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Journal of Virology, November 2000, p. 10293-10303, Vol. 74, No. 22
Department of Microbiology and Immunology and
Comprehensive Cancer Center, University of Michigan Medical School,
Ann Arbor, Michigan 48109-0620
Received 12 June 2000/Accepted 19 August 2000
Retrovirus plus-strand synthesis is primed by a cleavage remnant of
the polypurine tract (PPT) region of viral RNA. In this study, we
tested replication properties for Moloney murine leukemia viruses with
targeted mutations in the PPT and in conserved sequences upstream, as
well as for pools of mutants with randomized sequences in these
regions. The importance of maintaining some purine residues within the
PPT was indicated both by examining the evolution of random PPT pools
and from the replication properties of targeted mutants. Although many
different PPT sequences could support efficient replication and one
mutant that contained two differences in the core PPT was found to
replicate as well as the wild type, some sequences in the core PPT
clearly conferred advantages over others. Contributions of sequences
upstream of the core PPT were examined with deletion mutants. A
conserved T-stretch within the upstream sequence was examined in detail
and found to be unimportant to helper functions. Evolution of virus
pools containing randomized T-stretch sequences demonstrated marked
preference for the wild-type sequence in six of its eight positions.
These findings demonstrate that maintenance of the T-rich element is
more important to viral replication than is maintenance of the core PPT.
Upon entering cells, retroviruses
convert their single-stranded RNA genomes to double-stranded DNA.
As minus-strand DNA is synthesized, the RNase H activity of reverse
transcriptase (RT) degrades the RNA in the RNA/DNA duplex. However, one
portion of the RNA, the polypurine tract (PPT), is resistant to this
RNase H degradation. The PPT RNA is subsequently used as the primer for
plus-strand synthesis (2).
The region of the retroviral genome required for plus-strand priming
was initially characterized by Sorge and Hughes, who noted that more
than 9 but not more than 29 bases upstream of the primer cleavage site
are required for avian sarcoma virus replication (30). In a
previous study, we established that sequences as far upstream as Several reports have examined roles of sequences within the PPT in
plus-strand priming: most using model templates in purified reactions.
Rattray and Champoux demonstrated that when PPTs with mutations at
position Plasmid construction.
3' untranslated region mutations were
introduced into a Mo-MLV provirus plasmid, pMLV-neo (24), or
the packaging mutant pMLV
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Selection of Optimal Polypurine Tract Region
Sequences during Moloney Murine Leukemia Virus Replication
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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(where 1 refers to the base immediately upstream of the cleavage
site) are required for Moloney murine leukemia virus (Mo-MLV)
plus-strand priming and that a T-rich stretch in this region is
critical (24). Noad et al. have similarly established that
T-rich sequences upstream of the PPT are required for plus-strand
priming for the pararetrovirus cauliflower mosaic virus
(16). Additionally, Ilyinskii et al. have demonstrated that
the T stretch upstream of the simian immunodeficiency virus (SIV) PPT
is required for SIV replication (11), and a T stretch upstream of the Ty1 PPT is important for plus-strand priming and transposition of that yeast retroelement (33).
1, 2, 4, or 7 were tested, additional cleavage sites
appeared, suggesting that the integrity of these positions is necessary
for Mo-MLV cleavage specificity (22). Powell and Levin
showed that the cleavage site-proximal half of the human
immunodeficiency virus type 1 (HIV-1) PPT is required for plus-strand
priming, while the cleavage site-distal half of the PPT is expendable
in vitro (20). Similarly, the PPT's cleavage site-proximal G stretch is important for plus-strand priming
during the replication of both cauliflower mosaic virus and Mo-MLV,
although individual targeted mutations within this region are tolerated by Mo-MLV (16, 24). In this study, we examined how much
genetic variation within the PPT and its upstream T stretch was
compatible with replication by determining which sequences persisted in
replicating Mo-MLV populations when these regions initially contained
randomized sequences.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
(17). Mutations
were introduced using PCR-mediated site-directed mutagenesis and other
standard techniques. Sequences of all 3' untranslated regions were
confirmed by dideoxy sequencing (Sequenase II kit; U.S. Biochemical).
Oligonucleotides that were synthesized in the University of Michigan
Biomedical Research Core Facility and that contained the indicated
mixtures of nucleotides at specified positions were used to synthesize
the mutant pools. Ligation mixtures for generating plasmid pools were
introduced into Escherichia coli DH5
cells by
electroporation. Calculated pool sizes represent the total number of
colonies obtained multiplied by the percentage of colonies expected to
contain correct plasmids. This percentage was based on screening 10 to
30 individual colonies from the transformation plates for each pool to
determine the percentage that contained a pool plasmid instead of the
highly replication-defective parental plasmid. Bacterial colonies were
then pooled and propagated to produce the pooled plasmid preparations
subsequently used to transform mammalian cells. Note that several of
the pools used here contained significantly fewer members than would be
required to represent the theoretical genetic complexity possible from
the degenerate oligonucleotides used in mutagenesis. Instances where
this is the case are indicated in the text. In some instances,
degenerate positions within the oligonucleotides were inadvertently
biased toward particular residues rather than containing all intended substitutions at equal levels. However, high levels of sequence heterogeneity were nonetheless present within all pools, as confirmed by sequencing several individual members of each pool (Table
1, parts A, D, F, H, and
J, and data not shown). These sequencing analyses suggested that for
all pools, for every 20 pool members, there were at least 17 different
individual sequences, and in most instances even less sequence
repetition was observed. Note that two different degenerate
oligonucleotide preparations, each with its own biases in
substitutions, were used to generate the GATC PPT pools; one
preparation was used to synthesize the 530-member pool, and the other
was used to generate the larger pools.
TABLE 1.
Sequences of degenerate pools before and after passage
1 to 41) followed by a mutant att sequence. Deletions were introduced upstream of PPT1 or
PPT2 by PCR mutagenesis.
Cells. NIH 3T3 cells, Rat2 cells, and XC cells and derivatives were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Gibco). 293T cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (HyClone). Puromycin-resistant 3T3 cells were selected in puromycin (6 µg/ml; Sigma).
Replication assays of targeted mutants.
Forty percent
confluent 6-cm-diameter plates of 293T cells were cotransfected with
pMLV-neo derivatives and the LacZ reporter plasmid pCH110
(8), using CaPO4 (15) or
Lipofectamine as instructed by the manufacturer (Gibco). Two days
posttransfection, supernatants were harvested and cells were stained
with 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal) to determine transfection efficiency as previously described (17). Five to 20% of the cells typically stained blue in
this assay.
Replication assays of degenerate pools. Degenerate pool plasmids were transfected into 293T cells, and virus was quantified by RT activity as described above. The number of infectious virions per volume of supernatant was also determined by XC assay as previously described (25). Briefly, 10% confluent 10-cm-diameter plates of 3T3 cells were infected with serial dilutions of virus. After 2 days, supernatants were removed and infected cells were UV irradiated for 10 s by exposure to the sterilizing lamp in a biosafety cabinet. Media containing ~106 XC cells was added; 2 days later, cells were fixed by adding 0.5 ml of formaldehyde and stained with hematoxylin. Plaques were counted using a 10× magnification dissecting microscope.
After quantification of virus, 3T3 cells were infected as above with the equivalent of enough virus to saturate the pools (approximately three times as many PFU, as determined by XC assay, as the size of the pool used). Once cells infected with pooled viruses showed detectable virus spread by RT assay, subsequent infections with the surviving pool viruses were performed by infecting fresh 3T3 cells with the equivalent of 1 to 20 µl of wild-type virus and subsequent passaging of the cells every 3 days as described above. For larger pool sizes, it was necessary to infect multiple plates of 3T3 cells for the first passage in order to ensure complete representation of the pool. In this case, after the first passage, viruses from all plates were pooled before being quantified and analyzed.Preparation and analysis of viral DNA. Low-molecular-weight DNA was extracted from Rat2 cells 24 h postinfection (9). PPT region DNA was analyzed by PCR using primers specific for env and U3. For sequencing, ClaI-to-NheI restriction fragments of these PCR products were introduced into pUC19 and individual subclones were sequenced or, where indicated, PCR products were sequenced directly without subcloning.
PPT use for the tandem PPT constructs was analyzed by Southern blotting. Low-molecular-weight DNA was digested with EcoRV, phenol chloroform extracted, ethanol precipitated, and resuspended in 70% formamide-10 mM EDTA. After heating to 95°C, samples were separated on 5% polyacrylamide-8 M urea gels, electrotransferred to nylon (HyBond) at 80 V for 2 h, and hybridized with a 32P-labeled probe generated with a Rediprime II random primer kit (Amersham) under standard hybridization conditions (26). The probe was an NheI/XbaI restriction fragment from U3 of pMLV-neo.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Proviral clones with targeted alterations to the Mo-MLV PPT
region.
Most previously described Mo-MLV PPT point mutants
retained at least partial replication function, and even substitutions in the entire cleavage site-distal half of the PPT (the 7/17 substitution mutant) remained capable of limited replication
(24). Thus, with the aim of further defining which PPT
residues were mandatory for replication, additional targeted mutations
were introduced into the PPT of an infectious proviral clone. Each of
the mutations described in Fig. 1A was
studied both as a single mutation and in the context of the 7/17
substitution. Mutations tested included previously examined changes in
the highly conserved 2 and 4 positions, a point mutation at 1,
and blocks of mutations at 5 and 6 and at 3 to 6. In each of
these positions, the wild-type G was substituted with C. In the context
of the 7/17 substitutions, these reduced the purine content of the
remaining PPT to five, four, or two bases, respectively. An additional
mutation (the 1,3,5/17 mutation), with substitutions at 1,
3, 5, and 6, was introduced into the 7/17 background. This
mutation left only the highly conserved 2 and 4 positions of the
PPT intact.
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Some of the core PPT is required for viral replication.
To
examine replication efficiencies of these mutants, proviral plasmids
were transiently transfected into 293T cells. Virus was harvested and
used to infect NIH 3T3 cells, and viral spread was monitored every 3 days. Results are summarized in Fig. 1B. Consistent with earlier
findings (24), the 2C and 4C mutants replicated with
little or no delay relative to wild type, and the 7/17 mutant
replicated with an average delay of 4 days. When either 2C or 4C
was combined with the 7/17 mutation, virus spread remained
undetectable for more than 47 days. Similar results were observed with
the 1C and 5/6C mutants. These two mutants replicated with modest
delays when present alone, but replication was not detectable
throughout 47 days of infected cell passage for virus harboring these
changes in the context of the 7/17 mutation. Replication was not
detectable for virus with the 3/6C mutation either alone or in the
context of the 7/17 mutation. The 1,3,5/17 mutant also
failed to replicate. For mutants that did replicate, sequence analysis
of viral DNAs after replication revealed that no changes to the
original sequences had occurred within the sequenced 3' untranslated region.
Generation and evolution of random sequence PPT pools. Degenerate mutant pools were used to further examine sequence requirements within and upstream of the PPT. Table 1 presents a compilation of the genome regions subjected to randomization in this study. The table summarizes the sequence properties of these mutant pools both before and after virus passage, as determined in the experiments described below.
The first pools were created to address whether a specific sequence of purines is required in the PPT or if a structure conferred by any purine-rich sequence is sufficient (3, 20). Experimental evidence suggests that for both HIV-1 and Mo-MLV, RNase H cleavage specificity is determined by the structure of the nucleic acid substrate when it is bound by RT and that subtle sequence differences might affect the structure enough to alter substrate recognition by RNase H (5). In these randomized purine PPT pools, positions1 to 11
of the PPT core were randomized such that each position contained either a G or an A (Fig. 2, purine PPT).
Mutagenized plasmid pools of various sizes
some containing
significantly fewer members than would be required to account for the
total possible genetic complexity (2,048 different sequences)were
transiently transfected into 293T cells. Virus was collected and used
to infect NIH 3T3 cells, and viral spread was monitored as described
above. Once the culture showed detectable viral spread, virus was
harvested and quantified by RT assay, and equal amounts of wild-type
and pool virus were used to infect fresh cells. The time from infection
of NIH 3T3 cells until virus spread was detectable was considered a
single passage of the pool. The time required to complete a passage
under these conditions (roughly 5 to 8 days) suggests that each passage consisted of several successive rounds of viral replication. After each
passage, a PCR product of pooled surviving viral DNA was sequenced.
Pools were passaged in this way until no evidence of further evolution
of the initially randomized region was detectable.
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4 and
9 positions (Table 1, part C), and the same sequence predominated in
a second independent evolution of this pool.
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8
position (Table 1, part C).
As shown in Fig. 3B, the ~6,300-member purine PPT pool, which should
contain all possible sequences, evolved to wild type from positions 1
to 6. However, little sequence preference was detected from 7 to
11, with A and G both still present within the population in each of
these positions after five passages (Fig. 3B; Table 1, part C).
Sequencing gels that tracked the evolution of the pools were quantified
by phosphorimager. These data quantitatively confirmed visual
inspection-based interpretations of evolutionary trends in different
portions of the PPT during pool passage (data not shown).
Similar conclusions about the relative importance of wild-type
sequences in specific PPT positions could be made from two experiments
that examined individual purine PPT pool members: some that were
isolated from the pool that survived one replicative passage, and
others that were founding members of the plasmid pool. When
individual virus sequences were cloned after a single passage of
the 179-member pool and 21 of these clones were sequenced, over 80%
were found to contain the wild-type sequence at positions 1, 2,
5, 6, 10, and 11 (Table 1, part B). In a separate experiment,
the replication efficiencies of 27 founding members of the purine PPT
pool were examined individually. As evident from variations in
detectable virus spread (indicated in the data column for part A in
Table 1), sequences that were compatible with efficient replication
bore a stronger resemblance to wild type than those in viruses which
replicated poorly or not at all.
Since the 179-member pool was significantly smaller than the total
potential pool size of 2,048, it may not have included the wild-type
PPT. Therefore, to determine how well the predominant sequence from
this pool replicated relative to wild type, NIH 3T3 cells were
coinfected with equivalent amounts of virus containing the wild-type
PPT and virus containing this predominant sequence PPT. The sequence of
the surviving virus was examined through four passages (Fig.
4). The predominant sequence was present
in amounts equivalent to wild-type levels throughout four passages of
the virus. These findings suggest that virus with this sequence replicated with the same efficiency as wild type in this culture system. Note, however, that because these experiments were not carried
out at low multiplicity of infection, the possibility of
trans-acting differences could not be ruled out
conclusively.
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Requirement for purines in the PPT.
Some retroviruses,
including human adult T-cell leukemia virus, Mason-Pfizer monkey virus,
and simian retrovirus type 1, contain pyrimidines within their PPTs
(21, 28, 29). Thus, a second random sequence pool in which
PPT positions 1 to 11 were any of the four bases (Fig. 2, GATC PPT)
was created to address whether or not pyrimidines would be permitted at
any positions in the Mo-MLV PPT. Three mutant plasmid pools of
approximately 530, 4,000 and 5,000 members were generated. The
theoretical genetic complexity of this pool was ~4.2 × 106 sequences. Thus, each of the studied pools contained
only a small subset of possible sequences, and the pools may or may not
have included the wild type. PPT region sequences of randomly selected prepassage GATC PPT pool members are shown in Table 1 (part D).
2 and 10. However, the central part of the PPT, positions 4 to
9, were identical to wild type. Coinfection with equal amounts of
this virus and of wild-type virus yielded only wild-type
sequences after a single passage (data not shown), indicating that this
sequence was a poor competitor with the wild-type sequence.
In a second experiment that involved infection with the complete
530-member pool, spread of the pooled virus was detectable with a 3-day
delay relative to wild type. Unlike the severely delayed subpopulation
above, the virus which persisted was not homogeneous after the first
couple of passages of this larger pool (Fig.
5B). In this pool as well as in the
4,000- and 5,000-member pools (Fig. 5C and D), pyrimidines were not
detectable in most positions after a single passage of the virus.
Purines were strongly favored at all positions of the PPT, but
discrimination between the purines occurred more slowly than did
discrimination against pyrimidines. Predominant sequences of these
pools are shown in Table 1 (part E). The finding that each subset pool
generated the same trend in sequence selection suggests that the
conclusions drawn may hold true for the entire GATC PPT pool.
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Importance of the T-stretch upstream of the PPT.
We have
previously shown that a mutation which obliterates the T stretch
upstream of the Mo-MLV PPT eliminates viral replication and decreases
the efficiency of plus-strand priming (24). To examine how
much variation in this region was compatible with Mo-MLV replication, a
T-stretch degenerate pool was tested. A pool of approximately 7,000 sequences was created in an infectious proviral clone, in which each
position from 21 through 28 was replaced with any of the four bases
(Fig. 2, GATC T-stretch). The theoretical size of this pool was about
6.5 × 104. Note, however, that T was overrepresented
in all randomized positions of the pool (Fig. 5, original pool
sequences), presumably as an unplanned consequence of the
oligonucleotide synthesis process. Thus, T-rich sequences were more
prevalent in the population than would be predicted if the pool
were equally randomized. Nonetheless, sequencing individual
members of the prepassage pool revealed that the intended genetic
variation was well represented (Table 1, part F). These degenerate
T-stretch pools were passaged as described above for the PPT pools.
23/28 region of the first-passage
survivors were wild type in two independent experiments, suggesting
that very little sequence variation in this region was compatible with
replication. Somewhat more variation was observed in the 21 and 22
positions. The sequence of these positions remained heterogeneous, with
a T/C mix in the 22 position after passaging in one evolution
experiment and a T/A mix in the 21 position observed in an
independent evolution experiment (Fig. 6; Table 1, part G).
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23/28 region, two additional T-stretch pools were constructed. The
TA pool contained either A or T at every position from 21 to 28
(Fig. 2, TA T-stretch), while the TC pool contained an A or G at 24
and a T or C at every other position in this region (Fig. 2, TC
T-stretch). Both of these pools were genetically marked with a 1A
substitution to rule out the possibility that what appeared to be rapid
evolution could have been due to wild-type virus contamination. The
plasmid pools tested were severalfold larger than the theoretical
complexity (256 members). Sequencing the pooled plasmids before virus
passage as well as sequencing individual members of the prepassage
pools revealed that the intended genetic variation was well represented
(Table 1, parts H and J, and data not shown). These pools were passaged
as described above.
As with the completely degenerate pool, these two pools evolved to
essentially a single sequence within a single passage. In six of eight
single clones randomly selected from the first passage of the TA pool,
the T-stretch sequence was completely wild type (Table 1, part K). In
the other two clones, the only differences from wild type were that the
21 or 28 position was A instead of T. In five of five clones from
the TC pool, the T stretch was wild type in all positions except 21,
which was a C (Table 1, part I). These data are consistent with those
of the completely degenerate T-stretch pool and indicate that whereas the 21 and 22 positions of the T stretch can vary, there is strong
selective pressure for maintaining wild-type sequences in the 23/28 region.
Effects of T-stretch mutations on trans-acting
replication functions.
We previously reported that replication
defects of T-stretch mutants are greater than can be accounted for by
the magnitude of effects on plus-strand priming (24). Thus,
to test whether or not mutations in the T-stretch affected virus
protein production, we introduced the 21/28 mutation
(24) (Fig. 7), which contains a GC-rich sequence in place of the T stretch, into a
packaging-defective proviral plasmid. This construct was used to
examine possible effects of T-stretch alterations on helper functions.
This T-stretch-defective construct and a
wild-type provirus plasmid were individually
cotransfected with a puromycin resistance-conferring retroviral vector
into 293T cells to generate vector-containing virions. No differences
in virion content in the media of cells transfected with wild type versus those with the 21/28 mutant helper were detectable (data not
shown), suggesting that production of the Gag-Pol polyprotein was not
affected by the T-stretch mutation. To examine possible effects on
envelope production or other functional properties, the infectivity of
the virions was tested. Equivalent amounts of virus were used to infect
NIH 3T3 cells, and puromycin-resistant titers were determined.
Equivalent numbers of puromycin-resistant colonies were obtained from
vectors expressed with wild type or with 21/28 mutant helpers (Fig.
7). Thus, trans-acting helper functions were not affected to
a detectable extent by this T-stretch mutation.
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Effects of deletions upstream of the PPT on plus-strand
priming.
To test the importance of sequences upstream of the core
PPT for plus-strand priming and replication, five deletion mutations were introduced into the 3' untranslated region between the end of
env and the PPT (Fig. 8A). In
addition to the T stretch described above, this region of Mo-MLV
contains an A stretch from 31 to 39 (Fig. 8A). The deletions that
were constructed are shown in Fig. 8A. The replication efficiencies of
these deletion mutants were determined as described above. Consistent
with results published while this work was in progress (1),
the 
14/41 mutant, which contained a deletion of the entire
upstream sequence, replicated with a significant delay (12 days later
than wild type) without reversion in this region (data not shown).
Similar results were found with the 14/20 and 14/28
deletions. Replication time courses for 20/41 and 31/41
were similar to those of the other deletion mutants; however, PCR of
viral DNA revealed that these deletions were no longer present (data
not shown). This most likely indicates reversion by patch repair as
previously observed for different mutants in this region
(24). These putative revertants were not characterized
further.
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14/41, 20/41, and 31/41)
were introduced into PPT2 of the tandem PPT system that we have previously described, to examine the mutations' effects on plus-strand priming (24). In these tandem PPT vectors, a second PPT is
inserted into U3 downstream of the endogenous PPT (Fig. 8B). In this
system, the viral RNA contains two PPTs, either of which can prime
plus-strand synthesis. Because the choice of PPT determines the left
edge of the final double-stranded DNA, the relative use of the two PPTs
can be determined by examining the upstream long terminal repeat (LTR)
of reverse transcription products. The ectopic PPT, PPT2, includes 30 bases of sequence upstream of the core PPT. We have shown previously
that if both PPTs contain wild-type sequences, PPT1 is used about 70%
of the time and PPT2 is used 30% of the time (24). Since
the wild-type PPT1 was present in all constructs, all three PPT2
deletion mutants replicated with wild-type efficiency as expected, and
none contained changes to the original sequence in this region after replication.
To visualize the PPT1 and PPT2 products for all deletion mutants,
Southern blotting was performed on nonintegrated reverse transcription
products digested with EcoRV to generate LTR end fragments.
As shown in Fig. 8C, PPT2 use was detectable at a low level for the
parental construct but not for any of the deletion mutants, which
suggested that for each deletion mutant, sequences or structures
upstream of the PPT which were disrupted are important for plus-strand
priming. Note that because the level of priming from the wild-type PPT2
was low, the extent to which the deletions affected plus-strand priming
could not be quantified precisely. However, these observations were
consistent with confirmatory findings from experiments with reiterative
primer extension performed as previously described (24)
(data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study we examined the replicative advantages of
maintaining wild-type sequences within specific regions of the Mo-MLV 3' untranslated region. We observed that maintenance of the T-stretch core, which consists of nucleotides from 23 to 28 upstream of the
site of plus-strand cleavage, was highly selected during viral replication. Results indicated that maintenance of the 1/6 cleavage site-proximal half of the PPT conferred a selective advantage during
viral replication as well, albeit at a lower level than conservation of
the T stretch. High purine content in the cleavage site-distal half of
the PPT was also advantageous. However, this latter region was far more
tolerant of variation from the wild-type sequence than was the core
PPT's cleavage site-proximal half.
We have previously demonstrated that viruses which possess only the
cleavage site-proximal half of the PPT can replicate with relatively
modest delays (24). To assess the replication efficiency of
viruses with even fewer wild-type PPT residues, additional targeted changes were introduced in the context of the
7/17 mutation. The results suggested that whereas many individual
changes within the cleavage site-proximal half of the PPT are
tolerated, the entire cleavage site-proximal five or six bases are
necessary for replication in the absence of the cleavage site-distal
half of the PPT.
Evolution of randomized purine PPT pools further illustrated sequence
biases within the PPT. Wild-type sequences at positions 2, 5, 6,
10, and 11 were strongly preferred. Selection for wild type
at all other PPT positions appeared to be significantly weaker, as
these positions were slower to evolve toward wild type. A random
purine PPT mutant that differed from wild type at positions 4 and 9
was shown to replicate as well as the wild-type virus during extended
passage in mixing experiments. This demonstrates that PPT sequences
other than wild type can be fully infectious, at least under the
relatively high-multiplicity-of-infection conditions examined here.
Comparison of the PPT sequences of many different retroviruses and
retroelements reveals that the core PPTs of some of these include
pyrimidine residues (mostly T's) (22, 24). Thus,
replication was tested for Mo-MLV PPT pools in which all four bases
were included at each PPT position. Consistent with results from the
degenerate purine pools, these fully degenerate GATC PPT pools showed
rapid evolution toward sequences with wild-type residues at most
positions. After two passages of a small GATC PPT pool, T's remained
detectable at positions 2 and 10, while surviving members of larger
GATC pools contained no detectable PPT pyrimidines after a single
passage. These observations indicate a stronger preference for purine
content in the Mo-MLV PPT than is the case for many other retroviruses and retroelements. Not only do some of these other elements naturally possess pyrimidines within their core PPT regions, but tolerance during
replication for variation that includes pyrimidines has also been
reported (13).
It has been suggested that a unique structure within the PPT is responsible for its recognition during plus-strand primer generation (for a review, see reference 10). Although binding of RT to nucleic acids alters the nucleic acids' structures, at least in the case of DNA/DNA duplexes (12), inherent structural differences between PPT duplexes and other RNA/DNA duplexes that form during replication might contribute to the resistance of the PPT to RNase H cleavage. Findings here demonstrate that the specific sequence of purines within the PPT, and not mere purine richness, is important to Mo-MLV PPT function. Therefore, if the structure of the nucleic acids is important to the PPT's function during replication, it must be a structure more distinct than that which can form from any repeated purine sequence.
When RT binds nucleic acids, most contacts between the enzyme and nucleic acids occur in the DNA polymerase domain, with the limited contacts that form in the RNaseH domain itself less important to substrate positioning (12). Thus, it has been suggested that proper placement of the RNA/DNA substrate into the RNase H active site may be highly sensitive to subtle changes in substrate regions that are relatively free of protein contacts (5). In the context of RT bound for PPT primer generation, this region would roughly coincide with the cleavage site-proximal half of the PPT, which studies reported here found to be especially conserved.
Requirements for sequences upstream of the PPT in viral replication
were also examined in this study. Particular attention was paid to
investigating roles of the T stretch which we have previously shown to
be required for optimal replicative plus-strand priming
(24). In the present study, the striking extent to which selectivity for wild-type sequences was observed in the T stretch, relative to advantages for wild-type sequences in the core PPT, was
especially evident from comparisons of sequences that persisted through
passage of pooled viruses randomized in these regions. Even after
several passages, each consisting of several successive rounds of viral
replication, significant sequence variation was observed in randomized
PPT pools (for example, Fig. 5B). Among 21 individual survivors of a
randomized core PPT pool's first passage (Table 1, part B), only two
single positions (2 and 5) were conserved, with no contiguous
patches of even two wild-type residues found in common among all
survivors. In contrast, all 13 sequenced survivors of single passages
of T-stretch pools were identical to wild type in all six positions
from 22 to 27 (Table 1, parts I and K).
Deletions in the 30 bases upstream of the PPT, as shown in tests
involving the tandem PPT system, interfered with efficient plus-strand
priming. Nonetheless, the 14/41 mutant with a deletion in this
region was capable of supporting viral replication when present as a
single PPT. This is consistent with results that Bacharach et al.
published while this work was in progress (1). The results
of Bacharach et al. suggest that deletions in this genome region affect
the site of plus-strand priming and/or removal of the plus-strand
primer. We observed that alterations to the T-rich region did not
affect helper functions, suggesting that this sequence contributed to
viral replication solely in cis. These findings are also
consistent with the recent report by Bacharach et al. (1).
All replication roles of the 3' sequences upstream of the Mo-MLV PPT
are not yet understood, even for the core PPT and upstream T stretch. A
model has been proposed which suggests that the T stretch located in
PPT upstream sequences might function in a DNA polymerase-dependent
step of the plus-strand priming process (16). Mo-MLV
RT has been shown by footprinting to contact from 27 to +6 of
the template strand (numbered relative to the site of polymerization)
and from 26 to 1 of the primer strand when bound in the
configuration to polymerize DNA (34). Thus, the T stretch
studied here is likely to contact RT when it is bound to the PPT
region. Mutations in the DNA polymerase domain of RT have been shown to
affect RNase H cleavage (4, 6, 14, 19, 23, 27, 31). Thus, it
is reasonable to propose that contacts made by the DNA polymerase
domain upstream of the PPT during DNA polymerization might contribute
to the specificity or efficiency of RNase H cleavage at the PPT.
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ACKNOWLEDGMENTS |
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We acknowledge Monica Roth for providing XC cells and advice on their use; David Peterson, Alison Dormer, Deanna Kulpa-Stom, and Rachel Westfall for assistance at various stages of this project; and Julie Pfeiffer, Rosa Yu, and Terry Dixon for helpful reading of the manuscript.
This research was supported by NIH grant R29 CA69300.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology and Comprehensive Cancer Center, University of Michigan Medical School, 5614 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0620. Phone: (734) 936-6466. Fax: (734) 764-3562. E-mail: ateles{at}umich.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, November 2000, p. 10293-10303, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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