From the Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742
Received for publication, November 18, 2002, and in revised form, February 14, 2003
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ABSTRACT |
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An in vitro strand transfer
assay that mimicked recombinational events occurring during reverse
transcription in HIV-1 was used to assess the role of nucleocapsid
protein (NC) in strand transfer. Strand transfer in highly structured
nucleic acid species from the U3 3' long terminal repeats,
gag-pol frameshift region, and Rev response element were
strongly enhanced by NC. In contrast, weakly structured templates from
the env and pol-vif regions transferred well
without NC and showed lower enhancement. The lack of strong polymerase
pause sites in the latter regions demonstrated that non-pause driven
mechanisms could also promote transfer. Assays conducted using NC zinc
finger mutants supported a differential role for the two fingers in
strand transfer with finger 1 (N-terminal) being more important on
highly structured RNAs. Overall this report suggests a role for
structural intricacies of RNA templates in determining the extent of
influence of NC on recombination and illustrates that strand transfer
may occur by several different mechanisms depending on the structural
nature of the RNA.
All retroviruses are characterized by reverse
transcription, which involves the conversion of viral RNA into
double-stranded DNA that becomes integrated into the host cellular
chromosome. This process is initiated from a nucleoprotein complex in
the virion core that is primarily composed of the dimerized diploid RNA
genome coated with nucleocapsid protein
(NC)1 (1, 2). In addition the
viral enzymes such as reverse transcriptase (RT), integrase (IN), and
protease (PR) are present along with host tRNA molecules. RT carries
out DNA synthesis during reverse transcription whereas NC serves as a
co-factor in this process and others (see below). Nucleocapsid protein
is a highly basic, positively charged protein, comprised of 55 amino
acid residues. It is derived by proteolysis of the gag and
gag-pol precursor polyprotein (3, 4). All retroviral NC
proteins contain either one or two conserved 14-residue invariant
motifs,
Cys-X2-Cys-X4-His-X4-Cys, where X denotes variable amino acids. They are also known as
the zinc finger motifs, because they bind zinc ions strongly (5-7). The two zinc fingers of HIV are in close spatial proximity (8-10) and
have similar structures (11) but are not functionally equivalent (12,
13). NC exhibits a multitude of essential functions in the life cycle
of HIV (14-18) that make it an ideal target for drug therapy (19-21)
and vaccine development (22). It has been shown to unwind (17, 23) and
anneal the tRNALys primer to the RNA genome (24-26). NC
also participates in recognition and packaging of the viral genome (13,
27-29), in the maturation of genomic RNA dimer (30-32), and possibly
in integration of the double-stranded DNA into the host genome (33). In
addition, NC serves as a nucleic acid chaperone (14, 34) capable of destabilizing secondary structures and enhancing the annealing of
complementary nucleic acids.
Previous studies have demonstrated that one or more of this array of
functions enables NC to enhance strand transfer (a process that leads
to recombination) during HIV replication (35-37). Strand transfer
involves the switching of DNA being synthesized on one RNA template
(referred to as "donor") to homologous regions on the same or on a
second RNA template (referred to as "acceptor") where the synthesis
continues. There are two obligatory strand transfers that occur at the
termini of the viral RNA (38-40). These are integral steps without
which the viral replication cannot proceed to completion. In addition
to these vital events, the virus can also undergo strand transfers at
internal regions of the RNA template that can potentially occur at any
position along the genome and during the syntheses of both minus and
plus DNA strands (41-43). These are important steps that aid in
generating genetic diversity in the viral population (39, 40, 44, 45) and allow some viruses to evade host immune response and drug therapy.
They also may increase the probability of successful DNA synthesis by
providing a salvage pathway for broken or damaged genomes (38).
NC has been shown to enhance the two obligatory transfers (35, 37, 46,
47), as well as internal strand transfer events (15, 36, 48, 49). The
mechanism by which NC protein promotes these processes is not yet
completely understood but presumably stems from a combination of the
nucleic acid annealing and helix destabilization activities of NC (36,
37, 50-53). You and McHenry (37) have shown that NC stimulates the
annealing of the HIV repeat region sequences by 3000-fold and
consequently accelerates strand transfer of terminally redundant
sequences. Tsuchihashi and Brown (51) have shown that NC promotes
strand exchange between double-stranded and single-stranded molecules.
Others (15) have shown that NC facilitates strand exchange between
donor and acceptor RNAs using model retroviral replication
intermediates. A number of other nucleic acid-binding proteins have
been shown to facilitate melting and annealing reactions in a similar
fashion. Escherichia coli single-stranded
DNA-binding (SSB) causes a reduction in the electrostatic repulsion of
negatively charged DNA, melts out intramolecular structures, and hence
enhances renaturation (54). RNA-binding proteins like heterogeneous
nuclear ribonucleoprotein A1 also accelerate annealing and strand
exchange (55).
Though a majority of earlier reports on strand transfer have elaborated
upon the activity of NC in highly structured RNA templates, relatively
little is known about the effects on RNA templates with weak or no
structure. In this report we set out to address two specific questions.
(i) Presuming that strand transfers occur all along the genome (41),
does NC exhibit a consistent or a differential influence on strand
transfer along the length of the viral genome? For example, in regions
with strong secondary structures, NC may be required to unwind these
structures to facilitate association of the nascent DNA and acceptor
RNA, whereas in regions relatively devoid of stable structures, NC may
exert only a minimal influence on strand transfer. (ii) Do the zinc
fingers of NC contribute equally or differentially toward strand
transfer on various regions of the genome? This question stems from the
apparent functional non-equivalence of the two HIV NC zinc fingers
noted above. To understand these aspects of NC, strand transfers in
five different regions of the HIV genome were analyzed; three of these
regions were highly structured whereas two had relatively weak
structures. Two of the highly structured regions, the
gag-pol frameshift region and Rev response element (RRE),
are important HIV regulatory sites. Results demonstrated that even in
the absence of NC a relatively high rate of strand transfer occurred in
regions with little structure compared with more structured regions.
However, NC markedly stimulated the latter in comparison to the former.
To evaluate the contributions made by the zinc fingers, the activity
levels of mutant NC proteins containing duplicate copies of finger 1 (N-terminal zinc finger) or finger 2 (C-terminal zinc finger) on high
or low structure RNA was tested. Results were consistent with finger 1 possessing a larger portion of the protein's helix-destabilizing
activity. Overall, this report establishes the differential effect of
NC on strand transfer on various regions of the HIV genome.
Plasmid pNL4-3 obtained from the NIH AIDS Research and
Reference Reagent Program contains a complete copy of the HIV-1 genome derived from strains NY5 and LAV (56). PCR primers were obtained from
Integrated DNA Technologies, Inc. Recombinant HIV-RT was graciously
provided by Genetics Institute (Cambridge, MA). This enzyme has a
specific activity of ~40,000 units/mg (one unit of RT is defined
as the amount required to incorporate 1 nmol of dTTP into nucleic acid
product in 10 min at 37 °C using oligo(dT)-poly(rA) as
primer-template). The enzyme contained very low levels of
single-stranded nuclease activity, which was found to be inhibited by
including 5 mM AMP in the assays (57). At this
concentration, the AMP did not affect the polymerase or RNase H
activity of RT. Aliquots of HIV-RT were stored frozen at PCR Amplification of DNA Substrates--
Five different sets
(four primers per set) of PCR primers were specifically designed to
yield donor and acceptor RNAs that shared homology over a 150-base
region and amplified DNA from five different areas of the
pNL4-3 plasmid (see Table I).
An SP6 promoter sequence was included on some of the primers to allow transcription of the DNA by SP6 RNA polymerase. PCR reactions were
performed according to the enzyme manufacturer's protocol using the
provided buffer. One hundred pmol of each primer was used. The
following cycling parameters were used: 35 cycles of denaturation,
annealing, and extension at temperatures of 94, 50, and 72 °C,
respectively, for 1 min each, one cycle of extension at 72 °C for 5 min. The PCR products were purified on 8% native polyacrylamide gels
(29:1) (acrylamide:bisacrylamide) and used to prepare RNA as described
below.
Preparation of RNA Substrates--
Run-off transcription
(performed according to the enzyme manufacturer's protocol) was
conducted using ~5 µg of the purified PCR DNAs and SP6 RNA
polymerase enzyme to generate donor RNA transcripts of 175 nucleotides
and acceptor RNA transcripts of 177 nucleotides. The transcription
reactions were treated with 0.4 units/µl of DNase I-RNase-free enzyme
for 15 min to digest away the template DNA. Then they were extracted
with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with
ethanol. The RNA pellets were resuspended in 50 µl of RNase-free
water, loaded onto hydrated Sephadex G-25 spin columns, and processed
according to the manufacturer's directions. The amount of recovered
RNAs was determined spectrophotometrically from optical density.
RNA-DNA Hybridization--
DNA primers that bound specifically
to the donor RNA transcripts were 32P-labeled at the 5' end
with T4 polynucleotide kinase according to the manufacturer's
protocol. Each of the five donor RNAs was hybridized to a complementary
labeled primer by mixing primer:transcript at an ~5:1 ratio in 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 80 mM KCl, and 0.1 mM EDTA (pH 8.0). The mixture
was heated to 65 °C for 5 min and then slowly cooled to room temperature.
Time Course and NC Titration Reaction--
Donor RNA-primer DNA
hybrids (2 nM final concentration of RNA) were preincubated
for 3 min in the presence or absence of 10 nM acceptor RNA
template and NC (as indicated) in 42 µl of buffer (see below) at
37 °C. Two molecules of NC per nucleotide were used in the
reactions, i.e. final concentrations of 1.16 and 0.27 µM of NC were used in reactions with or without acceptor
RNA templates, respectively. The reactions were initiated by the
addition of 8 µl of HIV-RT at a final concentration of 2.5 units/µl. The following reagents at the indicated final
concentrations were also included in the reaction mixtures: 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 80 mM KCl, 0.1 mM EDTA (pH 8.0), 6 mM
MgCl2 (53), 100 µM dNTPs, 5 mM AMP (pH 7.0), 25 µM ZnCl2, and
0.4 units/µl RNase inhibitor. Reactions were allowed to incubate for
time points of 0, 2, 4, 8, 16, 32, and 64 min at 37 °C. At these
time points, a 6-µl aliquot of each reaction was terminated by mixing
with 4 µl of a solution containing 25 mM EDTA (pH 8.0),
2.5 ng of RNase-DNase-free enzyme and allowed to digest for 20 min at
37 °C. Two µl of proteinase K at 2 mg/ml in 1.25% SDS, 15 mM EDTA (pH 8.0), and 10 mM Tris (pH 8.0) was
then added to the above mixture, which was placed at 65 °C for
1 h. Finally, 12 µl of 2× formamide dye (90% formamide, 10 mM EDTA (pH 8.0), 0.1% xylene cyanol, 0.1% bromphenol
blue) was added to the mixture, and the samples were resolved on an 8%
denaturing polyacrylamide gel containing 7 M urea. Similar reactions were conducted during the analysis of mutant NC proteins (1.1 and 2.2). In the NC titration experiments the total reaction volume was
reduced to 12.5 µl, the amount of NC in the reactions was varied as
indicated in Fig. 7, and the reactions were allowed to proceed for 32 min. Extended DNA products were quantified by phosphorimager analysis
using a GS-525 phosphorimager from Bio-Rad.
Gel Electrophoresis--
Denaturing 8% polyacrylamide gels
(19:1) (acrylamide:bisacrylamide), containing 7 M urea, and
native 8% polyacrylamide gels (29:1) (acrylamide:bisacrylamide) were
prepared and subjected to electrophoresis as described (59).
Strand Transfer Assay--
The general approach used to test for
strand transfer in the different regions of the HIV genome is depicted
in Fig. 1. This assay is designed to
simulate internal strand transfer events occurring during minus strand
DNA synthesis. The donor (template on which DNA synthesis initiates)
and acceptor (template to which DNAs initiating on the donor can
potentially transfer) represent the two strands of viral genomic RNA in
the virion. DNA synthesis is initiated from a 5' end-labeled DNA primer
that was specifically designed to bind only to the 3' end of the donor
RNA. Strand transfer can occur over the transfer zone, which is the
region of homology between the donor and acceptor RNAs. Primer
extension to the end of the donor produces a 175-base full-length
donor-directed DNA product. Strand transfers result in 197-base
transfer DNA products that includes the additional non-homologous bases
at the 5' end of the acceptor RNA. The 5' end of the donor outside the
transfer zone is not homologous to the acceptor, which prevents DNAs
extended to this region from transferring. This limits the system to
analysis of internal transfer events between the donor and acceptor
rather than events occurring from the template termini. The difference in the lengths of the two DNA products allowed us to quantify strand
transfer events in donor-acceptor pairs from five different regions of
the HIV genome (Figs. 2). It is also
possible for strand transfer to occur between two donor RNAs in the
reaction. This type of transfer was not quantified in the experiments.
However, it probably represents only a small amount of the total
transfer, because reactions included a 5-fold excess of acceptor over
donor and also many of the donors would not be available, because they are used for DNA synthesis.
The percent transfer efficiency was defined as transfer DNA products
(T)/transfer + full-length donor-directed products (F), times 100 ((T/(T+F)) × 100). The number reflects the proportion of DNA primers extended to the end of the acceptor versus
those extended to the end of the donor. This representation of the
data, as opposed to simply determining the gross level of transfer
product, expresses transfer relative to total DNA extension. Therefore, differences in the total amount of primers extended with the various substrates used are compensated for.
Prediction of Secondary Structures of the RNA
Substrates--
RNAdraw (61)
(iubio.bio.indiana.edu/soft/molbio/ibmpc/rnadraw-readme.html)
and Zuker's RNA folding programs (62-64)
(www.bioinfo.rpi.edu/applications/mfold/) were used to
predict the secondary structures for the RNA substrates (both donor and
acceptor) utilized in this report. Fig. 2 shows the structures of only
the donor RNAs derived from the U3 3'LTR (A),
gag-pol (B, referred to as GagPol substrate), RRE
(C), pol-vif (2D, referred to as
PolVif substrate), and env (E, referred to as Env
substrate) regions. In each region, the acceptor RNAs were predicted to
have structures similar to the donor (acceptor structures not shown). A
highly negative Effect of NC Protein on Strand Transfer in Highly Structured RNA
Substrates--
In this section, autoradiograms of strand transfer
assays conducted on three RNA substrates are shown in Fig.
3, and their corresponding graphical
quantitations are shown in Fig. 4. The first pair of donor-acceptor RNA substrates we tested were from the U3
3'LTR region (corresponding to bases 9074-9265 of the HIV-1 genome as
derived from plasmid pNL4-3 (56); see "Experimental Procedures").
This region folded to form a complex structure with several strong stem
loops and a
Transfer efficiency results from the above experiment are shown
graphically in Fig. 4A. It is evident from this graph that NC enhances strand transfer significantly in this region of the genome.
Each of the experiments was repeated two or more times to confirm the
observed trends. Experiments presented are representative of the observations.
The second pair of donor-acceptor structured RNAs examined was from the
gag-pol region (bases 2009-2200,
The third donor-acceptor pair of structured RNAs examined was from the
RRE region (bases 7801-7992,
As with the other structured RNAs examined above, NC enhanced strand
transfer in the RRE region that was accompanied by chasing of the major
paused DNA products. The predicted secondary structures seemed to
obstruct RT as indicated by the presence of pause sites. Moreover, the
disappearance of pause sites correlated with the presence of acceptor
and was further enhanced by NC (Fig. 4C). The results
suggest that the pause sites are probably focal points for strand
transfers in these particular structured RNAs. Note that this does not
imply that all transfers from the RNAs occur from the pause sites, only
that transfer is exaggerated from these positions.
Effect of NC Protein on Strand Transfer in Weakly Structured RNA
Substrates--
In this section, autoradiograms of strand transfer
assays conducted on two RNA substrates are shown in Fig.
5, and their corresponding graphical
quantitations are shown in Fig. 6. The
first donor-acceptor pair of RNA substrates tested was from the
pol-vif region of the viral genome (bases 3419-3610,
Similar results were obtained with the second low structure
donor-acceptor pair of RNA substrates from the env region
(bases 7079-7270, Effect of Varying Concentrations of NC on Three RNA
Substrates--
We conducted strand transfer assays with increasing
concentrations of wild type NC protein on three of the substrates: U3 3'LTR, GagPol, and PolVif (Fig.
7A). NC concentrations were
increased from 0.0625 to 4 µM. Because 32 min was
sufficient to obtain adequate levels of reverse transcription and
strand transfer, reactions were allowed to proceed only to this time
point. The results are presented graphically in Fig. 7B.
Both the U3 3'LTR and GagPol regions showed a significant increase in
transfer efficiency as the concentration of NC was increased. The
efficiency of transfer approximately doubled with GagPol from 0 to 1 µM NC whereas U3 3'LTR increased by about one-third. No
further increase was observed beyond 1 µM NC. In
contrast, the less structured PolVif region showed a higher level of
strand transfer without NC and increased by a relatively small amount
when NC was included. Levels of efficiency at 1 µM NC
were comparable for PolVif and U3 3'LTR regions whereas the GagPol
region peaked at a lower level. These observations show that NC
enhances transfer in a concentration-dependent manner and
to a greater extent on RNAs with more structure.
Effects of Mutant NC Proteins on Env and GagPol RNA
Substrates--
Two different mutants of NC protein were tested to
determine their effect on transfer from structured (GagPol) and
non-structured (Env) regions. These were finger mutants in which one of
the two NC zinc finger was replaced with the other (12). HIV NC has two
non-identical zinc fingers, a N- and a C-terminal finger, denoted 1 and
2, respectively. In mutant 1.1, finger 1 replaces the finger 2 giving
this protein two copies of finger 1. In 2.2, finger 2 replaces finger 1 giving this protein two copies of finger 2. Recent work in our
laboratory2 has suggested
that the fingers possess biased functional activities with finger 1 containing more nucleic acid unwinding activity and finger 2 having
more annealing activity. Clearly, strand transfer is a complex process
that requires both activities for maximal stimulation (see
"Discussion"). However, by using substrates with vastly different
levels of structure we reasoned that it might be possible to uncover
the properties of the mutants. Presumably, transfer on the highly
structured GagPol substrate would require more unwinding activity to
destabilize structures in the transferring DNA and acceptor template.
Transfer on the Env substrates should be less dependent on unwinding
activity. Shown in Fig. 8A are strand transfer experiments with wild type or mutants 1.1 or 2.2 NC,
performed on the Env substrates. Plots from this experiment are shown
in Fig. 9A. Wild type and 1.1 showed comparable levels of transfer on this low structure substrate
whereas 2.2 enhanced transfer somewhat better than the others. This was
in contrast to strand transfer on the highly structured GagPol
substrate (see Fig. 8B and Fig. 9B). In this case
2.2 was the least stimulatory whereas 1.1 and wild type were
comparable. The results are consistent with 2.2 retaining annealing
activity but having less unwinding activity than 1.1 or wild type. This
supports a role for finger 1 in unwinding. The role of finger 2 in
annealing is less clear from these experiments, because the mutant
without finger 2 (1.1) was nearly as good as wild type on both
substrates.
In this study, an in vitro assay that simulates
internal strand transfer events occurring during minus strand DNA
synthesis was used. The substrates tested transfer over 150-base
homologous regions and were chosen to span a wide range with respect to
structural strength. This allowed a comparison of transfer on highly
structured versus relatively non-structured RNAs in the
presence or absence of NC. The level of transfer in the absence of NC
was higher on substrates with low structure and without prominent DNA
synthesis pause sites (see Figs. 5 and 6). NC only modestly stimulated
transfer on these substrates. In contrast, highly structured substrates with prominent pause sites transferred relatively poorly in the absence
of NC but were highly stimulated when NC was present (see Figs. 3 and
4). Much of the stimulation on the latter substrates appeared to result
from the "chasing" of paused products into transfer
products. Overall the results showed that strand transfer could occur
efficiently by mechanisms that do not involve pausing, although when
present, pause sites serve as focal points for transfer events. In
addition, NC stimulated transfer of highly structured regions, probably
by aiding in the unwinding of secondary structure, as mutant NC
proteins with lower unwinding activity were less stimulatory (see Figs.
8 and 9).
It was interesting that the low structure substrates showed high levels
of strand transfer even though very little pausing was observed. Both
pause-driven transfer (57, 69-71, 76) and transfer driven by
non-pause-related mechanism (based on interactions between template
structures for example) have been proposed (77-81). In pause-driven
strand transfer, paused DNA products are the substrates that ultimately
are used to produce transfer products. The pause-driven mechanism
proposes that when secondary structures stall the progression of RT, it
creates an opportunity for increased degradation of donor RNA by RT
RNase H activity (57, 82). The nascent DNA may be released from the
donor, or the acceptor RNA may invade and displace the donor RNA.
Eventually, the nascent DNA associates with the acceptor RNA, where
synthesis continues. Although NC has been shown to reduce pausing, it
may augment strand transfers under such a mechanism by accelerating
interactions between the acceptor and complementary DNA. Recently, it
has been shown that pausing not only results in transfer of the paused
DNA product but can also promote transfers downstream of the pause site
(70). In this case pausing apparently allows time for the acceptor to interact with the nascent DNA to form a trimeric complex consisting of
the acceptor, donor, and DNA. The acceptor then "zips" up
the complementary DNA and displaces the donor as RT continues extension of the paused DNA. This leads to transfer at a point beyond the pause
site. Alternatively, other reports (79, 80) have proposed that
interactions between the transferring DNA and specific structures on
the acceptor can trigger strand transfers in the absence of pausing. In
this mechanism specific structures on the acceptor interact with the
DNA after the formation of a trimeric structure. These interactions
lead to displacement of the donor as the DNA binds to the acceptor.
Ultimately, there may be several mechanisms by which strand transfer
can occur. These could depend on local structures or sequences. The
relative quantitative importance of the various mechanisms remains to
be determined.
For those regions with high structure in our experiments, at least part
of the transfer appeared to result from a standard pause-driven
mechanism where DNAs stalled at the pause site transferred to the
acceptor (see Fig. 3 and "Results"). The other regions (PolVif and
Env) showed very little pausing, so some other type of mechanism is
likely. One possibility is a non-pause-induced mechanism similar to the
"zipping" mechanism proposed above. In the above mechanism
pausing provides time for the formation of a trimer, which then
resolves at a point downstream of the pause site. In low structure
regions, pausing may not be necessary for trimer formation, because the
lack of secondary structures that impede hybridization allows rapid
association of the acceptor and nascent DNA. With this in mind, strand
transfer could be viewed as a compromise between opposing processes.
Pausing favors transfer by providing substrates that can readily
transfer to the acceptor. However, the high degree of structure that is
generally associated with pausing is not conducive to association
between the acceptor and transferring DNA, thus opposing transfer. If
most recombination events occurred by non-pause-induced zipping
mechanisms, then recombination would likely be fairly homogenously
spread over the whole HIV genome and may even focus in regions with low
structure. A recent report indicated that on average transfers occur
~three times during production of a single provirus and that apparent "hotspots" probably exist (43). Whether there are local
structures or pause sites in the hotspot regions will require a more
detailed analysis of these regions.
The effect of two NC finger mutants on strand transfer was also
examined. Several reports (17, 24, 83, 84) using NC finger deletion
mutants to try and understand the role of specific protein domains have
been published. Results show that the highly basic backbone of the
molecule is responsible for a portion of the annealing activity (25),
whereas zinc coordination by the finger domains is required for full
unwinding activity (83, 84). As was noted earlier, recent results in
our laboratory suggest that the two fingers may have disproportionate
roles in annealing and unwinding with finger 2 possessing more of the
former activity and finger 1 possessing more of the latter
activity.2 Results reported here showed that mutant 1.1 (two copies of finger 1) enhanced strand transfer to approximately the
same level as wild type on both the highly structured GagPol substrate
and low structured Env substrate (Fig. 9, A and
B). In contrast 2.2 (two copies of finger 2) showed greater
enhancement than wild type on the Env substrate whereas stimulation was
reduced on GagPol. These results are consistent with a role for finger
1 in unwinding as the loss of this finger resulted in reduced transfer
in the more structured substrate. The results are at least partly
consistent with a previous report (83) using these mutants. In that
study the mutants were evaluated using a system to test strand transfer of the transactivation response containing minus strand
strong-stop DNA ( Results presented here indicate that the role of NC in recombination is
likely dependent on the structure of the region from where strand
transfer is occurring. Unwinding activity is especially important in
this process, because regions with high structure appear to transfer
relatively poorly unless NC is present. Consistent with recent findings
(81), results also showed that strand transfer could occur in the
absence of strong polymerase pausing, perhaps because of more rapid
association of the acceptor and DNA in non-structured regions. The
emerging story for retrovirus recombination is one in which events can
occur by several potential mechanisms. Further experiments will be
required to uncover the details of the various mechanisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C,
and a fresh aliquot was used for each experiment. HIV NC clone was a
generous gift from Dr. Charles McHenry (University of Colorado). NC was
purified according to the protocol described (58). The purity of the protein was evaluated using Coomassie Blue staining of 17.5% SDS-PAGE gels (59). Quantification was by absorbance at 280 nm using an
extinction coefficient of 8350 cm
1
M
1 (58). Aliquots of NC were stored frozen at
70 °C, and a fresh aliquot was used for each experiment. NC finger
mutants 1.1 and 2.2 were a gift from Dr. Robert Gorelick (SAIC,
Frederick, MD). These proteins were expressed and purified as described
by Bushman and co-workers (60) and quantified by amino acid analysis on a Beckman Systems 6300 amino acid analyzer (Beckman Coulter, Inc., Fullerton, CA). Taq polymerase was from Eppendorf. SP6 RNA
polymerase, DNase I-RNase-free, and RNase-DNase-free were from Roche
Diagnostics. RNase inhibitor was from Promega. T4 polynucleotide kinase
was obtained from New England Biolabs. Proteinase K was obtained from Eastman Kodak Co. Radiolabeled compounds were as follows:
[
-32P]ATP was obtained from Amersham Biosciences,
Sephadex G-25 spin columns were from Amika Corp., and all other
chemicals were from Sigma or Fisher Scientific.
Primers used in PCR amplification of various segments of the HIV-1
genome
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the strand
transfer assay. Donor and acceptor RNA templates are labeled along
with their corresponding total lengths. The 150-nucleotide
(nt) boxed region enclosing the two templates is
the region of homology or the transfer zone. The asterisk
represents the 5'-labeled primer that is complementary to only the
donor template. The broken lines represent the full-length
DNA that is synthesized on the donor, and the dotted lines
represent the transfer DNA that has undergone a strand transfer event
to the acceptor. The lengths of full-length donor-directed DNA and
transfer DNA products are indicated at the bottom of the
figure.
View larger version (18K):
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Fig. 2.
Predicted secondary structures of highly and
weakly structured donor RNA templates. RNAdraw and Zuker's
RNA folding programs (see "Results") were used to predict the
secondary structures of donor RNA templates (at a temperature of
37 °C, using program default settings) derived from three regions of
the HIV genome. The structures show 155 bases of the donor templates
excluding the 20 bases that bind to the primer. The base that is encircled in two rings
is the 5' end. The probabilities for particular base pairs are
indicated by the thickness of the lines between them with
thicker lines representing base pairs with greater
probabilities of formation. The pause sites shown here were identified
by time course reactions and mapped by sequencing reactions as
explained under "Results." A, the U3 3'LTR donor
template is shown (bases 9096-9265, G =
49.30 kcal/mol).
65 marks the position of a major pause site at a U residue
(enclosed in the box) that is 65 nucleotides from the 3' end
of the donor template (note that the 20 nucleotides binding to the
primer are not shown). #1 and #2 denote the
boundaries of the 24-nucleotide potential hotspot (see "Results").
Pause sites within this region have not been mapped. B,
GagPol donor template is shown (bases 2031-2200,
G =
45.30
kcal/mol). 77 marks the position of a major pause site at an
A residue (enclosed in the box). SS refers to the
"slippery site" heptamer sequence (enclosed in the box).
C, RRE donor template is shown (bases 7823-7992,
G =
59.40 kcal/mol). 72 marks the position of a major pause
site at an A residue (enclosed in the box). D,
PolVif template is shown (bases 3441-3610,
G =
19.3
kcal/mol). E, Env template is shown (bases 7101-7270,
G =
15 kcal/mol).
G value and a high base pair melting temperature
indicated the presence of strong structures. For example, the donor
GagPol RNA had a predicted
G value of
45.3 kcal/mol, and the
predicted stem loops persisted even at temperatures above 55 °C
whereas the donor Env RNA showed a
G =
15.0, and the
structure melted out completely above 55 °C. Although the RNA
folding programs may not predict the structure of RNAs with 100%
reliability, a reasonable estimate of the strength and characteristics
of the RNA would be expected, especially because relatively small RNAs were used. In addition, the GagPol and RRE regions used have been shown
previously (65-68) to possess strong stem loop structures similar to
those predicted by the folding programs. For our purposes an exact
rendering of the structures is not necessary; just a general idea of
their relative strengths and the presence or absence of pause site is
required. The latter can be evaluated from RT primer extension reactions.
G =
49.3 kcal/mol (Fig. 2A). A
previous in vivo study identified a 24-base segment between bases 9158 and 9183 in this region as a potential hot spot for recombination (42). The study used two viral vectors based on different
strains of HIV-1 to analyze recombination and obligatory strand
transfers in HIV. The vector viruses were harvested from producer cells
(CD4
HIV-1 Env inducible cell line) and were used to
infect target cells (CD4+ HeLa T4 cells). Of the 86-target
cell clones analyzed, 11 clones were shown to undergo homologous
recombination during minus strand U3 synthesis. DNA sequencing analysis
of these clones revealed a 24-base segment as a potential hot spot for
recombination. The report also suggested that the region might have RNA
secondary structures that induce pausing. Donor and acceptor RNA
templates were generated from this zone and used in time course
reactions. Fig. 3A is an autoradiogram of a PAGE showing the
resolved DNA products. The bands at position 197, as indicated by the
molecular ladder on the leftmost lane, are products of strand transfer
from the donor to acceptor RNA. The bands at position 175 results from completed synthesis on the donor RNAs without any transfer events. Three distinct pause sites P1, P2, and P3 were observed between positions 65 and 90. P1 and P2 are within the 24-base region noted above, whereas P3 lies just outside at position 65. The major pause
sites were mapped using DNA sequencing gels in which sequencing reactions on DNA were run using the primer from these reactions (data
not shown). Interestingly, the characteristics of the position 65 site
are strikingly similar to those of another pause site characterized
previously (57, 69). Both sites are located within the stem of a strong
stem loop structure. In both cases, RT stalled at a U residue located
just behind a series of strong G-C pairs in the stem (Fig.
2A). In the absence of acceptor template the paused products
persist even at 64 min, especially the 65-base products. Including NC
protein without acceptor did not change the profile significantly. In
the presence of acceptor template but absence of NC a decrease in the
amount of the paused products was observed over time in comparison with
reactions without acceptor. An increase in the amount of transfer
product was also seen. The results are consistent with paused products
transferring to the acceptor template and being extended. Paused
products were "chased" more rapidly in the presence of NC
and acceptor, and the level of transfer product also increased to a
greater extent than in reactions with acceptor alone (Fig.
4A). Results here are in agreement with earlier reports (57,
70, 71) that have shown that paused DNAs can be focal points of strand
transfer. The stimulatory effect of NC on recombination has also been
supported by numerous experiments (15, 36, 46).
View larger version (85K):
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Fig. 3.
Time course strand transfer assays of highly
structured RNA substrates. Shown are autoradiograms of assays
performed using highly structured RNA templates under standard
conditions as indicated under "Experimental Procedures."
ML is the molecular ladder indicating the positions of bands
in the other lanes. The reactions were carried out in either
the presence or absence of NC and acceptor RNA templates
(Accp) as indicated above each set of
lanes. Donor RNA was used in all reactions. Each reaction
was carried out at time points 0, 2, 4, 8, 16, 32, and 64 min as shown
by the corresponding set of seven lanes from left
to right. The transfer and full-length donor-directed DNA
products, the pause sites, and the primers are indicated as
T, F, P, and PR,
respectively. A, assays conducted on U3 3'LTR substrate are
shown. P1, P2, and P3 are three
distinct pause sites located between positions 65 and 90. P3 alone was
mapped to a U residue at position 65 from the 3' end of the template
(see "Results"). B, assays conducted on GagPol substrate
are shown. A major pause site was mapped to an A residue at position 77 from the 3' end of the template. C, assays conducted on RRE
substrate are shown. A major pause site was mapped to an A residue at
position 72 from the 3' end of the template.
View larger version (21K):
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Fig. 4.
Graph of efficiency of strand transfer
versus time for the highly structured RNA substrates.
Graphs were derived from the experiments shown in Fig. 3. The
percent transfer efficiency was defined as Transfer DNA products
(T)/Transfer + Full-length donor-directed products (F), times 100 ((T/(T+F) × 100). The templates used are indicated
above each graph. The solid circles represent
time course reactions conducted in the absence of nucleocapsid protein
( NC) and the presence of acceptor RNA (+Accp).
The open circles represent time course reactions conducted
in the presence of nucleocapsid protein (+NC) and acceptor
RNA (+Accp).
G =
45.30
kcal/mol) of the genome (Fig. 2B). This region includes the
portion of genomic mRNA where the programmed
1 ribosomal
frameshifting event used to produce enzymatic proteins (RT, PR, IN)
from the pol region occurs. The mechanism allows the virus
to maintain a well regulated ratio of Gag proteins to GagPol proteins
in infected cells, which is vital for efficient assembly of infectious
particles. The segment of gag-pol region that we used
included the heptameric "slippery site" (UUUUUUA sequence)
and a downstream RNA secondary structure, both of which have been shown
to promote efficient frameshifting (72, 73). Earlier literature has
suggested that the RNA secondary structure is a simple stem loop (73,
74), but a recent report favors a more complex intramolecular triplex
RNA structure (75). In either case, the structure seems to cause the
ribosomes to pause and subsequently slip
1 base over the slippery
site (Fig. 2B). Interestingly, this structure also caused
the RT to pause as shown on Fig. 3B. This figure shows a
time course reaction conducted on the GagPol substrates. The gel
reveals a single strong pause site. Sequencing reactions mapped the
site to an A residue at position 77 from the 3' end of the donor RNA.
In this case the A was also part of a strong stem structure and was
followed by four G-C pairs. Once again NC stimulated transfer from this region significantly (see Fig. 3B and Fig. 4B),
and production of transfer products was accompanied by an apparent
chasing of the paused DNA product.
G =
59.40 kcal/mol) (Fig.
2C). The RRE has been characterized by Malim et
al. (68) as a 234-base region to which the Rev protein can bind.
Later on, Mann et al. (67) demonstrated that an extra 58 bases at the 5' end and 59 bases at the 3' end of the original 234 region (a total of 351 bases) was the complete biologically active RRE. The binding of Rev protein to this highly structured region is important for the nuclear export of unspliced and partially spliced HIV
mRNAs. A strong pause site was mapped to an A residue at position 72 in this template (see Fig. 2C and Fig.
3C).
G =
19.3 kcal/mol) (Fig. 2D). The experiment shown
in Fig. 5A demonstrates that "strong" pause sites
are absent on this donor. Surprisingly, the efficiency of strand
transfer on this substrate in the absence of NC was significantly higher than for the more structured substrates described above. In
addition, though NC did hasten the onset of strand transfer, the
overall enhancement was less as compared with the highly structured RNAs (Fig. 6A).
View larger version (43K):
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Fig. 5.
Time course stand transfer assays of weakly
structured RNA substrates. Shown are autoradiograms of assays
performed using weakly structured RNA templates under standard
conditions as indicated under "Experimental Procedures." The
transfer and full-length donor-directed DNA products and the primers
are indicated as T, F, and PR,
respectively. Other markings are as described in the legend for Fig. 3.
A, assays conducted on PolVif substrates are shown.
B, assays conducted on Env substrates are shown.
View larger version (13K):
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Fig. 6.
Graph of efficiency of strand transfer
versus time for the weakly structured RNA
substrates. Graphs were derived from the experiments shown in Fig.
5. The substrates used are indicated above each graph. All
the other markings are the same as in the legend for Fig. 4.
G =
15 kcal/mol) (Fig. 2E). The
region was predicted to have the weakest structure among the other four
substrates, as shown by the
G value. This region also showed an
absence of strong pause sites (Fig. 5B). Some weak pause
sites were observed, but they disappeared quickly even in the absence
of NC and acceptor RNA. Once again, the level of strand transfer was
relatively high in the absence of NC and NC moderately increased
transfer but to a lesser extent than on structured substrates (Fig.
6B).
View larger version (32K):
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Fig. 7.
NC titration assay conducted on the U3 3'LTR,
PolVif, and GagPol RNA substrates. A, shown is an
autoradiogram of assays that were conducted using standard conditions
are indicated under "Experimental Procedures" on three RNA
templates as indicated above each set of lanes.
ML is the molecular ladder that indicates the position of
bands in the other lanes. All lanes labeled A are
the control lanes with no NC protein and no acceptor RNA. Lanes
labeled B contain acceptor RNA but no NC protein. Lanes
labeled C to I contain acceptor RNA and NC protein. The
final concentrations of NC protein used ranged from 0.0625 to 4 µM, i.e. lanes C, 0.0625 µM; lanes D, 0.125 µM;
lanes E, 0.25 µM; lanes F, 0.5 µM; lanes G, 1 µM; lanes
H, 2 µM; and lanes I, 4 µM.
The reactions were conducted for 32 min. T, F,
and PR refer to the transfer DNA products, full-length
donor-directed DNA products, and the primers, respectively.
B, shown is a graph derived from experiments shown in
A of efficiency of strand transfer in U3 3'LTR, PolVif, and
GagPol templates versus NC concentration in
µM. The percent transfer efficiency was defined as
described for Fig. 4. The solid circles represent assays
conducted on U3 3'LTR RNA, the open circles are assays on
PolVif RNA, and the solid inverted triangles are assays on
GagPol RNA.
View larger version (54K):
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Fig. 8.
Time course strand transfer assays on Env or
GagPol substrates with wild type NC or mutant NC proteins. Shown
are autoradiograms of assays that were conducted using standard
conditions as indicated under "Experimental Procedures" for the Env
RNA substrate (A) or the GagPol RNA substrate
(B). Assays were conducted in the absence or in the presence
of wild type (Wt) NC, mutant 1.1, or mutant 2.2 NC as
indicated above each set of lanes. Lanes
labeled C are controls with no acceptor RNA or NC protein. All the
other markings are the same as in legend for Fig. 3.
View larger version (15K):
[in a new window]
Fig. 9.
Graph of efficiency of strand transfer
versus time, using mutant NC proteins. The
percent transfer efficiency was defined as described for Fig. 4.
Results were derived for the experiments shown in Fig. 8. The
substrates used are indicated above each graph. The
solid circles and the open circles represent
reactions conducted in the absence and in the presence of wild type
(Wt) NC protein, respectively. The solid and
open inverted triangles represent reactions conducted in the
presence of 1.1 and 2.2 mutant NC proteins, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ssDNA). In those experiments mutant 1.1 retained
50% of wild type activity, and 2.2 showed a complete loss of activity. The differences observed between these experiments and ours could have
resulted from the substrates used. The
ssDNA substrate forms a very
strong complex secondary structure, which may require a high degree of
unwinding and annealing activity to overcome the strong structure.
Also,
ssDNA has a tendency to self-prime, an effect that prevents
transfer but is inhibited by NC (47, 85). Therefore this substrate may
be very sensitive to small changes in NC that have an effect on
self-priming and/or annealing and unwinding. Overall, however, both
sets of experiments support a role for finger 1 in helix destabilization.
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ACKNOWLEDGEMENTS |
---|
We thank Genetics Institutes for the kind gift of HIV-RT and the AIDS Research and Reference Reagent Program for plasmid pNL4-3. We also thank Dr. Robert Gorelick from Science Applications International Corp., Frederick, MD for NC mutant proteins and Dr. Charles McHenry from the University of Colorado for the plasmid clone for wild type NC overexpression.
![]() |
FOOTNOTES |
---|
* This work was supported by NIGMS, National Institutes of Health Grant GM51140.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.
To whom correspondence should be addressed: Dept. of Cell Biology
and Molecular Genetics, University of Maryland, Bldg. 231, College
Park, MD 20742. Tel.: 301-405-5449; Fax: 301-314-9489; E-mail:
jd146@umail.umd.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M211701200
2 M. J. Heath, S. S. Derebail, R. J. Gorelick, J. J. DeStefano, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NC, nucleocapsid
protein;
RT, reverse transcriptase;
HIV, human immunodeficiency virus;
LTR, long terminal repeats;
ssDNA, minus strand strong stop DNA;
IN, integrase;
PR, protease;
RRE, Rev response element;
HIV-1, HIV, type
1.
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