate this
experimental strategy as a method to analyze the packaging signal.
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FIG. 4.
Randomization of O3 Base. (A) Diagram of the O3 element
and adjacent sequences. The nucleotides of the O3 Base stem are shown.
(B) Sequence randomized in the O3 Base experiment. Ten positions, shown
as N, were substituted with mixed nucleotides. The DNA pool containing
the randomized sequences was transfected into TEFs. The resulting virus
was used to infect a fresh culture. Virus from infected cells was
collected, and its RNA was subjected to RT-PCR and sequencing. (C)
Results from two experiments. The recovered virus showed the same
single sequence in each trial, with only two changes relative to the
wild-type sequence (underlined bases). (D) Result from a third trial
with the same pool. One position (underlined) differed from the wild
type.
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L3 Base and L3 Mid.
The predicted L3 element (nt 239 to 298)
is a 60-nt sequence downstream of O3 and containing three base-paired
stems, two internal loops, and a 7-nt loop at the end (Fig. 3). The
stems are highly conserved across the 15 viral strains for which
sequence information is available (see Materials and Methods). For
example, L3 Base shows a single change in two strains, the 5' half of
L3 Mid shows a single change in one strain, and the 3' half of L3 Mid
shows variability in two nucleotides, one of which is predicted to
bulge out of the stem. In contrast, several of the predicted internal
loops of L3 show multiple changes. To address the importance of the L3
element, we first constructed two viral DNA pools with randomized
sequences corresponding to L3 Base (nt 238 to 243 and 292 to 297, total
of 12 nt) and to L3 Mid (nt 248 to 255 and 281 to 289, total of 17 nt)
(Fig. 5). In each experiment, both sides of each stem are randomized; we would expect that if the stems are
required, only sequences that allowed base pairing of the two halves
would be recovered. Due to a bias in the oligonucleotide synthesis,
which was discovered after completion of the selections, the input
sequences had an excess of purines. For example, L3 Mid had 17 randomized positions; seven clones from the pool were sequenced, for a
total of 119 relevant nucleotides. The composition was 52% G, 30% A,
9.4% T, and 8.6% C. This bias is an artifact that must be considered
in interpretation of the results.
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FIG. 5.
Randomization of L3 Base (A) and L3 Mid (B). Diagrams of
the L3 element are shown. N indicates a randomized position.
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The L3 Mid pool was transfected into TEFs, and the resulting virus was
used for three consecutive infections of fresh TEF
cultures.
Viral RNA was recovered after the third passage and
subjected to
RT-PCR; the products were subcloned into pBluescript,
and
individual clones were sequenced. The results were striking.
We
recovered many different mutant sequences, all highly enriched
in G and
A due to the input bias. All of the sequences were very
different from
the wild-type sequence (Table
2).
Furthermore,
examination of the two halves of each sequence revealed
that the
mutants did not maintain base pairing: no stem can form at
this
position in any of the mutants. We conclude from these results
that neither the primary sequence nor the predicted secondary
structure
is essential for viral infectivity. The fact that no
single sequence
was recovered more than twice indicates that no
one sequence had a
large selective advantage over the others.
In a parallel experiment, the L3 Base pool was analyzed in the same
way, with virus being passaged seven times. Virus was
recovered after
passages 4 and 7. As in the case of the L3 Mid
experiment, the L3 Base
sequences recovered were varied, purine
rich, and very different from
the wild-type sequence. They also
did not base pair (Table
3). Several contained a single base
deletion in the 3' half. Two sequences were recovered multiple
times.
One was found in two clones from passage 4 and two clones
from passage
7, while the other was found in six clones from passage
4 and two from
passage 7. This may be an artifact of the PCR amplification,
or it may
indicate that those sequences were overrepresented in
the virus
population; however, the fact that the number of clones
with these
sequences did not increase from passages 4 to 7 indicates
that they do
not have a strong selective advantage over the other
sequences. These
data clearly show that the predicted stems are
dispensable for
infectivity. Furthermore, the large excess of
purines was not
detrimental at these positions, despite the dissimilarity
of this base
composition from the wild type.
Sequence analysis revealed that a spontaneous point mutation had been
selected in all 31 clones of L3 Mid and L3 Base mutants.
Nucleotide
213, which is in the O3 element, was changed from G
to U. Secondary-structure predictions for this change in the context
of the
wild-type sequence showed only a minor alteration, the
extension of a
small hairpin by 1 bp. In an attempt to understand
these results, we
generated several recombinant viruses of defined
sequence. A virus in
which the G213U mutation was placed in the
wild-type context replicated
normally (data not shown). Four constructs
which contained single
recovered L3 Base or L3 Mid mutant sequences
were made (Fig.
6A). Construct BR4-5 corresponds to a
particular
sequence recovered from passage 4 of the L3 Base virus pool,
including
the mutations in the stem and the G213U change. The
derivative
BR4-5 U/G has the same stem sequence but contains the
wild-type
G at position 213. Likewise, constructs M2 and M2 U/G contain
the stem sequences from a third-round L3 Mid virus, with and without
G213U, respectively. These defined DNAs were transfected into
TEFs, and
equal amounts (by RT assay) of the resulting viruses
were used for
infections. The two viruses with the U at position
213 replicated
normally, as assessed by RT assays of infected
cultures. The two
viruses with the G at position 213 were tested
twice. In one
experiment, both showed very low levels of virus
production; in a
second, both replicated with delayed kinetics
and then reached
wild-type levels, suggesting that a reversion
event had occurred (data
not shown). These experiments support
the idea that the single
nucleotide change had a very strong effect
on replication.
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|
FIG. 6.
Recombinant L3 Stem mutants. (A) Sequences of nt 213 and
239 to 298 in the wild-type virus and indicated mutants. Mutated bases
are underlined. (B) Kinetics of virus spread. Equal amounts of virus
particles produced by transfected cells were used to infect TEFs. Virus
production was monitored by RT assay. Each time point represents virus
from a confluent 10-cm-diameter plate; the medium was on the plate for
24 h prior to collection.  , mutant Double.U;  , mutant
Double.G.
|
|
The fact that individual mutants from the L3 Base and L3 Mid selected
pools could replicate normally indicates that each stem
alone is
dispensable. We considered the possibility that the virus
could
replicate with one of the two stems intact, but not if both
stems were
disordered. To address this possibility, we made a
recombinant which
contained one selected sequence from each pool
(Fig.
6B). In this viral
RNA, neither stem should be able to form.
We constructed two versions
of this viral DNA, one with the wild-type
G213 (called Double.G) and
one with G213U (called Double.U). Again,
equal amounts of virus
produced by transfected cells were used
for infections. We found that
Double.U, like the wild-type virus,
could replicate when both stems
were disrupted. Double.G showed
delayed kinetics but reached wild-type
levels after several days
(Fig.
6B), again suggesting a reversion
event. Seven days after
the Double.G infection, the virus was recovered
and the relevant
portion of the RNA was amplified by RT-PCR. Sequencing
showed
that it had indeed reverted to G213U. These data all strongly
support the idea that the point mutation is critical when the
L3
structure is lost.
O3 Mid.
The recurring selection of the spontaneous G213U
mutation would appear to be an important clue about the structure and
function of . Some but not all secondary-structure predictions show
a small stem-loop from nt 214 to 225 in the wild-type sequence, and the
G213U mutation is predicted to lengthen the stem by 1 bp. To better
understand the structural features surrounding nt 213, we randomized nt
212 to 225 and analyzed the recovered virus in the same manner as
described above. The pool size was estimated at 1.6 × 105, much smaller than the theoretical complexity of 3 × 108 for 14 nt. Hence, only about 1 in 1,000 of the
possible sequences would be sampled in this experiment. Virus was
recovered after the first and third rounds of infection, and cDNAs were
subcloned and sequenced (Table 4).
The sequence characteristics for the two rounds were similar. Several
sequences were observed more than once, indicating that
the population
of virus had limited diversity. The wild-type sequence
did not appear
in these clones, as expected given the sampling
size, but the observed
sequences share some general properties
with the wild-type sequence.
All are rich in G bases and have
few A bases. All but one can be
modeled as a stem-loop containing
at least two base pairs. Nucleotides
212 and 214 are invariably
G, the wild-type nucleotide. Position 213 is
U in all but one
sequence (it is A in clone O3MR1-14); no clones have
the wild-type
G. Thus, in the context of these mutations, as in the L3
stem
mutations, a U at that position is highly favored. No other
positions
are conserved relative to the wild-type sequence. One example
of base covariation is evident: nucleotide 216 is a G and 223
is a C in
SR-A and all other known strains. These bases were seen
in about
one-third of our recovered sequences, but in the remaining
clones, a C
at 213 and a G at 223 were seen. This covariation
indicates that a base
pair between these nucleotides is highly
selected. According to
secondary-structure predictions, all but
two of the sequences can form
a hairpin which includes this base
pair. We also found that nt 225 could be any base. This position
is the first nucleotide of the UAG
stop codon of ORF 3 in the
wild-type virus. The fact that the other
three bases are allowed,
despite ruining the stop codon, agrees with
the previous finding
that a U225G mutant showed wild-type infectivity
and packaging,
presumably due to the presence of an in-frame stop codon
15 nt
downstream of the mutation (
11). In addition, many of
the mutants
had altered amino acid sequences for the peptide encoded by
ORF
3; this also is consistent with previous studies which concluded
that the amino acid sequences of the ORFs are not important (
15,
18). In summary, these results indicate that the sequence of
nt
212 to 225 need not be conserved, although the local structure
may be
important; however, the data do not allow us to explain
the strong
selection for the G213U mutation. We can only speculate
as to the
effect of G213U on viral replication. It is possible
that this mutation
stabilizes the predicted small hairpin structure,
or all of O3. Perhaps
the stem is usually stabilized by the presence
nearby of L3, so that in
the absence of L3, the additional stability
imparted by G213U is
needed. The mutation might also prevent alternative,
incorrect foldings
with downstream sequences by stabilizing the
stem.
Kissing loop.
We next examined the loop at the end of element
L3. In the SR-A strain of RSV the loop has the sequence
AGGGCCC, while in other strains the loop contains
other 6-nt, GC-rich palindromes. The loop, called a kissing
loop, has been shown to promote dimerization of RNAs in vitro
(13). A kissing loop contains a palindromic sequence that
can base pair with the corresponding loop of an identical monomer.
Several studies suggest that a kissing loop initiates genome
dimerization in HIV-1 (5). Based on the in vitro data, a
similar role was proposed for the loop in RSV (13). To test
this hypothesis, we randomized the 7 nt of the loop. The pool size was
estimated at 5 × 105, greater than the theoretical
complexity of 1.6 × 104, and thus all possible
sequences of the seven 7-nt stretch should have been sampled in this
experiment. We recovered and sequenced viral genomes after two and five
passages. The sequences of the 24 clones recovered after passage 2 were
clearly biased toward palindromic symmetry (Table
5). Nine sequences, of which one was
repeated once, contained 6-nt palindromes and five contained 4-nt
palindromes. The remaining nine sequences were not palindromic and thus
could not serve as kissing loops. By contrast, the recovered sequences
after five passages were all palindromes (Table 5). Six of 24 clones
contained the wild-type sequence. Several other GC-rich palindromes
appeared multiple times. The fact that several sequences were seen more
than once in this small sample indicates that they are highly selected.
Three of the 24, including one of the wild-type clones, also contained
the G213U mutation. We interpret these data to mean that a palindromic
sequence in the loop is favored but not absolutely required for
replication.
L3.
Based on the findings that the predicted stems and loop
of L3 can be greatly altered, we chose to test a complete deletion of
L3. We constructed a mutant called L3, in which the entire L3
element is cleanly deleted. This mutant was transfected into TEFs, and
the resulting virus used to infect fresh cells. The infections were
followed for up to 13 days by RT assay. While cultures infected with
wild-type virus contained detectable RT activity within 3 to 5 days and
had sustained high levels after 5 to 7 days, L3 virus levels
increased slowly and never exceeded 5% of wild-type levels. The
infectivity experiment was repeated four times, and in each case
the RT activity was 20- to 300-fold lower than that of a fully infected
wild-type culture; a representative experiment is shown in Fig.
7. The virus was recovered from one of
the infected cultures, its RNA was subjected to RT-PCR, and the cloned
sequence was examined. It had maintained the deletion and, unlike the
L3 Base and L3 Mid mutants, did not contain G213U or any other
mutations in the leader. These results are consistent with the
hypothesis that L3 is not required for but does contribute significantly to packaging specificity or to other viral functions.
View larger version (16K):
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|
FIG. 7.
Kinetics of L3 virus replication. Equal amounts of
virus particles produced by transfected cells were used to infect TEFs.
Virus production was monitored by RT assay. Each time point represents
virus from a confluent 10-cm-diameter plate; the medium was on the
plate for 24 h prior to collection. wt, wild type.
|
|
| |
DISCUSSION |
In this study, we have used an in vivo selection scheme to examine
short sequences and predicted RNA secondary structures in the RSV
packaging signal. We were able to select functional sequences from
pools of virus with up to 17 randomized nt. Some experiments confirmed
the importance of known elements; others showed the nonessential nature
of several conserved sequences.
Our first experiment involved randomization of 10 nt including 8 nt of
the stem O3 Base. The input material was a large and diverse sequence
pool; the output, selected virus, was very restricted
only two
different sequences were recovered. The sequences were very similar to
the wild-type sequences and were selected after just one round of
infection. The strong selection for the stem structure points to
its importance for infectivity, in agreement with the data of Knight et
al. (20) and Banks et al. (2). The results gave
us confidence in the validity of the technique for examining the sequence and for the possibility of selecting functional sequences out
of pools of this size and complexity.
In contrast to the strong selection seen in the O3 Base
experiment, we found no selection for sequence or predicted structure in L3 Base and L3 Mid. The recovered sequences after many rounds of
infection were unlike the wild-type sequence in both sequence and base
composition and could not base pair to form stems. A recombinant virus
with both stems disrupted replicated like the wild type, a fact which
strengthens our conclusion: the predicted stems are dispensable for
infectivity. This result is surprising, considering the high sequence
conservation of the stems in natural strains of the virus. However, the
data do agree with the finding of Katz et al. (19) that a
deletion of 22 nt in L3 does not abrogate infectivity.
An unexpected result of the L3 Base and Mid experiments was the
discovery of the spontaneous mutation, G213U. This single nucleotide
change appears able to rescue infectivity of the L3 stem mutants: one
recombinant virus, constructed with both stems disrupted and the G at
position 213, replicated poorly and then reverted to U at position 213. This kind of rapid genetic change is common in retroviruses due to the
high error rate of RT. In an attempt to understand the phenotype of
this mutation, we probed the sequence and local structure around it. We
randomized nt 212 to 225. The recovered sequences, although different
from the natural sequence, had a base composition very close to that of
the wild type. Positions 212 and 214 were always G, as in the wild
type, and nearly all recovered clones had G213U, implying that this change may compensate for nearby mutations as well as the changes in
L3. One pair of bases covaried, making a putative GC or CG base pair.
All of the recovered sequences can be modeled as short stem-loops; we
interpret this to mean that the local structure is likely to be
important. Several sequences were found multiple times among the
recovered clones, indicating that the virus population had a limited
diversity and that some of the sequences may be more fit than others.
This experiment did not answer the question of why G213U is selected in
the presence of L3 stem mutants; however, it did reveal some of the
sequence requirements at that location.
The apparent dispensability of the stems led us to question
related function proposed for L3, dimerization. L3 was
shown to act as a kissing-loop dimer initiation signal in vitro
(13). A kissing loop is a loop containing a palindromic
sequence, which can base pair with the loop of a second identical
molecule. Fossé et al. (13) made transcripts in vitro,
corresponding to nt 1 to 626 of RSV, which dimerize spontaneously. They
found that an oligonucleotide which is complementary to nt 258 to 274 interfered with the dimerization reaction and that deletion of nt 207 to 270 reduced dimerization. This led to the suggestion that L3
contains a kissing-loop structure. We decided to test this hypothesis
in vivo by randomizing the 7-nt loop at the end of L3. A variety of
sequences were recovered. Over five rounds of infection, we observed a
dramatic change in the virus population. After two rounds, the majority
of sequences did not contain palindromes, although more were observed
(42%) than would be expected by chance (5%); after five rounds, all
contained palindromes. Compared to the case of O3 Base, in which the
stem was selected after only one round, these results suggest a weak
but significant selection pressure for a palindrome. From these
experiments, it is impossible to distinguish whether the advantage
imparted by the palindrome is due to an effect on dimerization,
packaging or both; this question awaits further study, using more
specific assays for those functions. The assertion that a palindromic
loop in the predicted dimer initiation sequence is not essential is
supported by a recent study on HIV-1. It was found that recombination
between genomes with different kissing-loop sequences occurred as
efficiently as recombination between identical genomes (35).
Recombination depends on the copackaging of two genomes into a single
virion; this result indicates that copackaging can occur in the absence
of a kissing-loop interaction.
Given the data on the various parts of L3 (the dispensability of the
stems and the loose requirement for a palindromic loop), we questioned
whether the L3 element was needed at all. The L3 mutant, missing all
60 nt of L3, was constructed to address that issue. This mutant
replicated, but did so quite poorly. The severity of the defect shows
that L3 has some function in replication; the fact that it replicates
at all, however, means that the function is not required or can be
carried out at least partially by other sequences. A quantitative
assessment of packaging and other functions might sort out which
functions are affected in this mutant.
We have shown that replication-competent sequences can be recovered
from pools containing up to 14 random nts in a row, or two stretches
totaling 17 random positions. Sample size may be a limitation when a
sequence has stringent requirements. For example, in the O3 Base
experiment, strong selection for the stem resulted in only two
sequences being selected out of roughly 105. If a longer
stretch had been randomized, probably no virus at all would have been
recovered due to the sampling problem. For the other experiments, the
selection was not as stringent, and we were able to recover fit
molecules when sampling a tiny fraction of the possible sequences.
Stretches even longer than those assayed here should be workable in
this system, if the requirements are not very strict for the sequence
in question.
Overall, our results demonstrate the power of in vivo selection as a
tool for studying packaging and other viral RNA functions. One
advantage of the method over the more traditional site-directed mutagenesis approach is that vast numbers of mutants are tested at one
time. One can simultaneously discover which of sequences are
allowed and which are not. Furthermore, one can follow the evolution of
a virus population over time, distinguishing weak and strong selection
pressures. A feature of the method which is both an advantage and a
disadvantage is the fact that the readout of the assay is infectivity,
while the actual selection may occur at the level of one or more viral
functions. Complementing the in vivo procedures with assays for
specific steps of replication would allow a more thorough
investigation. In sum, in vivo selection has proved to be a useful
approach for the study of packaging and should be applicable to many
other retroviral RNA sequences of limited complexity.
| |
ACKNOWLEDGMENTS |
This work was supported by grant CA20081 from the USPHS.
We thank Maxine Linial, Michael Sakalian, Lance Stewart, and Steinar
Johansen for many helpful conversations and suggestions. Thanks are due
to Stephen Hughes and Rebecca Craven for reagents.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca,
NY 14853. Phone: (607) 255-2443. Fax: (607) 255-2428. E-mail:
vmv1{at}cornell.edu.
| |
REFERENCES |
| 1.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1998.
Current protocols in molecular biology, vol. I.
John Wiley & Sons, New York, N.Y.
|
| 2.
|
Banks, J. B.,
A. Yeo,
K. Green,
F. Cepeda, and M. Linial.
1998.
A minimal avian retroviral packaging sequence has a complex structure.
J. Virol.
72:6190-6194[Abstract/Free Full Text].
|
| 3.
|
Berkhout, B., and B. Klaver.
1993.
In vivo selection of randomly mutated retroviral genomes.
Nucleic Acids Res.
21:5020-5024[Abstract/Free Full Text].
|
| 4.
|
Berkhout, B., and J. L. B. Van Wamel.
1996.
Role of the DIS hairpin in replication of human immunodeficiency virus type 1.
J. Virol.
70:6723-6732[Abstract/Free Full Text].
|
| 5.
|
Berkowitz, R.,
J. Fisher, and S. P. Goff.
1996.
RNA packaging.
Curr. Top. Microbiol. Immunol.
214:177-218[Medline].
|
| 6.
|
Berkowitz, R. D., and S. P. Goff.
1994.
Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein.
Virology
202:233-246[Medline].
|
| 7.
|
Burns, C. C.,
M. Moser,
J. Banks,
J. P. Alderete, and J. Overbaugh.
1996.
Identification and deletion of sequences required for feline leukemia virus RNA packaging and construction of a high-titer feline leukemia virus packaging cell line.
Virology
222:14-20[Medline].
|
| 8.
|
Clever, J. L., and T. G. Parslow.
1997.
Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation.
J. Virol.
71:3407-3414[Abstract].
|
| 9.
|
Coffin, J. M. (ed.).
1997.
Retroviruses.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 10.
|
Donze, O.,
P. Damay, and P. F. Spahr.
1995.
The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.
Nucleic Acids Res.
23:861-868[Abstract/Free Full Text].
|
| 11.
|
Donze, O., and P.-F. Spahr.
1992.
Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.
EMBO J.
11:3747-3757[Medline].
|
| 12.
|
Ellington, A. D., and J. W. Szostak.
1990.
In vitro selection of RNA molecules that bind specific ligands.
Nature (London)
346:818-822[Medline].
|
| 13.
|
Fossé, P.,
N. Motte,
A. Roumier,
C. Gabus,
D. Muriaux,
J. L. Darlix, and J. Paoletti.
1996.
A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.
Biochemistry
35:16601-16609[Medline].
|
| 14.
|
Hackett, P. B.,
M. W. Dalton,
D. P. Johnson, and R. B. Petersen.
1991.
Phylogenetic and physical analysis of the 5' leader RNA sequences of avian retroviruses.
Nucleic Acids Res.
19:6929-6934[Abstract/Free Full Text].
|
| 15.
|
Hackett, P. B.,
R. B. Petersen,
C. H. Hensel,
F. Albericio,
G. S. I.,
A. C. Palmenberg, and G. Barany.
1986.
Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus.
J. Mol. Biol.
190:45-57[Medline].
|
| 16.
|
Hayashi, T.,
T. Shioda,
Y. Iwakura, and H. Shibuta.
1992.
RNA packaging signal of human immunodeficiency virus type 1.
Virology
188:590-599[Medline].
|
| 17.
|
Joyce, G. F.
1989.
Amplification, mutation and selection of catalytic RNA.
Gene
82:83-87[Medline].
|
| 18.
|
Katz, R. A.,
B. R. Cullen,
R. Malavarca, and A. M. Skalka.
1986.
Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.
Mol. Cell. Biol.
6:378-379.
|
| 19.
|
Katz, R. A.,
R. W. Terry, and A. M. Skalka.
1986.
A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging.
J. Virol.
59:163-167[Abstract/Free Full Text].
|
| 20.
|
Knight, J. B.,
Z. H. Si, and C. M. Stoltzfus.
1994.
A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.
J. Virol.
68:4493-4502[Abstract/Free Full Text].
|
| 21.
|
Linial, M.,
E. Medeiros, and W. S. Hayward.
1978.
An avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: biological and biochemical characterization.
Cell
15:1371-1381[Medline].
|
| 22.
|
Mann, R.,
M. C. Mulligan, and D. Baltimore.
1983.
Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.
Cell
33:153-159[Medline].
|
| 23.
|
McBride, M. S., and A. T. Panganiban.
1996.
The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.
J. Virol.
70:2963-2973[Abstract].
|
| 24.
|
McBride, M. S., and A. T. Panganiban.
1997.
Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.
J. Virol.
71:2050-5058[Abstract].
|
| 25.
|
McBride, M. S.,
M. D. Schwartz, and A. T. Panganiban.
1997.
Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation.
J. Virol.
71:4544-4554[Abstract].
|
| 26.
|
Mortlock, D.,
E. B. Keller,
C. J. Ziegra, and M. M. Suter.
1994.
High efficiency transfection of monkey kidney COS-1 cells.
J. Tissue Culture Methods
15:76-180.
|
| 27.
|
Mougel, M., and E. Barklis.
1997.
A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions.
J. Virol.
71:8061-8065[Abstract].
|
| 28.
|
Mougel, M.,
Y. Zhang, and E. Barklis.
1996.
cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA.
J. Virol.
70:5043-5050[Abstract/Free Full Text].
|
| 29.
|
Oertle, S., and P.-F. Spahr.
1990.
Role of the Gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA.
J. Virol.
64:5757-5763[Abstract/Free Full Text].
|
| 30.
|
Paillart, J. C.,
L. Berthoux,
M. Ottmann,
J. L. Darlix,
R. Marquet,
B. Ehresmann, and C. Ehresmann.
1996.
A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis.
J. Virol.
70:8348-8354[Abstract].
|
| 31.
|
Rein, A.
1994.
Retroviral RNA packaging: a review.
Arch. Virol.
9:513-522.
|
| 32.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 33.
|
Sonstegard, T. S., and P. B. Hackett.
1996.
Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag.
J. Virol.
70:6542-6552[Abstract/Free Full Text].
|
| 34.
|
Stewart, L.,
G. Schatz, and V. M. Vogt.
1990.
Properties of avian retrovirus particles defective in viral protease.
J. Virol.
64:5076-5092[Abstract/Free Full Text].
|
| 35.
|
St. Louis, D. C.,
D. Gotte,
E. Sanders-Buell,
D. W. Ritchey,
M. O. Salminen,
J. K. Carr, and F. E. McCutchan.
1998.
Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro.
J. Virol.
72:3991-3998[Abstract/Free Full Text].
|
| 36.
|
Swanstrom, R.,
H. E. Varmus, and J. M. Bishop.
1982.
Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus.
J. Virol.
41:535-541[Abstract/Free Full Text].
|
| 37.
|
Tuerk, C., and L. Gold.
1990.
Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.
Science
249:505-510[Abstract/Free Full Text].
|
| 38.
|
Walter, A. E.,
D. H. Turner,
J. Kim,
M. H. Lyttle,
P. Mueller,
D. H. Mathews, and M. Zuker.
1994.
Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.
Proc. Natl. Acad. Sci. USA
91:9218-9222[Abstract/Free Full Text].
|
| 39.
|
Wang, L.-F.,
R. Voysey, and M. Yu.
1994.
Simplified large-scale alkaline lysis preparation of plasmid DNA with minimal use of phenol.
BioTechniques
17:26-28.
|
| 40.
|
Whitcomb, J. M.,
B. A. Ortiz-Conde, and S. H. Hughes.
1995.
Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers.
J. Virol.
69:6228-6238[Abstract].
|
| 41.
|
Wills, J. W.,
R. C. Craven, and J. A. Achacoso.
1989.
Creation and expression of myristylated forms of Rous sarcoma virus Gag protein in mammalian cells.
J. Virol.
63:4331-4343[Abstract/Free Full Text].
|
| 42.
|
Yang, S., and H. M. Temin.
1994.
A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.
EMBO J.
13:713-726[Medline].
|
| 43.
|
Zuker, M.
1989.
On finding all suboptimal foldings of an RNA molecule.
Science
244:48-52[Abstract/Free Full Text].
|
Journal of Virology, October 1998, p. 8073-8082, Vol. 72, No. 10
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Abstract
Introduction
