From the McGill University AIDS Centre, Lady Davis
Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada
and Departments of ¶ Medicine and
Microbiology, McGill
University, Montreal, Quebec H3A 2B4, Canada
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ABSTRACT |
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We have investigated the role of sequences that
surround the primer binding site (PBS) in the reverse
transcriptase-mediated initiation of () strand DNA synthesis in human
immunodeficiency virus type 1. In comparisons of reverse transcription
initiated from either the cognate primer tRNALys.3 or a DNA primer
D-Lys.3, bound to PBS sequences, we observed that a
+3 pausing site occurred in both circumstances. However, the initiation
reaction with tRNALys.3 was also characterized by a pausing event after
incorporation of the first nucleotide. Alteration of sequences at the
5'-end instead of the 3'-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template
sequence-dependent. In contrast, the pausing event at the
+1 nucleotide position was still present in experiments that employed
either of these mutated RNA templates. The mutations at the 5'-end of
the PBS also caused a severely diminished rate of initiation and the
strong arrest of reactions at the +1 stage when tRNALys.3 was used as
primer. Therefore, we propose that the +1 pausing event is an
initiation-specific event in regard to reactions primed by tRNALys.3
and that sequences at the 5'-end of the PBS may facilitate the release
of reverse transcription from initiation to elongation.
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INTRODUCTION |
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Retroviruses employ specific tRNA molecules as primers to initiate
the synthesis of () strand DNA (1-5). These primer tRNAs bind to an
18-nt1 segment of viral
genomic RNA termed the primer binding site (PBS). Although such binding
is principally mediated by a stretch of 18 nt close to the 3'-end of
tRNA, other sequences within viral genomic RNA and the tRNA primer are
also involved and can modulate the initiation of (
) strand DNA
synthesis, as shown in both retroviral (6-10) and other (11-16)
systems. In HIV-1, this includes a stretch of four nucleotides,
i.e. 622AAAA625 (the A-rich loop)
that interacts directly with positions 33USUU36
of the anticodon loop of primer tRNALys.3. This A-rich loop is important for initiation of (
) strand DNA synthesis and generation of
progeny virus (17).
In HIV-1, the initiation stage of synthesis of () strand DNA, primed
by tRNALys.3, can be distinguished from subsequent strand elongation in
regard to both the binding and kinetic properties of reverse
transcriptase (RT) (18-20). These reports showed that initiation was
characterized by both early short (
) strand DNA products, resulting
from pausing at the +3 or +5 nt positions, and rapid dissociation of RT
from the initiation complex. The secondary structure formed between
tRNALys.3 and the viral RNA template may play an important role in
initiation of reverse transcription.
To pursue this subject, we developed an in vitro reverse transcription system in which low concentrations of dNTPs (i.e. 160 nM) were used to enhance our ability to detect very early pause sites, e.g. +1 and +3, in reactions primed with the tRNALys.3 cognate primer. To study the roles of sequences flanking the PBS, we generated a series of mutated HIV-1 RNA templates that contained mutations at both the 5'- and 3'-ends of the PBS that may potentially disrupt the secondary structure of complexes of tRNALys.3 and viral RNA template. Our data provide the first evidence for a +1 pausing event in reverse transcription initiated from primer tRNALys.3. We have also demonstrated on the basis of mutagenesis studies that the +3 pausing site depends on the nature of sequences at the 5'-end of the PBS and that deletion of an A-rich loop in this region or of substitutions at the 5'-end of the PBS may make it difficult for primer tRNALys.3 to be extended beyond the +1 pausing site.
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MATERIALS AND METHODS |
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Plasmid Construction-- DNA sequences at nt positions 473-1417 of HIV-1 (HxB2D) were cloned into the RNA expression vector pSP72 (Promega, Madison, WI) through use of BglII (473) and PstI (1412). The deletion of the A-rich loop (HIV/del-A) and the construction of mutation HIV/A2 have been described previously (17, 21). We also engineered a substitutional mutation (HIV/HUA) by replacing the sequence 624AATCTCTAGCAG635 with 5'-GAACACCCAACATT-3'; this was done by PCR using an antisense primer 5'-CCTGTTCGGGCGCCAAATGTTGGGTGTTCTTCCACA CTGACTAAAGG-3' (positions 650-606), containing the above substitutions (underlined) and a NarI restriction site (GGCGCC), together with a sense primer, 5'-AGACCAGATC TGAGAATGG-3' (positions 468-486), that contained a BglII restriction site (AGATCT) (see Fig. 3A) (22). Partial deletions of sequences downstream of the PBS, i.e. HIV/LD1, HIV/LD2, and HIV/LD3, were generated as described (23).
In Vitro Reverse Transcription-- These reactions were performed as described using RNA template that was generated through use of an Ambion Mega-scripts kit (Austin, TX) (see Fig. 1A) (17). tRNALys.3 prepared from human placenta (24), or a synthetic DNA primer complementary to the PBS, i.e. D-Lys.3 (5'-GTCCCTGTTCGGGCGCCA-3') (positions 653-636), was annealed onto RNA template by denaturing at 85 °C for 5 min and annealing at 55 °C for 10 min in a reaction mixture containing 83 mM Tris-HCl (pH 7.5) and 125 mM KCl. Reverse transcription reactions were performed in a volume of 20 µl containing 1 pmol of primer:RNA template complex, 50 mM Tris-HCl (pH 7.5), 75 mM KCl, 5 mM MgCl2, 10 mM DTT, dNTPs, 20 units of RNA guard ribonuclease inhibitor (Amersham Pharmacia Biotech) and reverse transcriptase at 37 °C for various times, after which reactions were terminated by adding EDTA to a final concentration of 50 mM. In some experiments, reverse transcription reactions were performed in the presence of HIV-1 nucleocapsid (NC) protein (30 pmol for each reaction) as described (21). The cDNA products were fractionated on 8% denaturing polyacrylamide gels containing 7 M urea. The RT preparations used included wild-type HIV-1 enzyme (p66/51) prepared as described (25) and mutated HIV-1 RT containing a mutation at codon 478 (i.e. E478Q), responsible for defective RNase H activity (26).
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RESULTS |
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The Initiation of HIV-1 () Strand DNA Synthesis from tRNALys.3 Is
Characterized by Rate-limiting Pause Sites at nt Positions +1 and
+3--
Initiation of HIV-1 reverse transcription is a distinct step
from that of elongation (18-20). To further investigate this subject, we developed an in vitro reaction system that employed a PBS
that contained only G, A, and C among the 5 nt at its 5'-end (Fig. 1A). Therefore, when dCTP,
dTTP, and dGTP were included in these reactions, only early products of
reverse transcription were generated. Reactions were also performed
with only dCTP (Fig. 1A, lane 1) or both dCTP and dTTP (Fig.
1A, lane 2) to provide information on the positions of the
first and second bands seen on gels. The results of Fig. 1A
show that reaction products were observed at both the +1 and +3
positions in addition to a final expected product at the +5 site
(lane 3). These reactions did not pause at either the +2 or
+4 positions, indicating that they were rate-limiting only after the
first and third nt were added. To prove that the above-mentioned pause
sites (i.e. +1 and +3) were not due to an absence of dATP in
the reactions, experiments performed with all four dNTPs were
terminated at different times (5, 15, or 45 min), with the result that
the +1 and +3 pause sites were still present (Fig. 1A, lanes 4, 5, and 6).
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The +3 Pause Site Is Dependent on Sequences at the 5'-End but not
the 3'-End of the PBS--
To study the role of sequences at the
5'-end of the PBS, we generated a mutated RNA template termed HIV/del-A
that contained a deletion of the A-rich loop
(622AAAA625) and a mutated RNA template HIV/HUA
described above (Fig. 2A). The
latter construct contained only T, A, and C within the 13 nt at the
5'-end of the PBS; hence, when only dATP, dTTP, and dGTP are included
in reactions with the HIV/HUA template, extension should only proceed
to the +13 stage. In reactions performed with 45 ng of HIV-1 RT,
tRNALys.3 as primer, and wt RNA genome, i.e. HIV/WT, three
bands at positions +1, +3, and +5 were clearly observed (Fig.
2A, lane 9). However, when either HIV/del-A or
HIV/HUA served as RNA template, only one band at position +1 was seen,
even when three different dNTPs were included (lanes 1-6);
furthermore, this band was weaker than that seen at the same position
with the wt RNA template HIV/WT. Considering the () strand DNA
products at the +3 and +5 positions in the case of wt RNA template
HIV/WT, deletion of the A-rich loop or substitutions within the region nt 624-635 led to both greatly diminished efficiency of initiation, as
well as an arrest of extension at the +1 nt position.
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Reverse Transcription Does Not Pause at the +1 Site when Initiated from a DNA Primer-- As shown above, reverse transcription initiated from tRNALys.3 still paused at the +1 position, even when sequences at both the 5'- and 3'-ends of the PBS were changed. When the first nt at the 5'-end of the PBS was changed from G to T (i.e. template HIV/HUA), the addition of the first dATP still represented a rate-limiting step (Fig. 2A, lanes 4-6). To study whether this was unique to RNA primers, reactions were also performed with an 18nt DNA primer, i.e. D-Lys.3, bound to the PBS.
When only dCTP was included in the reaction mixture, only one band was seen at position +1 (Fig. 4A, lane 1). When both dCTP and dTTP were present, a band corresponding to nt position +2 was observed, and that at the +1 position had disappeared (lane 2). When dCTP, dTTP, and dGTP were included, two bands corresponding to the +3 and +5 nt pause sites were observed (lane 3). When all four dNTPs were present, extension occurred to beyond the +5 position, although a strong pause site at nt position +3 was still present, regardless whether reactions were run for 5, 15, or 45 min (Fig. 4A, lanes 4, 5, and 6). Thus, when a DNA oligomer was utilized as primer, reactions did not pause at the +1 site, but they did pause at position +3.
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NC Protein Helps Reverse Transcription Escape from the +1 Pausing Event-- To study whether NC protein could affect the +1 and +3 pausing events in reverse transcription reactions primed with tRNALys.3, 30 pmol of NC protein was added to our 20-µl reaction system, and reactions were terminated at various times (1, 4, 16, 32, and 64 min) (Fig. 6, lanes 1-5). A similar time-course without NC protein was also performed as a control (Fig. 6, lanes 6-10). The data show that inclusion of NC resulted in significantly less pausing at the +1 nt position and did not affect the +3 nt pause site. Furthermore, more +5 nt product was generated in the presence of NC protein, suggesting that NC had contributed to more efficient elongation of reverse transcription.
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Pausing at A-rich Loop Positions during Synthesis of () Strand
DNA--
When a still higher dNTP concentration was used,
i.e. 2.56 µM, four additional pause sites were
observed at positions +11, +12, +13, and +14, corresponding to the four
As found within the A-rich-loop (622AAAA625).
These four bands diminished in intensity when reactions were incubated
for longer times, e.g. 45 min (Fig.
7, lanes 4- 6).
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DISCUSSION |
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Biochemical analysis has shown that the initiation of HIV-1
reverse transcription can be distinguished from subsequent elongation (18-20). The biological relevance of this observation is suggested by
the fact that RT can lose its ability to discriminate against a nonself
tRNA primer when the latter was extended by two nt (27). Second,
in vitro labeling revealed that primer tRNALys.3 was
extended by two nt within virus particles that had engaged in synthesis of () strand DNA (28). In our system, initiation has been
characterized on the basis of several early pause sites
(e.g. +1 and +3) that add substantially to our understanding
of early events in reverse transcription.
This is the first demonstration that pausing at the +1 nt site
represents a rate-limiting step in reverse transcription reactions performed with an HIV-1 RNA template and the cognate primer tRNALys.3. This is not an unexpected finding because HIV-1 RT possesses both RNA-dependent DNA polymerization and
DNA-dependent DNA polymerization activities. During reverse
transcription, the enzyme is bound to either RNA-DNA or DNA-DNA hybrids
during RNA-dependent DNA polymerization and
DNA-dependent DNA polymerization, respectively, except at
the initiation of synthesis of () strand strong-stop DNA ((
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ssDNA), when the enzyme is bound to a RNA-RNA hybrid and employs
tRNALys.3 as primer for production of cDNA. After initiation takes
place, the role of primer is effectively replaced by the newly made
DNA, from which further extension will occur (5). Therefore, the
initiation of (
) ssDNA synthesis is a distinct stage of reverse
transcription, especially in regard to incorporation of the first
dNTP.
When the first C deoxyribonucleotide from the dNTP pool is added to the
3'-OH of the A ribonucleotide at the 3'-end of tRNALys.3, displacement
of a ribonucleotide-ribonucleotide pair (A-U) must occur in favor of a
newly formed deoxyribonucleotide-ribonucleotide pair (dC-G) at the RT
polymerization active site. Due to the absence of a 2'-OH residue in
dC, the two nucleotide pairs (A-U and dC-G) may assume different
conformations. Therefore, a structural rearrangement of the
polymerization active site is required for the RT enzyme to adapt to
the new dC-G pair and to add the next deoxyribonucleotide (dT) to the
one-base extended primer. It is generally believed that a
conformational change of RT must precede the chemical step, resulting
in a rate-limiting event (29-32). Our results show that this
rate-limiting step results in the pause site at the +1 position. Because the RT enzyme was originally bound to a RNA-RNA helix, it may
require a conformational rearrangement to switch its binding mode from
a RNA-RNA duplex to a DNA-RNA hybrid during synthesis of () ssDNA.
Our results demonstrate that this conformational rearrangement may
begin to occur as soon as the first dC has been added. In contrast, the
use of the DNA primer, D-Lys.3, necessitates that the
duplex to which RT is bound will always be a DNA-RNA hybrid, thus
permitting the enzyme to catalyze the extension reaction without a
change in conformation. Consequently, reactions primed by the DNA
primer, D-Lys.3, do not need to pause after addition of the
first dC.
Other studies have also indicated that a conformational rearrangement might be required for RT to proceed after addition of the first nt. Direct evidence for this comes from studies of the relationship between the RNase H cleavage site and the polymerization active site (26). Generally, a constant distance of 18 nt must exist between the RNase H cleavage site and the nascent primer 3' terminus. However, a distance of 19 nt instead of 18 nt was observed between these sites after incorporation of the first dC at the 3'-end of primer tRNALys.3 (26). Because the spatial relationship between the functional RNase H cleavage site and the polymerization active site serves as an indication of RT conformation, the 19-nt distance suggests a novel conformation for RT after incorporation of the first nt. Other evidence for this has been provided by studies of the binding and kinetic properties of HIV-1 RT during initiation and elongation that showed that RT dissociated approximately 200 times faster from the initiation (RNA template/tRNALys.3) than from the elongation complex (19). Further supporting this notion, mutagenesis studies of the HIV-1 RT palm subdomain have shown that RNA and DNA primers may be differentially recognized by RT, i.e. RT may be associated with RNA versus DNA primers in different conformations (33). This also helps to account for the importance of the +1 pause site demonstrated in this paper.
We have also noted that synthesis of () strand DNA paused at the +3
nt position, regardless whether tRNALys.3 or a DNA primer was employed.
This suggests that the secondary structure of the complex formed
between the viral RNA template and the primer must play a role in the
specification of this pause site. The PBS and its flanking sequences
constitute a highly ordered secondary structure. The binding of the
tRNALys.3 primer to the PBS results in a complex with altered secondary
structure in the region of the PBS, in which the three nt (GAC) at the
5'-end of the PBS are looped out and the eight nt just at the 5'-end of
the PBS bind to other complementary sequences to form a stable stem
structure (8, 9). Therefore, when extension from the primer reaches the
+3 stage (dG), the stem structure has to be disrupted before the fourth
nucleotide (dC) can be added, resulting in pausing at the +3 nt
position (Fig. 8A). A similar
secondary structure is probably formed when the DNA primer,
D-Lys.3, is bound to the PBS, such that pausing at the +3
stage also occurs in this circumstance.
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This hypothesis is supported by our experiments in which a mutated RNA
template, HIV/HUA, containing substitutional changes at the 5'-end of
the PBS (624-635) was employed (Fig. 2B). In this
situation, the stem structure shown in the wt RNA template (Fig.
8A) was disrupted (Fig. 8B). As a consequence, we
found that pausing no longer occurred at the +3 position. In addition, reactions that employed the mutated RNA template HIV/del-A (Fig. 2B) paused less frequently at the +3 nt site than did those
performed with wild-type RNA template (HIV/WT). This result might also
be attributable to a destabilization of the stem-loop caused by
deletion of the A-rich loop, because mutations in this region can cause disturbances in secondary structure of complexes between tRNALys.3 and
viral genomic RNA (34). Therefore, template structure may be
responsible for the type of pausing seen at the +3 position during
initiation of synthesis of () strand DNA.
Our data add significantly to the notion that the initiation of HIV-1 reverse transcription represents a distinct phase in RT reactions (18-20). First, we have documented that a pause site at the +1 position is rate-limiting. The failure of other groups to have previously detected this site might be due to their use of higher concentrations of dNTPs (e.g. 50 µM). Also, the tRNALys.3 employed in our experiments is from human placenta rather than from other animal species. Because all retroviruses use tRNAs as primers, the +1 pausing site may be common to all RTs.
Second, the transition from initiation to elongation probably occurs at the +1 site as well as at the previously observed +3 and +5 positions. On the basis of our studies, the +3 pause site strongly depends on the secondary structure of the viral RNA template. Conceivably, the +1 position may represent the actual point of transition from initiation to elongation, and the +3 pause site may be involved in the arrest of reactions at other early stages. Finally, the A-rich loop, as well as other sequences at the 5'-end of the PBS, may be involved in both the efficiency of initiation as well as release from the +1 pause site.
The +1 and +3 pausing events in viral reverse transcription may play a
regulatory role similar to that observed in the case of E. coli RNA polymerase, for which initiation of gene transcription begins in an abortive mode and pauses near the start site, and for
which a regulatory subunit, , is required for transcription to
proceed to elongation (35, 36). In HIV-1, the NC protein may play a
role analogous to that of the
factor, because NC can help to
overcome the +1 pausing event (Fig. 6). NC protein is also able to
unwind the secondary structure of template RNA, facilitating the
synthesis of long products of reverse transcription (37), and can
stimulate the annealing of complementary RNA and/or DNA sequences to
stabilize hybrids (38). We believe that NC helps to form and stabilize
complexes between primer tRNALys.3 and viral genomic RNA and that the
+3 nt pause site is a consequence of the secondary structure of the
complex thus formed. In this context, the +3 nt pause site should be
observed both when NC protein is present or absent, as observed in the
results of Fig. 6.
The role of the A-rich loop in HIV-1 reverse transcription and viral
replication is controversial (39). Mutations in the A-rich loop
resulted in both severely diminished initiation efficiency of HIV-1
reverse transcription and defective transition from initiation to
elongation (Refs. 17 and 18 and this report). However, our use of viral
RNA template deleted of the A-rich loop, HIV/del-A, resulted in only a
modest decrease in amount of final () strand DNA product (171 nt)
(Fig. 7). Consistently, viruses containing a deletion of the A-rich
loop were only moderately impaired in replicative capacity (17). The
present studies show that the A-rich loop is responsible for four
strong pause sites, +11 through +14, that limit processivity and that
deletion of these four As eliminated these pause sites and yielded
longer (
) strand DNA products. Thus, the initial deficit in viral
replication caused by deletion of the A-rich loop may have been
partially compensated by elimination of the pause sites at positions
+11 through +14.
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FOOTNOTES |
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* This research was supported by grants from Health and Welfare Canada and by the Medical Research Council of Canada.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.
§ These two authors contributed equally to this research; their work has been performed in partial fulfillment of the Ph.D. degree, Faculty of Graduate Studies and Research, McGill University, Montreal, Quebec, Canada.
** Holder of a National AIDS Scientist Award from Health Canada. To whom correspondence should be addressed: McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260; Fax: 514-340-7537; E-mail: mdwa{at}musica.mcgill.ca.
The abbreviations used are: nt, nucleotide(s); NC, nucleocapsid; PBS, primer binding site; HIV, human immunodeficiency virus; RT, reverse transcriptase; ssDNA, single-stranded DNA; wt, wild-type.
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REFERENCES |
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