From the Unité de Regulation Enzymatique des Activités Cellulaires, CNRS-FRE 2364, Département de Biologie Structurale et Chimie and ¶ CNRS-URA 1960, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France and § Uppsala Universitet, Department of Cell and Molecular Biology Biomedical Center, SE-75124, Uppsala, Sweden
Received for publication, December 3, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Genetic recombination is a major force driving
retroviral evolution. In retroviruses, recombination proceeds
mostly through copy choice during reverse transcription. Using a
reconstituted in vitro system, we have studied the
mechanism of strand transfer on a major recombination hot spot we
previously identified within the genome of HIV-1. We show that on this
model sequence the frequency of copy choice is strongly influenced by
the folding of the RNA template, namely by the presence of a stable
hairpin. This structure must be specifically present on the acceptor
template. We previously proposed that strand transfer follows a
two-step process: docking of the nascent DNA onto the acceptor RNA and
strand invasion. The frequency of recombination under copy choice
conditions was not dependent on the concentration of the acceptor RNA,
in contrast with strand transfer occurring at strong arrests of reverse
transcription. During copy choice strand transfer, the docking step is
not rate limiting. We propose that the hairpin present on the
acceptor RNA could mediate strand transfer following a mechanism
reminiscent of branch migration during DNA recombination.
By reshuffling large regions of genetically distinct genomes,
recombination speeds up the rate of evolution (1, 2). Recombination
constitutes the most frequent genomic aberration in retroviruses; its
frequency of occurrence is equal to the cumulative frequency of all the
other types of mutations (3). The most intensively studied member of
this group of viruses, the human immunodeficiency virus type 1 (HIV-1),1 illustrates the
impact of recombination on the dynamics of retroviral evolution. In
this case, at the very least 10% of the circulating strains have been
generated by genetic recombination among different HIV-1 subtypes (4).
In retroviruses, recombination occurs mainly during reverse
transcription (5). Each viral particle contains two copies of
single-stranded positive genomic RNA (6). If two different variants of
a virus infect the same cell, as recently documented for HIV-1 (7), the
viral progeny will be constituted by homozygous and heterozygous
virions. Recombination can then occur when a heterozygous virus infects
a new cell. Indeed, during synthesis of the ( Despite the dramatic impact of recombination on the evolution of
retroviruses, the underlying mechanisms are not yet understood. Based
on the observation that the purification of viral RNA from retroviral
particles led to the isolation of fragmented molecules, it was
suggested that the genomic RNA is not intact within the viral particle.
It was therefore proposed that the switch would be a consequence of a
block of reverse transcription caused by a break on the RNA, a model
called "forced copy choice" (9). In this case the stalling of the
RT would constitute the crucial step of the process by allowing an
extensive degradation of the RNA template by the RNase H activity
carried by the RT itself, as demonstrated for ( This idea has, however, recently been challenged by increasing evidence
demonstrating that pausing during reverse transcription and strand
transfer are not necessarily coupled (13-15). In parallel, studies
carried out on RNA templates containing hairpin regions have suggested
that such structures could favor template switching by RTs (13, 16,
17). In these cases it was proposed that the hairpin structures enhance
the probability of strand transfer by mediating an interaction between
donor and acceptor RNA that increases their spatial proximity (13, 16,
17). Extensive random searches for the occurrence of recombination hot
spots during in vitro reverse transcription by HIV-1 RT had
revealed the correlation between the location of these hot spots and
the presence of predicted hairpin regions in the RNA template (14, 18).
Based on this observation, it was proposed that template switching
proceeds through a two-step mechanism: docking of the acceptor RNA onto
the nascent DNA and displacement of the donor RNA by the acceptor RNA
(14). The latter step would be guided by the folding of the RNA.
A recent study on the primer binding site of the equine
infectious anemia virus has shown that the hairpin present in that
region induces a strong pause of reverse transcription that increases
the efficiency of the docking step (15). In addition, in
vitro studies on the effect on strand transfer of the nucleocapsid
protein (NC), a major co-factor of the reverse transcription complex
(19), have suggested a mechanism of recombination governed by the
structures of the RNA rather than by pausing of reverse transcription
(reviewed in Refs. 3 and 20). Indeed, although NC enhances strand
transfer in vitro (reviewed in Ref. 3) it does not lead to a
parallel increase of pausing during reverse transcription, as predicted
for a mechanism of template switching governed by pausing of DNA
synthesis (14). Because the NC is a RNA chaperone (21, 22), it was
suggested that the enhancement of strand transfer observed in its
presence was because of its ability to modulate the structures of the
RNA templates (14).
Among the several recombinant HIV-1 strains isolated to date, a well
defined case is constituted by chimerical genomes between subgroups A
and either C or D, generated by recombination on the region coding for
the constant portion C2 of the envelope glycoprotein gp120 (23).
Recombination on this segment of genome allows reshuffling of the
portions of gp120 coding for the variable regions V1 and V2, relative
to regions V3 through V5. The spatial arrangement of regions V2 and V3
with respect to the constant regions of the protein has been shown to
be critical for allowing the virus to escape neutralizing antibodies
raised by the immune system of the host (24). In a previous report we
used several RNA sequences issued from the HIV-1 genome to investigate
the mechanism of template switching by HIV-1 reverse transcriptase
in vitro. We observed that the genomic sequence coding for
the C2 region constituted, indeed, the most important hot spot we found
during that work (18). Interestingly, a subsequent study on
recombination during infection of cells in culture with different HIV-1
subtypes has also shown the occurrence of frequent recombination in the
same region (25). In our previous study, the portion of 200 nt that constituted the hot spot within the C2 domain was called "Eb" and
was initially included in a model template where it was surrounded by
the sequences that flank it on the viral genome (Fig. 1A,
RNA E2). It was subsequently observed that by changing the
surrounding sequences (Fig. 1, RNA G1Eb) the frequency of
strand transfer on Eb was decreased by a 4- to 5-fold factor (18). We
referred to this effect as "context effect." In this work, we took
advantage of the context effect to investigate the role of the RNA
structure in the transfer process and to address the question of the
mechanism responsible for copy choice by HIV-1 RT.
Labeling of RNA and Determination of the Secondary
Structures--
To determine their folding, the various RNA templates
were labeled at their 3'-end as follows. A 21-mer oligonucleotide with a sequence complementary to the 3'-end of the RNA to label was annealed, at a molar ratio of 4:1 (oligo:RNA) and in a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl,
1 mM EDTA, by heating the mixture for 30 s at 95 °C
followed by a slow cooling to 30 °C. The oligonucleotide
carries at its 5'-end a non-hybridizing tail of six nucleotides
constituted by the sequence 5'-CTTTTT-3'. The annealed RNA was then
incubated for 20 min at 37 °C in a final volume of 55 µl in a
buffer containing 7 mM dithiothreitol, 20 mM Tris-HCl, pH 7.5, 12.5 mM MgCl2,
20 mM NaCl, 40 units of RNasin (Promega), 5 units of
Sequenase (United States Biochemicals), and 0.05 mCi of
[ RNA Synthesis and Recombination Assays--
The various
constructs used for RNA synthesis were generated following standard
cloning procedures (28). Each construct was systematically sequenced
before its use in RNA synthesis. RNA synthesis was performed as
previously described (29). Reverse transcription of the donor RNA was
carried out in the presence of the acceptor RNA (at a final
concentration of 100 nM each, unless otherwise stated)
after annealing an oligonucleotide specifically onto the donor template
(Fig. 2A). Annealing was performed at a molar ratio of
primer to donor RNA of 10:1 in 50 mM Tris-HCl (pH
7.8), 75 mM KCl, 7 mM MgCl2 at
65 °C for 5 min followed by a slow cooling to 40 °C.
Dithiothreitol (1 mM final concentration), the four dNTPs
(1 mM each), and RNasin (100 units; Promega) were added
after incubation for 5 min on ice. For the experiments with NC (55 amino acids, synthesized as described in Ref. 30), the protein was
added at this step at a ratio of 1 molecule of NC/8 nt of total RNA and
incubated for 10 min at 37 °C. Reverse transcription was started by
the addition of HIV-1 RT at a final concentration of 400 nM
and carried out for 90 min. The reaction was stopped by extraction with
phenol-chloroform. The samples containing NC were treated, before
phenol-chloroform extraction, for 1 h at 56 °C with proteinase
K (8 mg/ml), 0.4% (w/v) SDS, and 50 mM EDTA (pH 8.0). The
phenol-chloroform-extracted samples were then submitted to RNase
treatment. Purification of the reverse transcription product and
synthesis of the second DNA strand were performed as previously
described (14). BamHI and PstI digestion,
ligation, and Escherichia coli transformation were also
carried out as previously described (18) and as shown in Fig. 2. For
the experiments where the concentration of acceptor template was
varied, the procedure of reverse transcription of a fixed amount (100 nM) of donor RNA was identical to the one described above,
but the concentration of acceptor included in the assay was varied as
detailed in Fig. 5. In all cases HIV-1 RT was used at a
concentration of 400 nM.
Estimation of Recombination Rates per Nucleotide--
The
recombination frequency in the various intervals of the model templates
was calculated as follows. In the case of recombination between dE2 and
aE2 RNAs, as an example, a NcoI restriction site was present
at the boundary between Ea and Eb on the donor RNA, and an
ApaLI site marked the transition between Eb and Ec on the acceptor RNA (Fig. 3A, column I). All recombinant
molecules NcoI+/ApaLI+
were considered to issue from template switching within Eb. We define
F as the overall frequency of recombination observed in the
experiment, as given in Fig. 2, left panel. If b
is the number of blue colonies whose restriction pattern has been
analyzed and c is the number of recombinant colonies
NcoI+/ApaLI+, the
frequency of recombination within Eb (f) is given by
F(c/b). Recombination rates per nt were calculated by
dividing the frequency of recombination within a given region by its
size in nt (in the example given here, Eb is 200 nt long, and the
recombination rate is therefore given by f/200).
Primer Extension Assays--
Reverse transcription was primed
using a 5'-terminal-labeled deoxyoligonucleotide and carried out under
the same buffer and RT conditions as above. The templates used in these
assays consisted in truncated versions of dG1Eb or dE2, devoid of the
sequences coding for the reporter gene and therefore including only the viral sequences. Similarly when the experiments were performed in the
presence of the acceptor RNAs, modified versions of aG1Eb and of aE2
RNAs were used, constituted only by the viral sequence. The complex
between HIV-1 RT and the primer/template was pre-formed by incubation
for 10 min in the same reaction buffer as described above, devoid of
dNTPs and MgCl2. The reaction was started by the addition
of dNTPs and MgCl2 and stopped at various time intervals by
addition of EDTA to a final concentration of 15 mM. All
samples were ethanol-precipitated before electrophoresis on 8% (w/v)
polyacrylamide gel containing 8 M urea in a loading buffer
containing formamide at a final concentration of 22.5%. The intensity
of each band was estimated by phosphorimaging as described above.
Role of the Secondary Structure of the Templates--
To study the
potential correlation between the frequency of recombination on the Eb
region and its structure, we determined its folding in solution when it
is included either in E2 RNA (condition under which Eb is a
recombination hot spot) or in G1Eb RNA (where Eb was not a
recombination hot spot). In these two cases we call this region Eb* and
Eb°, respectively. The determination of the folding of the RNAs was
performed under the same buffer and temperature conditions as the
recombination assay (see "Materials and Methods"). These two RNAs
were labeled at their 3'-end, subjected to enzymatic probing (Fig.
1, panels B and C),
and their secondary structures were assessed by including the
constraints revealed by these probing assays into the m-fold program
(27). The most significant difference found between the folding of Eb*
and Eb° was in the 3'-terminal region of the sequences. In this part,
we identified a stem-loop motif, called here SL (Fig. 1D),
present exclusively in the case of Eb*. As shown in Fig. 1, the SL
hairpin is constituted at its 3'-end by a portion of the region Ea, the
sequence downstream Eb* in E2 RNA. When Eb is part of G1Eb RNA (Eb°),
Ea is replaced by the region G1a and the formation of the SL hairpin is
no longer possible (Fig. 1, panels B and C,
bottom). No other stable hairpins were found within
Eb°.
Which Template Drives the Switch?--
The influence of
these structural differences in the strand transfer reaction was then
investigated by using a recombination assay previously described (18)
and outlined in Fig. 2, left panel. Strand transfer involves two types of RNA templates, the donor and the acceptor. The correlation between the folding of the Eb
region and the frequency of strand transfer observed during its reverse
transcription was exploited to assess the role played by each of these
templates. The experiments were first performed on naked RNA templates.
The rationale of the experiment is outlined in Fig.
3A. In the two cases outlined
in Fig. 3A, columns I and IV, the
donor and the acceptor RNAs share complete sequence homology on the
viral sequence, apart from the presence of the restriction sites
indicated in the figure. We refer to these conditions as "symmetric," because the folding of the region Eb is the same on
the donor and the acceptor RNAs (either Eb* on both or Eb° on both).
In contrast, under the conditions depicted in columns II and III
("asymmetric conditions") Eb adopts a different folding between the
donor and the acceptor RNAs. Furthermore, under the asymmetric
conditions the region Eb constitutes the only region of homology
between the two RNAs (in black in the figure). The frequency of
template switching within Eb was determined by restriction analysis of
the recombinant products, as detailed under "Materials and
Methods." When Eb was in the Eb* conformation on the acceptor RNA,
strikingly close frequencies of recombination were observed on the Eb
interval, independent of which donor RNA was used (Fig. 3A,
columns I and II, naked RNAs).
Conversely, the frequency of strand transfer on Eb was similar when it
was in the Eb° conformation on the acceptor RNA, independent from the
use of dE2 or of dG1Eb as donor RNA (Fig. 3A, columns
III and IV). Therefore, it was evident that the
conformation of Eb on the acceptor RNA determined the frequency of
template switching. We then tested whether these conclusions reached on
experiments on naked RNAs could apply to recombination occurring in the
presence of the NC protein. Also, in this case the frequency of
recombination on Eb was found to depend on the folding of the acceptor
RNA (Fig. 3A, RNA·NC complex).
Strand Transfer within SL--
To document the role of the SL
hairpin in recombination taking place within Eb, we decided to
distinguish between the events of strand transfer occurring within SL
itself from those taking place outside the hairpin in the Eb interval.
We employed aE2 as the acceptor RNA and either dE2 or dG1Eb as donor
templates, the conditions under which the highest frequency of transfer
was observed on Eb (Fig. 3A, columns I and
II). For these experiments, a point mutation generating an
EcoRI site was introduced on both types of donor RNAs, dE2
and dG1Eb, creating dE2-eco and dG1Eb-eco RNAs, respectively (Fig.
3B). This EcoRI site is located immediately 5'
with respect to the base of the SL hairpin and allows mapping of strand
transfer within Eb. As shown in Fig. 3B (naked
RNA), the use of either dE2-eco or dG1Eb-eco as donor RNAs yielded
a recombination rate higher within the hairpin itself than in the downstream portion. Furthermore the rates of recombination were very
close when dE2-eco or dG1Eb-eco was used (15.1 and 14.0 × 10 Analysis of Pausing Pattern during Reverse
Transcription--
The observation that the type of donor RNA used
does not modify the frequency of template switching strongly suggests
that pausing of reverse transcription on the donor RNA is not the
trigger for strand transfer. It cannot be ruled out, though, that the pausing pattern on the donor templates is modified when reverse transcription is performed in the presence of aG1Eb or aE2 as acceptor templates. To investigate this point we first carried out a
labeled primer extension analysis on dG1Eb and dE2 RNAs (Fig.
4A, lanes
1-4, and B, lanes 1-5). Despite the
different folding of the SL region on these RNAs, a prominent pause
site was found in both cases at the same position, corresponding to a
stretch of four uridine residues. To check whether the presence of an
acceptor RNA could induce a change in the pausing pattern on the donor
template, reverse transcription of dG1Eb (Fig. 4A) or of dE2
(Fig. 4B) was then performed in the presence of either aG1Eb
or aE2. As indicated at the bottom of Fig. 4, these conditions reproduce those employed for the recombination assays depicted in Fig.
3A. In no case could a significant change in the pausing pattern be detected. The 4- to 5-fold difference in the frequency of
recombination observed, depending on the use of E2 or G1Eb as acceptor
RNA (Fig. 3A), was therefore not associated with an increased stalling of the reverse transcription.
Setting Up a System to Investigate Copy Choice and Forced Copy
Choice in Parallel--
To better evaluate the effect of strong
arrests of DNA synthesis on template switching, we have developed an
experimental system that reproduces the situation described in the
forced copy choice model (Fig. 2, right panel). In this
system reverse transcription was performed in parallel under two
different experimental conditions, referred to in Fig. 2 as "strand
transfer" and "standard" samples. In the strand transfer sample,
a donor template ("FCC donor RNA," for forced copy choice) is
reverse-transcribed in the presence of an acceptor RNA. The FCC donor
RNA is truncated within the region of homology with the acceptor RNA.
This system allows strand transfer to occur either from internal
positions of the region of homology or at the very 5'-end of the donor
template. The reverse transcription products are then treated as for
the copy choice experiments and cloned in E. coli (see the
Fig. 2 legend and "Materials and Methods"). Because only the
reverse transcription products that have reached the 5'-end of the
acceptor RNA will be converted into double-stranded molecules, this
assay allows cloning specifically of the products of strand transfer
(see the Fig. 2 legend). The number of blue colonies
generated is therefore proportional to the number of samples that
underwent strand transfer. However, to obtain a frequency of occurrence
of strand transfer under these conditions one needs to determine the
amount of full-length molecules that would have been obtained if no
obligatory strand transfer were required. For this reason, and with the
aim of comparing these frequencies with those found under copy choice
conditions, reverse transcription was performed in parallel on the same
donor RNA used for the copy choice experiments (Fig. 2, right
panel, standard sample). The resulting reverse
transcription products were then treated as described above and cloned
in E. coli. Because the amount of RNA employed in
this sample was the same as the forced copy choice (FCC) donor RNA used
in the strand transfer sample, the ratio of colonies found
in the strand transfer sample to colonies found in the
standard sample gives the frequency of strand transfer under
forced copy choice conditions (Fig. 2, right panel). This
system allows a strict comparison with the frequencies found for copy
choice because the same recombinant and parental molecules,
respectively, are generated in the two cases.
Effect of the Concentration of the Acceptor RNA--
The influence
of the concentration of the acceptor RNA on the strand transfer
reaction was then studied in parallel under the copy choice and the
forced copy choice conditions. This study was carried out not only on
E2 RNA but also on two other sequences we previously studied for their
ability to promote strand transfer in vitro: the region R
and a 400-nt segment of the gag gene we called "gag1"
(18). These two sequences were chosen as representative of another
recombination hot spot (the sequence R) and of a region where strand
transfer is a rare event (the gag region). The recombination assays were performed at a constant concentration of donor template (100 nM), varying the concentration of acceptor RNA from 25 nM to 1 µM (Fig.
5). Under the copy choice conditions, for
all the three sequences studied the frequency of strand transfer
remained constant for ratios higher than 1:1 (Fig. 5A). This
result is in sharp contrast with the one found under forced copy choice conditions where, for the three sequences studied, the frequency of
transfer was clearly dependent on the concentration of acceptor template in the whole range of concentrations tested (Fig.
5B).
We have studied here the mechanism of copy choice recombination
during in vitro reverse transcription by HIV-1 RT. In a
previous work we conducted a random search for recombination hot spots among various sequences of the HIV-1 genome. That analysis revealed the
presence of two strong hot spots, one constituted by the
transactivation response element (TAR) hairpin region, the other by a
200-nt-long region coding for the C2 domain of the glycoprotein gp120.
Here we have mostly focused on the latter region. We have first
determined the folding of this hot spot in solution and shown the
direct correlation existing between the high frequency of recombination on this region and the presence of a stable hairpin structure. The ability of the same sequence to promote recombination at
different rates depending on the structure adopted has provided the
means to assess, for the first time to our knowledge, the respective roles played by stem-loop structures on the donor and on the acceptor RNAs. Indeed, the comparison of the results obtained in the symmetric and asymmetric experiments (Fig. 3) demonstrates that on this RNA
sequence the presence of the stem-loop structure on the acceptor RNA
appears to be the most important structural element for this region to
constitute a recombination hot spot. Strand transfer occurred at high
and perfectly comparable frequencies when the hairpin was present on
the acceptor RNA regardless of its presence or absence on the donor
template (Fig. 3A, columns I and II). It is noteworthy that this observation stands true for naked RNA templates, conditions under which we have determined the folding of the
RNA in solution, as well as in the presence of the NC protein, conditions closer to the situation found in vivo. The
finding that the folding of the acceptor RNA constitutes the crucial
parameter for strand transfer is in line with previous observations on
the role of NC in the process. In fact, it was shown that the NC, which
modulates the folding of the RNA (21, 22), enhances copy choice through
its binding onto the acceptor RNA (14).
We previously proposed that the transfer process follows two steps,
docking of the nascent DNA onto the acceptor template and displacement
by the acceptor RNA of the donor RNA from the 3'-end of the nascent DNA
(14). The first step (docking) is expected to depend on the probability
of encounter of the acceptor RNA and of the nascent DNA, which is a
function of the concentration of these two moieties in solution. The
displacement step, in contrast, would involve an acceptor RNA already
docked onto the reverse transcription complex and would therefore not
depend on the concentration of the acceptor RNA in solution. To
distinguish between these two steps we have investigated the response
curves of recombination as a function of the concentration of the
acceptor RNA on three different sequences. For ratios of acceptor to
donor RNAs higher than one, no increase in the frequency of copy choice
was observed (Fig. 5A). The fact that recombination on the
gag sequence, even for high concentrations of acceptor RNA, never
reached the values found for Eb or R (Fig. 5A) indicates
that the efficiency of the process depends on intrinsic features of the
RNA considered rather than on the concentration of the acceptor RNA.
Because docking is expected to be sensitive to the concentration of RNA
in solution, we reason that the different efficiencies of strand
transfer are likely not because of the efficiency of docking. The
parallel observation, made in this work, that the frequency of
recombination observed on the same sequence (Eb) varies as a function
of its folding strongly suggests that the structure of the RNA template is rather responsible for the efficiency of copy choice.
The involvement of hairpin structures in strand transfer has been the
object of several studies. It was first suggested that the hairpins
formed by these regions could favor template switching by increasing
the spatial proximity between the donor and the acceptor RNAs through
an intermolecular interaction occurring within the hairpin region (13,
16, 17). Our results do not support such an interpretation because here
the hairpin structure is not required on the donor RNA and, therefore,
cannot act by mediating an interaction with the acceptor RNA.
Furthermore, the response curves given in Fig. 5A for copy
choice suggest that the spatial proximity of donor and acceptor RNAs is
not rate limiting. A recent work on strand transfer on a hairpin region
of the genomic RNA of the equine infectious anemia virus has suggested
an implication for hairpin structures in the two-step model discussed
above (15). In this new model ("dock and lock" model) the docking
of the nascent DNA on the acceptor RNA constitutes the limiting step.
In this case stalling of reverse transcriptase at the base of the
hairpin on the donor RNA improved its degradation by the RNase H
activity, thereby increasing the accessibility of the nascent DNA to
the annealing for the docking step. In the present work, the hairpin cannot act in a similar way because, if this were the case, its presence would be required on the donor RNA and not on the acceptor. However, the analysis of the pausing pattern during reverse
transcription of the SL hairpin has highlighted the presence of a
strong pause site in the descending portion of the stem (Fig. 4). This
pause might, therefore, act in a similar way by favoring the annealing of the nascent DNA onto the acceptor RNA. The contribution of slowing
down reverse transcription in the enhancement of strand transfer both
in vitro and in vivo has, in fact, been
previously described (31-33). However, although the presence of this
pause site most likely favors strand transfer, it is unlikely that it constitutes the crucial step for the reaction. In fact, the observation that the intensity of pausing on a given donor RNA is not affected by
the type of acceptor used (Fig. 4), although the frequency of
recombination is clearly different (Fig. 3A), manifestly
argues against this idea. Furthermore, if stalling of reverse
transcription constituted the trigger for the transfer event during
copy choice, the response curves as a function of the concentration of
acceptor RNA would be expected to be similar to those found under
forced copy choice conditions, where a manifest strong pause site is present. Fig. 5 shows instead that this is not the case for any of the
sequences studied. We conclude therefore that a pause site is not
sufficient to generate recombination hot spots and that it is
consequently impossible to predict recombination hot spots simply on
the analysis of the pausing pattern found during reverse transcription
on the donor RNA.
In light of these results, a possible mechanism accounting for template
switching on hairpin regions of the template is proposed in Fig.
6 ("hairpin-mediated strand
transfer"). Although this model is elaborated focusing on the most
stringent conditions under which Eb constituted a hot spot, the case
where the SL hairpin is present only on the acceptor RNA (Fig.
3B, column II), obviously it also applies when
both donor and acceptor RNAs contain a hairpin region. Under the
conditions of Fig. 3B, column II, because the homology between donor and acceptor RNAs begins in the mounting portion
of the stem of the SL hairpin, docking must necessarily occur within
SL. Furthermore, under these conditions the frequency of strand
transfer within SL was as high as when an extended homology between
donor and acceptor RNAs was present even before reverse transcription
reached the SL hairpin (compare frequency in the hairpin region in Fig.
3B, columns I and II). This
observation indicates that the SL region is sufficient to allow
efficient docking. As mentioned above, this step could be favored by
stalling of the polymerase at the pause site in the descending portion of the stem (Fig. 4). Once docking is achieved, the 3'-end of the
nascent DNA has to be transferred onto the acceptor RNA. How the
hairpin on the acceptor template is opened to accept the invading strand of the nascent DNA constitutes the main problem in understanding the mechanism of template switching in hairpin regions. Even in the
dock and lock model recently proposed, it is difficult to see how the
hairpin on the acceptor RNA could be opened and, especially, why
invasion should be favored within hairpin regions rather than on poorly
structured templates. In this regard, in our model the generation of a
structure equivalent to the intermediate that facilitates branch
migration during DNA recombination (Fig. 6D) offers a
plausible solution to this problem. As in that well established case
(34), here the gradual opening of the stem portion of the hairpin on
the acceptor would be favored by the concomitant formation of
alternative double-stranded structures, a feature only possible when a
hairpin is present on the acceptor RNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) DNA strand the reverse
transcriptase (RT) can switch template and, guided by the local
sequence homology, transfer DNA synthesis from one genomic RNA molecule
(the donor) onto the other (acceptor RNA). In a heterozygous virion
this process, known as copy choice, leads to genetic recombination
(8).
)DNA strong stop
strand transfer (10). The resulting single-stranded DNA would then be
available for annealing onto the complementary sequence provided by the
acceptor RNA. A similar situation can be encountered if stalling is
generated by strong pause occurring during reverse transcription of an
intact template. Indeed, a prominent pause site detected during
in vitro reverse transcription of a stretch of the HIV-1
nef gene was shown to increase significantly the local
frequency of strand transfer (11, 12). In these instances the stalling
of reverse transcription is regarded as the limiting step for strand transfer.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
32P]dideoxyATP (Amersham Biosciences). Labeled
RNAs were purified on 7% polyacrylamide gel and eluted by passive
diffusion at 4 °C in a buffer containing 10 mM magnesium
acetate, 500 mM ammonium acetate, 0.1% SDS, and 1 mM EDTA. The eluted RNA was extracted with
phenol-chloroform and precipitated in ethanol; the dried pellet was
conserved under ethanol at
20 °C. For determination of the
structure of the RNA, 8 pmol of the labeled RNA were heated in the
reverse transcription buffer (50 mM Tris-HCl, pH 7.8, 75 mM KCl, 7 mM MgCl2) at 65 °C for
5 min, slowly cooled to 40 °C, and transferred on ice. The RNA was
then treated with either T1 (0.3 and 0.15 units) or T2 (0.04 and 0.02 units) RNases for 5 min at 37 °C. T1 and T2 RNases cleave
single-stranded RNA molecules with preference for guanine residues for
T1 and adenine residues for T2 (26). The reaction was stopped by
phenol-chloroform extraction followed by ethanol precipitation. The
products were analyzed by autoradiography after electrophoresis on 7%
polyacrylamide gels (see Fig. 1). Quantification was performed using
phosphorimaging apparatus (Molecular Dynamics). The positions of
enzymatic cleavage were identified by reference to a ladder generated
by extensive T1 digestion of the same RNA molecule. The residues
identified as single-stranded in four independent experiments were
introduced as constraints in the structure prediction analysis by the
m-fold program (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, location of the main sequence studied
in this work, E2, within the HIV-1 genome. The three portions
constituting the E2 RNA (bottom drawing: Ec,
Eb, and Ea) are aligned with respect to the
primary structure of the glycoprotein gp120 (middle drawing:
SP, signal peptide; C1-C5, constant regions;
V1-V5, variable regions). B-D, folding of the
conditional hot spot Eb. In each panel the drawings at the
bottom indicate the model templates used. The names of the
templates are given in gray and italic on the
right of the drawing and those of the subregions that
constitute them below the drawings. B and
C, analysis by sequencing gel electrophoresis of the
cleavage pattern, by RNases T1 and T2, of the Eb region either in G1Eb
(B) or in E2 (C) RNAs. Individual residues are
numbered starting from the 5'-border of the region Eb. Consequently, Eb
spans from 1 to 200 nt on both RNAs (see diagrams). The
letters and numbers indicate the bases sensitive to cleavage by T1
(left) or T2 (right). B, lane
1, mock sample. Lanes 2 and 3, samples
treated with 0.3 and 0.15 units of T1, respectively. Lanes 4 and 5, samples treated with 0.04 and 0.02 units of T2,
respectively. C, lane 1, mock sample. Lanes
2 and 3, samples treated with 0.3 and 0.15 units of T1,
respectively. Lanes 4 and 5, samples treated with
0.02 and 0.04 units of T2, respectively. C, the
bars on the right indicate the participation of
the corresponding bases to a stem (S) or loop (L)
region as detailed in panel D. D, folding of the
3'-portion of the Eb region in the model template E2 (conformation Eb*
in the text). White and black
arrows indicate the residues whose conformation has been
determined empirically by T2 and T1 RNases, respectively, as shown in
panel C. The sequence Eb is given in black
letters; gray letters indicate residues belonging to
Ea, the region preceding Eb on E2 RNA, following the sense of reverse
transcription. L1-L3, loop regions; S1 and
S2, stem portions. The dashed box SL in the
bottom drawing of panel D indicates the location of the
hairpin structure depicted above.
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Fig. 2.
Experimental systems used to study copy
choice or forced copy choice. Left panel, copy choice.
Reverse transcription is selectively primed on the donor RNA in the
presence of the acceptor RNA. The two RNAs share a region of homology
constituted by a viral sequence (vir), followed, in the
sense of reverse transcription, by a genetic marker, different on the
two RNAs. Although both templates carry a PstI site near
their 5'-terminus, only the donor template possesses a BamHI
site at the 3'-end. Processive reverse transcription of the donor RNA
leads to the synthesis of lac molecules,
whereas template switching on the region of homology generates
molecules carrying the sequence of a functional lacZ' gene.
The single-stranded DNAs possess the same sequence at the 3'-end
(black box) that will be used to prime synthesis of the
second DNA strand using Taq polymerase (this is not a PCR
reaction). The resulting double-stranded products are cloned in
E. coli using the BamHI and PstI
sites, which will be present on both parental and recombinant
molecules. On the appropriate dishes, recombinant and parental
molecules will generate blue (lac+) and white
(lac
) colonies, respectively. As a control, an
equivalent amount of plasmid vector used for the cloning of the reverse
transcription products was ligated and used for transformation,
providing an estimate of the background of the white colonies resulting
from transformation with circularized vectors. The background value
never exceeded 10% of the white colonies recovered from the
recombination samples and was systematically subtracted for computation
of the frequency of recombination. The ratio of blue colonies to the
sum of blue plus white colonies, corrected for the background value,
gives the frequency of copy choice recombination. When comparing
regions of different size the frequency of recombination was
measured in rate (per nt), following the procedure described under
"Materials and Methods." Right panel, the system used to
study forced copy choice. The symbols used are the same as for the
panel on the left. See "Estimation of
Recombination Rates per Nucleotide" under "Materials and Methods"
for details.
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Fig. 3.
Role of the acceptor RNA in template
switching. Inset, schematic depiction of the
conformations of the viral sequence referred to in text as Eb* and
Eb°. The Eb region, drawn in black, is encompassed
by red sequences in the E2 template and by light
blue ones in G1Eb RNA. The sizes of the different regions are:
Ea, 100 nt; Eb* (or Eb°) 200 nt; Ec,
100 nt; G1a, 150 nt, G1b, 150 nt. The green
arrow shows the direction of reverse transcription on the donor
template. A and B, the letters "a" and
"d" preceding the name of the RNA template stand for acceptor and
donor RNAs, respectively. A, symmetric (columns
I and IV) and asymmetric (columns II and
III) experimental conditions. The folding of the donor and
the acceptor RNAs in the SL region is schematically indicated
above each model template. Gray arrows indicate
the location of specific restriction sites that, after restriction
analysis of the recombinant DNA molecules, allow mapping of strand
transfer events along the sequence of homology as described under
"Materials and Methods." These sites were generated by the
introduction of point mutations. The frequency of recombination
observed under the different experimental conditions is given below.
B, mapping of strand transfer events at the level of the SL
region under the two experimental conditions that yield a high degree
of transfer (panel A, columns I and
II) was performed by introducing the additional restriction
site EcoRI on the donor RNA. This mutation did not affect
the overall frequency of recombination. The region Eb is therefore
subdivided into the hairpin and the downstream region. To normalize for
the different size of the two regions, the frequency of recombination
is given as rates per nt.
4 per nt), confirming that the type of donor RNA used
does not influence the distribution of the positions of strand transfer within Eb. We then evaluated whether the same conclusions can be drawn
from experiments performed in the presence of the NC protein by
performing the recombination assays on RNA·NC complexes. Also,
in this case strand transfer occurred at high rates on SL, regardless
of the type of donor template employed.
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Fig. 4.
Primer extension assays. Denaturing
polyacrylamide gel analysis of primer extension products. A,
synthesis of DNA was initiated specifically on the dG1Eb RNA in the
absence (1-4) or in the presence of an equimolar amount of
either aG1Eb (5-8) or aE2 templates (9-12) as
acceptor RNA. B, DNA synthesis was primed on dE2 in the
absence (1-5) or the presence of either aG1Eb
(6-10) or aE2 (11-15). The reactions were terminated at different incubation times: 1, 3, 10, and 30 min
(A) and 1, 3, 5, 10, and 30 min (B). The position
of the pause site on the sequence corresponding to SL is shown in
gray. The drawings at the bottom schematically indicate
which donor and acceptor RNA templates were employed in the assays
shown above, using the same representation as in Fig. 3.
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Fig. 5.
Effect of the concentration of the acceptor
RNA on copy choice and forced copy choice. A, copy
choice; B, forced copy choice. Only the viral portion
("vir" in Fig. 2) of the model templates used in these assays is
shown in each panel. A detailed description of these templates is given
in Ref. 18. The recombination rates refer to recombination occurring on
the regions shown in gray on the donor RNAs (the size of
which is given in the table). Error bars were calculated as
(b1/2/b), where b is the
number of blue colonies, and r is the recombination rate.
The standard sample is the one indicated in Fig. 2 for the
forced copy choice conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
The hairpin-mediated strand transfer model
for copy choice by RTs. A, the case presented is the
one where the donor template is dG1Eb and the acceptor is aE2 (see Fig.
3A, column II). Donor RNA, red;
acceptor RNA, blue; nascent DNA, black (the
arrow indicates the direction of reverse transcription).
A, the stem of the SL hairpin on the acceptor RNA is
represented as constituted by a lower and an upper part, a
and b, respectively, annealed to their
complementary sequences, a' and b'. The loop
region is indicated as c. Region a
corresponds to the Ea sequence in Fig. 1D and is therefore
absent from the donor RNA. B, reverse transcription
progresses on the donor RNA. C, because annealing of the
nascent DNA is likely favored on a single-stranded region of the
acceptor RNA, docking might occur once the loop region c is
reverse-transcribed (generating the complementary sequence
c'). D, the hybridization of the nascent DNA
onto the acceptor RNA then invades the stem region of the hairpin on
the acceptor RNA, generating the maximum possible extent of
double-stranded regions. The resulting intermediate structure redrawn
on the right of the panel resembles an intermediate of
branch migration occurring during DNA recombination (see supplementary
information). A migration downward of the position of the crossover
would lead to the transfer of the 3'-end of the nascent DNA on the
acceptor RNA.
In conclusion, this study provides direct evidence for the
correlation between recombination hot spots and the structure of the
viral genomic RNA. Which specific features of the hairpin structure
described here are crucial remains an open question. However, the model
proposed here could account for recombination occurring on most hairpin
regions, including strand transfer within the transactivation response
element hairpin. Obviously this does not exclude that template
switching also follows alternative mechanistic paths as shown, for
instance, by the residual frequency of recombination found within Eb
when both templates are devoid of the SL hairpin (Fig. 3, column
IV). However, the observation that the main recombination hot
spots issued from random searches correspond to hairpin regions (14,
18) suggests that such structures constitute preferential sites for
frequent template switching. This study provides a basis for the
dissection of the mechanism of template switching at such sites along
the HIV-1 genome.
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ACKNOWLEDGEMENTS |
---|
We thank Chantal Ehresmann, Roland Marquet, and Eric Westhof for valuable insights during the elaboration of the model proposed in this work. We are also grateful to Bernard Roques for the generous gift of the NC protein.
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FOOTNOTES |
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* This work was supported by Grant ANRS 02172 from the Agence Nationale pour la Recherche sur le SIDA (ANRS) (to M. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplementary figure.
Recipient of fellowships from ANRS and, currently, from SIDACTION.
To whom correspondence should be addressed. Tel.:
33-1-4568-8505; Fax: 33-1-4568-8399; E-mail: matteo@pasteur.fr.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212306200
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; nt, nucleotides; RT, reverse transcription; NC, nucleocapsid protein; SL, stem-loop.
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REFERENCES |
---|
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---|
1. | Coffin, J. M. (1992) Curr. Top. Microbiol. Immunol. 176, 143-164[Medline] [Order article via Infotrieve] |
2. | Temin, H. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6900-6903[Abstract] |
3. | Negroni, M., and Buc, H. (2001) in Annual Review of Genetics (Campbell, A., ed), Vol. 35 , pp. 275-302, Annual Reviews, Palo Alto, CA |
4. |
Sharp, P. M.,
Bailes, E.,
Robertson, D. L.,
Gao, F.,
and Hahn, B. H.
(1999)
Biol. Bull.
196,
338-342 |
5. | Hu, W. S., and Temin, H. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1556-1560[Abstract] |
6. | Vogt, V. M. (1997) in Retroviruses (Coffin, J. M. , Hughes, S. H. , and Varmus, H. E., eds) , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
7. | Jung, A., Maier, R., Vartanian, J. P., Bocharov, G., Jung, V., Fischer, U., Meese, E., Wain-Hobson, S., and Meyerhans, A. (2002) Nature 418, 144[CrossRef][Medline] [Order article via Infotrieve] |
8. | Vogt, P. K. (1973) in Possible Episomes in Eukaryotes (Silvestri, L., ed) pp. 35-41, North Holland |
9. | Coffin, J. M. (1979) J. Gen. Virol. 42, 1-26[Medline] [Order article via Infotrieve] |
10. | Telesnitsky, A., and Goff, S. P. (1993) EMBO J. 12, 4433-4438[Abstract] |
11. | DeStefano, J. J., Mallaber, L. M., Rodriguez-Rodriguez, L., Fay, P. J., and Bambara, R. A. (1992) J. Virol. 66, 6370-6378[Abstract] |
12. |
Wu, W.,
Blumberg, B. M.,
Fay, P. J.,
and Bambara, R. A.
(1995)
J. Biol. Chem.
270,
325-332 |
13. |
Kim, J. K.,
Palaniappan, C.,
Wu, W.,
Fay, P. J.,
and Bambara, R. A.
(1997)
J. Biol. Chem.
272,
16769-16777 |
14. |
Negroni, M.,
and Buc, H.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6385-6390 |
15. |
Roda, R. H.,
Balakrishnan, M.,
Kim, J. K.,
Roques, B. P.,
Fay, P. J.,
and Bambara, R. A.
(2002)
J. Biol. Chem.
277,
46900-46911 |
16. |
Berkhout, B.,
Vastenhouw, N. L.,
Klasens, B. I.,
and Huthoff, H.
(2001)
RNA
7,
1097-1114 |
17. |
Balakrishnan, M.,
Fay, P. J.,
and Bambara, R. A.
(2001)
J. Biol. Chem.
276,
36482-36492 |
18. |
Moumen, A.,
Polomack, L.,
Roques, B.,
Buc, H.,
and Negroni, M.
(2001)
Nucleic Acids Res.
29,
3814-3821 |
19. |
Welker, R.,
Hohenberg, H.,
Tessmer, U.,
Huckhagel, C.,
and Krausslich, H. G.
(2000)
J. Virol.
74,
1168-1177 |
20. | Negroni, M., and Buc, H. (2001) Nat. Rev. Mol. Cell. Biol. 2, 151-155[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Clodi, E.,
Semrad, K.,
and Schroeder, R.
(1999)
EMBO J.
18,
3776-3782 |
22. |
Williams, M. C.,
Rouzina, I.,
Wenner, J. R.,
Gorelick, R. J.,
Musier-Forsyth, K.,
and Bloomfield, V. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
6121-6126 |
23. | Robertson, D. L., Sharp, P. M., McCutchan, F. E., and Hahn, B. H. (1995) Nature 374, 124-126[Medline] [Order article via Infotrieve] |
24. |
Ye, Y.,
Si, Z. H.,
Moore, J. P.,
and Sodroski, J.
(2000)
J. Virol.
74,
11955-11962 |
25. |
Quinones-Mateu, M. E.,
Gao, Y.,
Ball, S. C.,
Marozsan, A. J.,
Abraha, A.,
and Arts, E. J.
(2002)
J. Virol.
76,
9600-9613 |
26. | Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J. P., and Ehresmann, B. (1987) Nucleic Acids Res. 15, 9109-9128[Abstract] |
27. | Zuker, M. (1989) Science 244, 48-52[Medline] [Order article via Infotrieve] |
28. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
29. | Negroni, M., Ricchetti, M., Nouvel, P., and Buc, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6971-6975[Abstract] |
30. | De Rocquigny, H., Gabus, C., Vincent, A., Fournie-Zaluski, M. C., Roques, B., and Darlix, J. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6472-6476[Abstract] |
31. | DeStefano, J. J., Buiser, R. G., Mallaber, L. M., Fay, P. J., and Bambara, R. A. (1992) Biochim. Biophys. Acta 1131, 270-280[Medline] [Order article via Infotrieve] |
32. |
Svarovskaia, E. S.,
Delviks, K. A.,
Hwang, C. K.,
and Pathak, V. K.
(2000)
J. Virol.
74,
7171-7178 |
33. |
Hwang, C. K.,
Svarovskaia, E. S.,
and Pathak, V. K.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
12209-12214 |
34. | Hiom, K. (2001) Curr. Biol. 11, 278-280 |