(Received for publication, February 20, 1997, and in revised form, May 4, 1997)
From the Departments of Biochemistry and Biophysics
and ¶ Medicine and the
Cancer Center, University of
Rochester, Rochester, New York 14642
We previously found that strand transfer by human
immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is
promoted at sites where RT pauses during synthesis. In this report,
strand transfer is measured within the 5 transactivation response
region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin
structure of TAR would induce RT pausing, promoting RNase H-directed
cleavage of the template and subsequent transfer at that site. We
further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created
a strong pause site for RT, but NC significantly promoted strand
transfer. The effect of NC is specific, since other single strand
binding proteins failed to stimulate transfer. In another unexpected
outcome, preferred positions of internal transfer were not at the pause
site but were in the upper stem and loop of TAR. Thus, we propose a new
mechanism for transfer within TAR described by an interactive hairpin
model, in which association between the donor and the acceptor
templates within the TAR stem promotes transfer. The model is
consistent with the observed stimulation of strand transfer by NC. The
model is applicable to internal and replicative end transfer.
Strand transfer is an essential step in the replication of retroviruses, including that of the human immunodeficiency virus type 1 (HIV-1).1 It involves the movement of a primer from one position on the viral genome and reannealing to a different position. In addition to transfer reactions from the ends of the viral genome that are required for replication (1-3), there is evidence that strand transfer occurs from internal regions of the viral sequences resulting in recombination (4, 5). This recombination occurs with high frequency during viral replication (4-7). We have previously demonstrated a positive correlation between internal strand transfer and mutagenesis during synthesis by the HIV reverse transcriptase (HIV-RT) in vitro (8). It appears that strand transfer resulting in mutations or homologous recombination is a likely source of some of the genetic variation that produces quasispecies of HIV. This genomic alteration impacts the efficacy of antiviral therapy against HIV, presumably by initiating the emergence of drug-resistant viral strains carrying mutations in their genome.
Internal strand transfers require the RNase H activity of HIV-RT to degrade the RNA template, called the donor, originally hybridized to the extended primer (9). This degradation promotes release of the primer terminus from the donor template so that it can transfer to an acceptor (10, 11). We have previously shown that there are "hot spots" in the viral sequence that promote pausing of HIV-RT, leading to increased strand transfer (8).
The process of strand transfer entails orderly reactions of dehybridization and reannealing of the partially elongated primer. We had previously proposed that a secondary structure on a template may pause the RT, causing more extensive degradation of RNA template around the pause site, thus allowing invasion of acceptor templates and leading to transfer (12). Therefore, it would be of interest to measure strand transfer from a donor template with a large hairpin structure.
It was also important to explore the role of nucleocapsid protein (NC) in the reaction. NC is closely associated with the genomic RNA in virions (13) and was found to strongly stimulate hybridization of single-stranded RNA and complementary DNA (14). Yet, NC was also found to be capable of destabilizing the double helix region of oligonucleotides (15). Therefore, given the opposing functions of NC on nucleic acids, we postulated that NC would be a physiological modulator of strand transfer, especially for templates with extensive secondary structures.
We chose the TAR region of HIV as the substrate for these measurements.
TAR is part of the repeat region (R region) of the HIV long terminal
repeat duplicated at the 5 and 3
end of the viral RNA. TAR is 59 nucleotides long and forms a stable hairpin structure. The 3-nucleotide
bulge on the upper stem of the hairpin binds an important viral
transactivator, Tat (16). Upon binding TAR, Tat substantially increases
the efficiency of transcription (17-19). Since the 5
TAR constitutes
the 5
end of the genome, TAR participates in the first strand transfer
reaction during reverse transcription. In this reaction, the nascent
strong stop DNA strand containing TAR is dissociated from the 5
end of
the (+)-strand RNA and rehybridized to the complementary sequence present at the 3
end. There is also evidence that primers that have
not fully copied TAR can transfer during viral replication, suggesting
an internal strand transfer mechanism (20). The latter is only a minor
pathway for replication (20, 21), but its frequency is high compared
with recombination in other parts of the genome (5).
Previous studies of the effect of NC on templates containing the R region (14) demonstrated major effects of NC on the kinetics of annealing between a single-stranded DNA and a single-stranded RNA. Additionally, substrates with the partial sequence of TAR were shown to be stimulated for strand transfer (22). Thus, features of the TAR region make it an ideal substrate for investigation of the effects of secondary structure on strand transfer.
Our results show that the structure of TAR causes a major pause in synthesis by RT, as we predicted. However, a surprising outcome from our experiments is that the pause site did not serve as a preferred transfer site, providing evidence for an alternative strand transfer mechanism. We offer an explanation for the stimulatory effect of NC and show that it cannot be produced by other single-stranded binding proteins. Based on the current data, we present a new model for the mechanism of strand transfer in TAR.
Materials
Recombinant HIV-RT (p66-p51 heterodimer) was provided by the
Genetics Institute (Cambridge, MA). The enzyme had a specific activity
of 40,000 units/mg. One unit is defined as the amount required to
incorporate 1 nmol of dTTP into nucleic acid product in 10 min at
37 °C using poly(rA)-oligo(dT) as template primer. HIV-NC was
chemically synthesized by the Louisiana State University Medical Center
Core Laboratories. The sequence of the mature NC used for synthesis was
that of the first 55 amino acids of the NC precursor protein described
by Khan and Giedroc (23). The peptide was kept under reducing
conditions, and aliquots were stored in 10% -mercaptoethanol. The
peptide concentration was determined by quantitative amino acid
analysis, performed by the Cornell University Peptide Facility. The
identity of the peptide was confirmed by amino acid composition
analysis. A molecular mass of 6444 daltons was determined by
electrospray mass spectrometry. Aliquots of both HIV-RT and NC were
stored at
80 °C, and a fresh aliquot was used for each experiment.
Escherichia coli single-stranded binding protein (SSB) was
obtained from U.S. Biochemical Corp. The recombinant human replication
protein A (RPA) was expressed and purified as described previously
(24). The Expand High Fidelity PCR system, used for all of our PCR
reactions, was purchased from Boehringer Mannheim.
pUC-BS-WT plasmid, which contains the HIV-1 long terminal repeat sequences, was given to us by Dr. Malcolm A. Martin (NIAID, National Institutes of Health). T4 DNA ligase, Taq DNA polymerase, T7 RNA polymerase, placental RNase inhibitor, RNase-free DNase I, dNTPs, rNTPs, restriction enzymes, and G25 and G50 Sephadex (RNA) columns were obtained from Boehringer Mannheim. T4 polynucleotide kinase and the DNA sequencing kit were obtained from U.S. Biochemical Corp. Radiolabeled compounds were from NEN Life Science Products. Epicurian Coli® SURE2 Supercompetent Cells and pBluescript® II SK+ vectors were obtained from Stratagene (La Jolla, CA). pGEM vectors were from Promega (Madison, WI). The plasmid purification kit was from Qiagen (Chatsworth, CA). The DNA primers were synthesized by Genosys, Inc. (Houston, TX). All other chemicals were from Sigma.
Methods
Generation of Donor and Acceptor RNA TemplatesThe donor template, BS-TAR, was made as follows: The PvuII-HindIII fragment of pUC-BS-WT plasmid was ligated into pBluescript II SK+, which previously had been digested with XhoI followed by a fill-in reaction with Klenow fragment and then digested with HindIII. The resulting plasmid, pBS-TAR, was transformed into competent cells. White colonies were selected, and the plasmid was amplified, harvested with a Qiagen plasmid purification kit, and quantitated by spectrophotometry. The plasmid with the correct insert containing the wild type TAR was identified by sequencing. pBS-TAR then was linearized by digesting with HindIII and transcribed in vitro, according to the Promega Protocols and Applications Guide as described before (8). The 166-mer RNA was gel-purified and was quantitated by hybridizing with a labeled DNA primer of known quantity, as described previously (30).
The acceptor template, GEM-TAR, was created by ligating the PvuII-HindIII fragment into a SmaI- and HindIII-digested pGEM®-7z+ vector. The plasmid construct, pGEM-TAR, was sequenced, amplified, purified, and quantitated as described above. It then was transcribed in vitro to create a 155-mer RNA that contains the TAR sequence.
The second donor template, TAR-PBS, was created by first amplifying the
plasmid, pBGUR, given generously by Dr. Stephen Goff, with two primers,
JK8 and JK9. The sequences for the two primers are as follows: JK8,
5-GGCCA GTGAG CGCGC GTAAT ACGAC TCACT ATAGG GTCTC TCTGG TTAGA CCAGA
TCTGA GCCTG GGAGC TCTCT GGCTAA-3
; JK9, 5
-CCTCG CCTCT TGCCG TGCGC
GCTTC AGCAA-3
. The amplified product contained a T7 promoter and the
wild type viral sequence from +1 to +276. The PCR product then was
digested with BssHII and cloned into
BssHII-digested pBluescriptIISK+. The resulting plasmid, pTAR-PBS, was then amplified and harvested as described above. The
template was designed such that the viral RNA to be used as a donor
template, TAR-PBS, started with the natural 5
end of TAR with no
plasmid sequence present.
The TAR-mtI acceptor template was made to contain point mutations
within TAR to probe the point of strand transfer. The point mutations
were designed such that the secondary structure of TAR was preserved.
To make such a template, four single-stranded DNAs were synthesized.
JK10 contains the T7 promoter, part of U3 and the first 34 nt of TAR
(5-CGGCC TAATA CGACT CACTA TAGGG TTGCC TGTAC GGGTC TCTCT GGTTA
AACCA GATCT GAGCC GCCCA GCTC-3
). JK12 has the
following sequence: 3
-TCA GCCGG ATTAT GCTGA GTGAT ATCCC AACGG
ACATG CCCAG AGAGA CCAAT TTGGT CTAGA-5
. The underlined
sequences indicate a region of complementarity between the two primers.
The two primers were annealed to create a double-stranded DNA with two
sticky ends. JK11 has the remaining TAR sequence and the additional 19 nt of the R region (5
-TCTGG TTAAC TAGGG
ATACCC ACTGC TTAAG CCACA ATAA-3
). JK11 was
then annealed to JK13 (3
-CTCGG ACCCT CGAG AGACC AATTG
ATCCC TATGGG TGACG AATTC GGTGT TATT TCGA-5
), whose sequence is partially complementary to JK11. Again, the
underlined regions indicate complementarity. The boldface letters are
point mutations, and the italicized letters indicate a point insertion.
The annealed JK10/JK12 and JK11/JK13 were ligated to each other, since
the last 14 nt at the 3
end of JK10 were complementary to the first 14 nt of the 3
end of JK13. The annealed and ligated product formed a
double-stranded DNA fragment that included a T7 promoter and the TAR
region with point mutations, with 5
and 3
ends ready to be ligated to
SfiI and HindIII sites, respectively. The
resulting DNA, dTAR-mtI, was inserted into a SfiI- and
HindIII-treated pGEM-9Zf(
) plasmid. Then the ligated plasmid pTAR-mtI was linearized by digesting with HindIII
and then transcribed in vitro to produce the 93-nt-long
acceptor RNA template with five specific point mutations.
A DNA primer
(5-CTAGTGGATCCCCCGGGCTG-3
), named JK1, was labeled at the 5
end
using T4 polynucleotide kinase and [
-32P]ATP (3000 Ci/mmol). Free, unincorporated nucleotides were separated by using
Quick Spin 228 G25 Sephadex RNA purification columns (Boehringer
Mannheim).
The labeled DNA primer was mixed with the RNA template at a 2:1 (primer/template) molar ratio in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 80 mM KCl. The primer and the RNA template were heated to 65 °C for 10 min and then slowly cooled to room temperature for annealing.
Primer Extension and Internal Strand Transfer AssayPrimer extension was performed in a final volume of 12 µl. Two units (50 ng) of reverse transcriptase (final reaction concentration of 35.6 nM) was preincubated for 5 min with 2 nM, in template termini, of primer-template at 37 °C in 50 mM Tris-HCl, pH 8.0, 80 mM KCl, and 1 mM dithiothreitol. The reaction was started by adding MgCl2 and dNTPs to a final concentration of 6 mM and 50 µM, respectively. It was terminated at various times by adding 1 volume of gel-loading buffer (90% formamide, 10 mM EDTA, pH 8.0, and 0.1% each of xylene cyanole and bromphenol blue).
Strand transfer reactions were also carried out in a final volume of 12 µl. The amount of primer-template and of RT was kept the same as in the primer extension reaction. The primer-template, and the acceptor in 40-fold excess, were preincubated for 5 min at 37 °C in the absence or the presence of NC in 50 mM Tris-HCl, pH 8.0, 80 mM KCl, 1 mM dithiothreitol. The amount of HIV-NC necessary to coat 100% of nucleic acids present in the reaction was calculated based on one molecule of NC binding to every seven nucleotides (25). HIV-RT was added and preincubated for 5 min further. The reaction was started, incubated for 20 min, and terminated as described above. In the reactions with RPA or SSB, the amount of RPA or SSB needed for 100% coating of templates in the reaction was calculated based on one molecule of SSB binding to every 65 nucleotides (26) and one molecule of RPA binding to every 30 nucleotides (24). The preincubation and reactions with SSB or RPA were carried out in the same way as with NC.
Analysis of Reaction ProductsExtended primers were separated electrophoretically on 8% denaturing polyacrylamide gels (19:1 acrylamide/bisacrylamide) with 7 M urea as described (27), visualized on a Kodak BioMax film (Eastman Kodak Co., Rochester, NY), and quantitated by using a PhosphorImager with ImageQuant software (Molecular Dynamics).
Experiments were designed to test the hypothesis that the unique secondary structure in the TAR region template influences the position and efficiency of internal strand transfer. Additionally, they test whether NC protein, known to stimulate annealing of TAR sequences, has a major impact on this process.
Primer Extension Catalyzed by HIV-RTWe have primed the
166-nt-long donor template, BS-TAR (Fig. 1), with a 5
end-labeled DNA primer and analyzed both the efficiency of primer
extension on the donor template and that of strand transfer to the
155-nt-long acceptor template, GEM-TAR. Both templates include the
complete TAR sequence, which serves as a region of homology between the
two templates. Homologous strand transfers are most likely to occur
within this region. In order for RT to synthesize a full-length
donor-directed product, it has to extend the primer through the
double-stranded stem-loop of TAR. During that process, encountering the
stable stem-loop structure was expected to make the RT pause, and
subsequently transfer the primer strand to the acceptor without
disturbing the stem region. Likewise, in order for the RT to transfer
the nascent DNA strand and to continue synthesis on the acceptor
template, the double-stranded region of TAR on the acceptor template
needs to be destabilized.
To examine whether the stem-loop structure of TAR affects
polymerization by RT, we analyzed the synthesis pattern by primer extension, in which the 5-end-labeled DNA primer is extended on the
RNA template, BS-TAR, in the absence of the acceptor (donor template in
Fig. 1). Results in Fig. 2 show a major pause site near
the base of the stem as we predicted. This was confirmed by sequencing
with the same primer in parallel. At earlier time points during the
extension, the major products formed by RT were those elongated up to
the base of the TAR hairpin loop (Fig. 2, P in lanes
1 and 2). Even at the later time points where the
full-length products were formed, the short products prematurely halted
at the base persisted (lanes 3, 4, and
5). We concluded that the secondary structure of TAR caused
RT to pause, which should promote strand transfer in the presence of
acceptor templates. Incidentally, we saw two other strong pause
products near the 166-nt full-length product. However, these pauses
occurred in the plasmid-derived sequences near the 5
end of the RNA
template, far past the region of homology containing the viral
sequence. In addition, the 5
regions of the donor and acceptor
template containing plasmid sequences do not share homology with each
other. Therefore, these pauses seen at the plasmid-derived sequences
were not likely to have affected the TAR-specific polymerization or the
strand transfer reactions below, catalyzed by HIV-RT.
Remarkably, in the presence of the trapping polymer heparin, which prevents rebinding of templates by RT, products included one that was 166 nt long, representing full-length synthesis (data not shown). This indicates that HIV-RT, although it usually pauses at the base of the helix stem and may dissociate, can at times synthesize completely through the TAR region at a single binding event.
Effect of NC on Strand TransferWe then examined the strand
transfer reaction in the presence and absence of NC (Fig.
3). NC coats the single-stranded RNA genome in a virion,
in a similar fashion as other single-stranded binding proteins. It was
shown to promote strand exchange, favoring the most stable duplex (15);
participate in dimerization of the viral RNA (28); unwind
tRNALys (23); and anneal tRNALys to viral RNA
(29). We reasoned that the known ability of NC to unwind and melt the
secondary structures would result in destabilization of the TAR
hairpin. The melting of the stem-loop in TAR should diminish the pause.
This would promote unhindered, continued synthesis on the donor, and
consequently there would be less opportunity for transfer.
To test this hypothesis, we performed strand transfer reactions using the donor BS-TAR primed with the same DNA primer mentioned above and the acceptor GEM-TAR. The acceptor template was designed so that the transfer product would be longer than the full-length extension product on the donor template. In this way, the two products could be readily differentiated by gel mobility.
We titrated the concentration of NC in strand transfer reactions, and calculated the transfer efficiency. The efficiency is calculated by dividing the amount of transfer product (Fig. 3, T) by the sum of products of strand transfer (T) and of full-length donor-directed synthesis without transfer (F): T/(T + F). At a high concentration of NC, formation of both F and T are inhibited (lane 1). This inhibition, observed at a level of NC greater than that necessary for 100% coating of the templates, has been observed previously (30). It is thought to result from co-aggregation of excess NC and templates, which may impede primer association or polymerization by RT. As we predicted, the pause site at the base of TAR faded as the concentration of NC increased (compare lanes 2 and 5, e.g.). This indicated to us that NC was active in its role of altering the template structure. The observation is also supported by previous studies by other investigators, who reported reduction of pausing in the presence of NC (35, 36).
The efficiency of strand transfer was greatly elevated at low concentrations of NC (lanes 2-5). The highest transfer efficiency was achieved at the concentration of NC that would coat 50% of the templates present. Particularly striking in the data presented in Fig. 3 is the degree of strand transfer enhancement. In the previous study using templates lacking large secondary structures, the magnitude of increase in the transfer efficiency by NC, when compared with reactions in the absence of NC, was approximately 50% (30). The quantitation of the strand transfer efficiency using the TAR-containing templates showed that NC stimulated the transfer by 300% in comparison with transfer in the absence of NC.
In our original hypothesis, the mechanism of strand transfer was based on sequential events of pausing of RT at the base of stem and degradation of the donor template at the pause site, followed by reannealing of the nascent DNA onto the acceptor. Accordingly, diminished pausing of RT in the presence of NC should lead to continuation of the donor-directed synthesis downstream of the pause site, thereby reducing the probability of transfer. The unexpected outcome of the increased transfer concurrent with the fading of the pause site on the donor led us to reconsider the fate of the DNA products whose synthesis was prematurely halted at the hairpin base. We considered two interpretations of this interesting observation. 1) NC could have promoted extension of the paused primer through the hairpin on the donor template. This would suggest that the increase in transfer is not related to the pause. 2) NC could have facilitated transfer of the primer from the pause site. The presence of NC would then stimulate the transfer of the paused product at the base of the stem and continued extension on the acceptor. This would reconcile the disappearance of the prematurely terminated products at the pause site with an accompanying increase in transfer.
Time Course of NC-stimulated Strand TransferWe conducted a
time course experiment of strand transfer in the presence and the
absence of NC to further characterize the kinetics of the NC-mediated
strand transfer within TAR (Fig. 4). The reaction
conditions were kept the same as before, and the efficiency of strand
transfer in the presence of NC was compared with that in the absence of
NC. Throughout the time course, the amount of transfer product in the
presence of NC was consistently higher than in its absence. The
increase in the formation of transfer product in the presence of NC
could be based on various effects of NC on the kinetics of the transfer
reaction. The general stimulatory effect of NC on synthesis might be
its predominant effect during the reaction. This would result in a
general increase in the number of primers initiated and, accordingly,
in the number of synthesis products overall, including the transfer
product. In such a case, the ratio of T to T + F would remain the same, i.e. the efficiency of
strand transfer would be unaffected with or without NC. However, we
found that the efficiency of strand transfer in the presence of NC was significantly higher at all time points when compared with
that in the absence of NC. Of the primers initiated for synthesis, more
transferred onto the acceptor instead of completing synthesis on the
donor template in the presence of NC throughout the time course.
An alternate explanation for the observed increase in the amount of the transfer product could be that the effect of NC was specific to the process of strand transfer, possibly facilitating the rate-limiting step(s) of strand transfer. If NC accelerates specifically the process of the strand transfer, the stimulatory effect of NC should be the greatest in the earlier time points. At these times, in the absence of NC, full-length donor-directed product would appear before the transfer event and subsequent transfer product synthesis could be completed. If NC eliminates a delay in the transfer event, both donor- and acceptor-directed full-length products would appear at similar rates. However, at later time points, RT would have had a sufficient time to synthesize full-length transfer product, so the increase in transfer efficiency caused by NC would be less pronounced. In fact, the NC-mediated transfer enhancement was more pronounced at earlier time points. For example, at 5 min, the efficiency of transfer in the presence of NC (lane 8) was more than 7 times higher than that in the absence of NC (lane 18), whereas after 20 min the efficiency was 3-fold greater in the reaction with NC (lane 10) than without NC (lane 20). At the two earliest time points, 40 s and 2 min, the transfer product is barely formed; thus, the efficiency of strand transfer could not be accurately measured.
RNase H Activity of HIV-1 RT on TAR-derived Templates during PolymerizationWe have shown that the secondary structure of TAR
pauses the HIV-RT during polymerization and that NC specifically
increases the efficiency of strand transfer catalyzed by HIV-RT. If
transfer occurs at the base of the hairpin, there should be enhanced
RNase H-directed cleavage at this site. The coupled RNase H activity of
the RT would cleave approximately 18 nucleotides upstream from the
polymerase active site (31). Assuming the polymerase contacts and
pauses adjacent to the base of the TAR structure, a primary cleavage product should be formed that is approximately 125 nucleotides long, from the 5-end-labeled donor template (Fig.
5A).
Fig. 5B shows a result consistent with our prediction. In
fact, the band corresponding to a 123-nt-long segment was the major product of RNase H activity. The reaction conditions were unchanged from the strand transfer reactions, except that the radioactive label
was on the 5 end of the donor RNA instead of the DNA primer. One can
see from the data that the template in the presence of NC is degraded
faster than in the absence of NC. This finding is consistent with a
previous report by Benkovic and colleagues (22), who showed that NC
enhanced the RNase H activity of the RT during strand transfer.
However, in contrast to their result regarding the specificity of RNase
H activity, we did not see any change in the cleavage pattern in the
presence of NC, measured within the limits of our assay system.
The increase of strand transfer in the presence of NC
might be due solely to the helix destabilization effects exhibited by all single-stranded binding proteins or a result of binding features unique to NC. To distinguish these possibilities, we measured the
effects of two other well characterized single-stranded DNA binding
proteins, E. coli SSB and human RPA, in the strand transfer reaction (Fig. 6). The concentrations of the proteins
were titrated down from 100 to 0% coating level. The efficiency of
strand transfer was then quantitated and compared with that of strand
transfer in the presence of NC. The results showed that neither RPA nor SSB stimulated strand transfer (lanes 1-6 and
7-12). In fact, the presence of either protein was
inhibitory to strand transfer. As the concentration of each protein was
decreased, the transfer efficiency improved. However, the amount of
primers fully synthesized to the end of the donor template remained
little affected or slightly increased, since the single-stranded
binding proteins are generally known to be stimulatory to DNA
synthesis. It is also interesting to note that the pause site near the
base of the stem remained unchanged at different coating levels of RPA
or SSB, whereas the increase in the NC concentration corresponded with
the decreased pause site. This may be due to specific interactions of
NC with the viral sequence, affecting the secondary structures of TAR, that are not exhibited by RPA or SSB. Although RPA and SSB were reported to enhance strand displacement from templates containing the
HIV-1 long terminal repeat (32), the two proteins were unable to
increase the strand transfer reaction of TAR. The inability of the
either protein to affect the transfer process indicates that a simple
destabilization or displacement of hybrid strands within the TAR
structure cannot be solely responsible for the observed enhanced rate
of transfer by NC.
Determination of Points of Transfer
It was important that we
define the molecular basis for strand transfer reactions within TAR to
further elucidate the positive effect of NC on the reaction. In an
attempt to understand how the transfer actually occurs, we mapped the
sites of transfer. Results described above were consistent with
transfer driven by the structure-related pausing of RT, followed by
degradation of the donor and invasion of the acceptor at the cleavage
site. To demonstrate that the base of TAR at which HIV-RT paused during synthesis correlated with the point of transfer of the elongated primers, we modified and applied the system used by Wu and colleagues, in which one could determine where the transfer occurred by sequencing the transfer product (8). First, we created a two-template system,
similar to the one used for the experiments above, but the new acceptor
now contained point mutations throughout the template, which would
allow us to determine the point of transfer by examining the sequence
of the hybrid transfer product, as described under "Experimental
Procedures" (Fig. 7A). The mutations were carefully chosen so that the overall secondary structure of TAR could
be maintained (Fig. 7B).
Using the new templates, we performed strand transfer and isolated the
transfer product. The single-stranded DNA product was converted to
double-stranded DNA, amplified by PCR, and cloned into a vector,
according to procedures previously reported (8). To decrease possible
mutations made by the Taq polymerase during PCR, a
commercially available "high fidelity" PCR set of Taq
combined with a 3
5
exonuclease was used. Thirty clones
representing individual strand transfer products were sequenced. We had
expected that most of the transfer products would be chimeras
consisting of the donor sequence from the 5
end of the product up to
the base of TAR and then the acceptor sequence from the base of TAR to
the 3
end, as a result of transfer at the pause site near the
base.
However, sequencing of the transfer products revealed yet another set
of surprising outcomes (Table I). Out of 30 transfer products sequenced, a majority (22 clones) were a result of transfer between positions 4 and 5, suggesting an end transfer. This is a
reaction that occurs during viral replication and is expected to be
efficient (2, 20, 21). Despite the presence of the secondary structure
at the 5 end of the donor, RT seems to transfer very efficiently after
synthesizing up to the end. The remaining seven clones, representing
about 23% of the transfer products, were a result of internal
transfer. Unexpectedly, there was no strong correlation between the
major pause site and the point of transfer. If the pause site were the
site of transfer, then we would have seen most of the transfer products
with marker mutations at positions 2, 3, 4, and 5. However, such a
product was seen in only 1 of 30 clones. Most of the internal transfer
products were found to contain markers at positions 3, 4, and 5. These data tell us that although the stem-loop of TAR created a very strong
pause site for the RT, most of the internal transfer occurred beyond
that site and well into the TAR structure, spanning the region around
the loop and the upper stem. This result suggested a different
mechanism altogether for the internal transfer of the TAR-derived
templates. Based on the new findings, a mechanistic model for the
transfer within TAR is proposed and discussed below.
|
We previously found a positive correlation between sequence-dependent pausing of RT and the frequency of strand transfer, using HIV-nef-derived templates (8). Based on this observation, our original hypothesis for the mechanism of internal strand transfer in the HIV TAR region was that the stable secondary structure halts the RT, which then extensively degrades the RNA donor around the pause site, freeing the nascent DNA primer for transfer. In this report, we demonstrate that strand transfer involving the TAR structure is not correlated with the pausing of RT. Most likely, pausing-related transfer still occurs but accounts for a minority of transfer events in this case. This surprising and revealing outcome suggests that an alternative mechanism is driving most strand transfer from this region.
When transfer positions were determined in TAR by point mutant analysis, only 1 of 30 clones represented the transfer product derived from internal transfer occurring near the pause site. Most of the internal transfer took place in the upper stem region of the prominent hairpin. The new finding suggests that the main impetus for strand transfer is most likely a direct interaction between the donor and the acceptor templates via complementarity of their stem structures. This assertion is also consistent with the decreased intensity of the product P in parallel with the increased efficiency of strand transfer in the presence of NC, as we observed above. The known ability of NC to melt and reanneal nucleic acids may help association between the complementary regions of the stem in the two templates. Upon binding of the acceptor to the donor, the local concentration of the acceptor is greatly increased, promoting the transfer.
Additionally, our results show that the dominant position of strand
transfer in the R region is at or near the very end of the donor
template, in accordance with the accepted model of reverse transcription (2). This observation is consistent with recent results
from the cell culture-based study of the minus strand strong stop
transfer of Moloney murine leukemia virus (21), showing the great
majority of transfer from the very 5 end.
Based on our sequencing data and the observations we have made with NC
above, we propose a model for the mechanism of strand transfer in TAR
(Fig. 8). In this "interactive hairpin model" the
intimate interaction between the two hairpins is the driving force
facilitating the transfer. As the primer is elongated through the TAR
region, the double strand of the stem opens up (Fig. 8, step
B). The displaced 5 single-stranded region of the donor hairpin
then binds the complementary sequence on the acceptor stem (step
C). The association between the donor and the acceptor templates
increases the local concentration of the acceptor. As the growing DNA
primer unwinds the hybrid between the two templates (step
D), the displaced acceptor strand binds this nascent DNA region
via complementarity (step E). Binding of DNA and the
acceptor RNA results in the transfer of the growing end of the nascent strand (step F). Thus, because of the extensive
intermolecular binding between the two hairpins, the internal transfer
site mainly occurs within the upper stem region of TAR.
The modest stimulation of strand transfer of about 50% by NC on substrates lacking large hairpins was interpreted to be the result of two opposing phenomena (30). NC stimulated synthesis on the donor template and was thought to cause each RT to have a shorter time to carry out the transfer reaction. However, the strand exchange properties of NC were expected to promote the transfer step. In TAR, the large 300% stimulation by NC is consistent with a different mechanism of transfer, as outlined in our proposed model.
Dynamic melting and annealing steps are central elements of the model.
Therefore, NC, which facilitates these reactions, should have a
substantial influence. For example, a report from De Rocquigny et
al. (29) demonstrates that NC stimulates binding of the tRNA primer to PBS. A similar type of activity in promoting RNA-RNA association can be at work during step C, in which the donor and the
acceptor bind. In addition, You and McHenry (14) have reported that NC
also greatly enhances hybridization between the DNA and RNA strands
containing TAR. The transition from step D to E, where the first
association between the nascent DNA and the acceptor RNA occurs, can be
facilitated by the same type of annealing activity. In addition, it is
possible that NC stimulates step C to precede step B; in other words,
the equilibration and base pairing of the donor with the acceptor may
occur before RT reaches the stem, leading to the displaced 3 segment
of the donor stem, and facilitating synthesis on the donor up to the
loop region. Therefore, the contribution of NC in this model is that it
reinforces the central feature of the strand transfer reaction, namely
the interaction between the hairpins.
The interactive hairpin model can also be offered to provide
mechanistic details for the first jump in the endogenous reverse transcription of HIV-1 in an infected cell. This replicative end transfer is the dominant reaction in vivo (20, 21). One of the advantages of the second set of our substrates, TAR-PBS and TAR-mtI, is that they simulate the parts of the viral genome
participating in the first strand transfer. TAR-PBS as the donor
represents the 5 end of the viral RNA from which the minus strand
strong stop DNA is synthesized, and TAR-mtI as the acceptor represents, despite the five marker mutations, the viral sequences of the 3
end of
the genome, to which the minus strand strong stop DNA transfers. The
first strand transfer, in addition, is also reported to be mainly an
intermolecular event in vivo (33). Thus, the information we
have extracted from the study with our TAR-derived, two-template system
serves as a good model for the first jump reaction in vivo.
Accordingly, our model can offer possible mechanistic steps of the
dominant end transfer as well; after the donor and the acceptor have
been brought together by base pairing at step C, the RT can copy all of
the way to the 5
end of the donor, completely displacing the acceptor
from the donor. Primer transfer can occur immediately after the
acceptor is displaced from the donor. While the donor and acceptor
templates are no longer annealed at the moment of transfer, they should
be in close proximity. This proximity could last for the appropriate
period of time for transfer in the confined space within which
replication occurs in vivo.
The TAR region has a central role in the viral life cycle. First, TAR
RNA acts as a cis-acting regulatory element that binds the
viral transactivator, Tat (16). As the cellular RNA polymerase transcribes the 5 TAR, the binding of Tat to the stem-loop
dramatically increases the rate of transcription of the viral genome
(34). Second, the TAR region is involved in the first strand transfer in the early steps of the viral replication. It is located within the R
regions that are duplicated in the 5
and 3
end of the genome. During
the first strand transfer, the nascent strong stop DNA is relocated
from the 5
end of the viral RNA to the R at 3
end to complete the
synthesis of the minus strand DNA. This translocation allows
duplication of the U5 sequence at both ends of the viral DNA. Given the
vital roles of TAR, it seemed remarkable that TAR consists of a
stem-loop structure that can potentially hinder efficient
template-directed synthesis and reannealing after the first jump. In
fact, we see evidence that the presence of TAR causes the polymerase to
pause and compromises the efficiency of synthesis (Fig. 2). However,
the interactive hairpin model suggests that the stem-loop at both ends
of the genome has evolved to facilitate the strand transfer and
subsequent viral replication by orderly base pairing among the involved
strands.
Construed from the model is that this stepwise association between the templates and the nascent DNA is further expedited by specific functions of NC. The specificity of the role of NC in the transfer reaction is supported by the observations that other well characterized single-stranded binding proteins failed to produce a similar stimulatory effect on the efficiency of transfer (Fig. 6). E. coli SSB and its human homolog, RPA, are known to melt secondary structures and stimulate polymerases. However, strand transfer of TAR does not seem to depend simply on helix destabilization. There must be an active association between the two templates, accomplished by the inherent sequence complementarity of TAR as well as concerted actions of the HIV-RT and NC.
We feel that our proposed model correctly describes parameters of strand transfer observed in TAR in vitro and is sufficiently flexible to account for recombination in TAR and steps in the first jump in vivo. It is also a general mechanism for recombination that can occur anywhere in the viral genome where there are large hairpins. Although fully consistent with our results, the model is new and requires additional testing to verify the proposed steps. We are currently devising experiments to detect intermediate recombination structures predicted by the model. We are also attempting to dissect the contribution of the TAR sequence and that of the TAR structure in determination of pause sites and the efficiency of strand transfer. It would be interesting to see whether the model can predict the site of pausing and transfer in other substrates with similar secondary structure. Variation of the stem size, thus modulating the degree of interaction between the donor, the acceptor, and the nascent DNA, should titrate the efficiency and sites of transfer.