From LaboRetro ENS, INSERM U412, 46 allée
d'Italie, 69364 Lyon cedex 07, France, § Institut de
Biologie Moleculaire et Cellulaire, CNRS UPR 9002, 15 rue Descartes,
67084 Strasbourg cedex, France, and the ¶ Division of Infectious
Diseases, Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106
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ABSTRACT |
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During HIV reverse transcription, (+) strand DNA
synthesis is primed by an RNase H-resistant sequence, the polypurine
tract, and continues as far as a 18-nt double-stranded RNA region
corresponding to the 3' end of tRNALys,3 hybridized
to the viral primer binding site (PBS). Before (+) strand DNA transfer,
reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS
RNA in order to reverse-transcribe the 3' end of primer
tRNALys,3. Since the detailed mechanism of (+) strand DNA
transfer remains incompletely understood, we developed an in
vitro system to closely examine this mechanism, composed of HIV
5' RNA, natural modified tRNALys,3, synthetic unmodified
tRNALys,3 or oligonucleotides (RNA or DNA) complementary to
the PBS, as well as the viral proteins RT and nucleocapsid protein
(NCp7). Prior to (+) strand DNA transfer, RT stalls at the
double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe
modified nucleosides of natural tRNALys,3. Modified
nucleoside m1A-58 of natural tRNALys,3 is only
partially effective as a stop signal, as RT can transcribe as far as
the hyper-modified adenosine (ms2t6A-37) in the
anticodon loop. m1A-58 is almost always transcribed into A,
whereas other modified nucleosides are transcribed correctly, except
for m7G-46, which is sometimes transcribed into T. In
contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA
PBS primer are completely reverse-transcribed. In the presence of an
acceptor template, (+) strand DNA transfer is efficient only with
templates containing natural tRNALys,3 or the RNA PBS
primer. Sequence analysis of transfer products revealed frequent errors
at the transfer site with synthetic tRNALys,3, not observed
with natural tRNALys,3. Thus, modified nucleoside
m1A-58, present in all retroviral tRNA primers, appears to
be important for both efficacy and fidelity of (+) strand DNA transfer.
We show that other factors such as the nature of the ( The distinctive characteristic of retroviruses is the conversion
of their RNA genome into double-stranded DNA, which is subsequently integrated into the cellular genome. Reverse transcription is a complex
process composed of multiple steps catalyzed by the viral enzyme
reverse transcriptase (RT)1
(reviewed in Ref. 1). The general model of reverse transcription includes two obligatory DNA transfer reactions (Fig. 1A). RT
initiates minus strand DNA synthesis by elongation of a specific
cellular tRNA primer annealed to the primer binding site (PBS) and
continues as far as the 5' end of the RNA genome, generating the
so-called strong stop cDNA (ss-cDNA). The concomitant
degradation of the RNA template by the RNase H activity of RT makes the
ss-cDNA single stranded. ss-cDNA is transferred by NC protein
(2, 3) to the 3' end of either the template RNA or the other RNA
molecule present in the virus particle to complete ( In this report, we analyzed the following factors that influence
the efficiency of HIV-1 (+) strand DNA transfer: the modified nucleosides of primer tRNALys,3, the RNase H activity of
RT, and deletions in the ( Oligonucleotides--
All DNA and RNA oligomers are listed in
Table I and were purchased from
Eurogentec. Radiolabeled oligonucleotides (primers SA-12, R5', and
SA-18) were prepared using T4 polynucleotide kinase and
[ Proteins and Enzymes--
T7 RNA polymerase, T4 polynucleotide
kinase, Taq DNA polymerase, and terminal transferase were
purchased from Promega. Sequenase was purchased from Amersham.
NCp7 (72 residues) generated by peptide synthesis (12) was
provided by D. Ficheux (IBCP, Lyon, France). Recombinant
HIV-1 reverse transcriptase (RT p66/p51) and HIV-1 RNase H( tRNALys,3--
Natural tRNALys,3 was
purified from beef liver as described (15). Synthetic
tRNALys,3 was generated in vitro using T7 RNA
polymerase (16).
Cloning--
PCR products were directly cloned into pGEM-T
(Promega), which possesses 3'-T overhangs (the pGEMTM-T
kit provides an exonuclease-free T4 DNA ligase).
RNA Template--
HIV-1 MAL 5' RNA corresponding to nucleotides
1-311 was generated in vitro as described (17), phenol- and
chloroform-extracted, and purified by gel filtration.
tRNALys,3 Reverse Transcription--
Reverse
transcription of natural tRNALys,3 or synthetic
tRNALys,3 was performed using 5'-32P-labeled
primer SA-12. 5'-32P primer SA-12 (3 pmol) was annealed to
tRNALys,3 (2 pmol) by incubating with NCp7 (22 pmol; NCp7
to nucleotide ratio was 1:7) for 10 min at 37 °C in 10 µl
containing 30 mM Tris-HCl (pH 7.5), 5 mM
dithiothreitol, 30 mM NaCl, 0.2 mM
MgCl2, 1 µM ZnCl2, and 2 units of
RNasin. Next, RTp66/p51 (5 pmol) was added and the reaction incubated
with 0.25 mM each dNTP for 15 or 45 min at 37 °C in 30 mM Tris-HCl (pH 8), 5 mM dithiothreitol, 60 mM NaCl, 3 mM MgCl2 in 25 µl.
After phenol and phenol/chloroform extraction, nucleic acid was
precipitated with 3 volumes of ethanol, recovered by centrifugation,
and resuspended in 6 µl of 87% formamide, Tris borate-EDTA buffer,
0.1% xylene cyanol, and 0.1% bromphenol blue (sample buffer). Half of
the mixture was heat-denatured for 2 min at 95 °C and resolved by 7 M urea, 20% PAGE.
tRNALys,3 Gene Sequencing--
Plasmid carrying
tRNALys,3 gene (7.5 µg) was alkaline-denatured and
ethanol-precipitated in the presence of 5'-32P primer SA-12
(30 pmol). The precipitate was resuspended in H2O (6 µl)
and added to a solution containing 10 mM dithiothreitol, 1× Sequenase buffer and 3 units of Sequenase. Sequencing reactions were initiated by addition of this mixture (3.5 µl) to 0.8 µl of
dNTP (2.5 mM of each) and 0.5 µl of ddNTP (1 mM). This reaction was performed for each ddNTP. Mixtures
were incubated at 37 °C for 15 min and then quenched by addition of
4 µl of sample buffer.
Amplification of Natural tRNALys,3 cDNA--
The
cDNA obtained from natural tRNALys,3 was purified
following 7 M urea, 16% PAGE. The eluted cDNA was
amplified by the RACE (rapid amplification of cDNA ends) method as
described (18), 3' tailed with poly(dA) by terminal transferase, and
PCR-amplified by using poly(dT) oligonucleotide and the primer
SA-12.
Plus Strand DNA Transfer Assay--
The reactions were performed
basically as described above for tRNALys,3 reverse
transcription. The HIV-1 MAL 5' RNA (311 nt; 1 pmol) was annealed to
natural tRNALys,3, synthetic tRNALys,3, RNA PBS
primer, or DNA PBS primer (2 pmol) by incubating with NCp7 (44 pmol;
NCp7 to nucleotide ratio was 1:7) for 10 min at 37 °C in a total
volume of 10 µl. Next, RTp66/p51 (8 pmol), dNTP (0.25 mM
of each), 5'-32P primer R5' (3 pmol), and SA-2 MOD or SA-5
MOD (1 pmol) were added and samples incubated 20, 40, or 60 min at
37 °C in a total volume of 25 µl. Products were resolved by 7 M urea, 6% PAGE.
RT RNase H( tRNALys,3 as Template for Reverse
Transcription--
Plus strand DNA synthesis, which is primed from a
PPT, is believed to proceed as far as the first modified nucleotide of
natural tRNA (1-methyladenosine). This nucleoside is localized just
downstream of the PBS complementary sequence of tRNALys,3
and may constitute the stop signal for RT preceding (+) strand DNA
transfer (Fig. 2B). To investigate this hypothesis, we
compared reverse transcription in vitro of the natural
tRNALys,3 containing modified nucleosides with that of
synthetic tRNALys,3, devoid of modifications. An
oligonucleotide (primer SA-12), complementary to the 10 last
nucleotides of tRNALys,3, was used to prime reverse
transcription of the tRNA by HIV-1 RT in the presence of NCp7. The
first three stops observed with both tRNAs (Fig. 2A) are
probably due to highly stable tRNA structure (Fig. 2B).
Other internal stops are detected only with the natural tRNALys,3; RT is partially stopped by m1A at
position 58 and Tm at position 54 and is almost completely blocked by
the first hypermodified nucleoside present at position 37 in the
anticodon loop: ms2t6A. A high degree of steric
hindrance generated by this nucleoside could explain the extent of this
block. After 45 min of incubation, a cDNA extended by one
nucleotide beyond position 38 can be detected. This may result
from the 3' terminal transferase activity of RT, resulting in
nontemplated blunt-end 3' addition of nucleotides (19-21). In contrast
to natural tRNALys,3, the synthetic tRNALys,3
does not impede RT progress. cDNA synthesized from the synthetic tRNALys,3 is the same length as tRNA and we suppose this
product can be self-primed by RNA folding back at the 3' end (22, 23)
to yield the additional 125-nucleotide cDNA.
In the absence of NCp7, natural tRNALys,3 reverse
transcription is almost completely halted by m1A-58,
whereas reverse transcription of synthetic tRNALys,3
remains complete but less efficient (data not shown). These results in
the absence of NCp7 are similar to those of Yusupova et al. (24). The data suggest that modified nucleosides stabilizing the
natural tRNALys,3 structure (25) impede RT progression.
Sequence Analysis of cDNA from Natural
tRNALys,3--
We sequenced the cDNA obtained from
reverse transcription of natural tRNALys,3 to determine RT
fidelity when reading modified nucleosides. The longest cDNA
obtained by reverse transcription of natural tRNALys,3 was
purified, amplified, and then cloned for sequencing. Table II reports
36 sequences thus obtained. The most frequently observed error
was the reverse transcription of m1A-58 into A (32/36).
Surprisingly, Tm-54, which partially arrests RT, analogous to
m1A-58 (Fig. 2A), is correctly transcribed into
an A. We also found a few errors at m7G-46 (transcribed
into T, 4/36) and possible random errors: m5C-50
transcribed into A (1/36) and A-51 transcribed into C (1/36). In
addition to these errors, we found three cases of single nucleotide additions at the 3' end of the cDNA (Table
II), confirming our hypothesis of
nontemplated blunt-end addition of nucleotides by RT. These results
indicate that, among modified nucleosides present between positions 38 and 76 of natural tRNALys,3, only m1A-58 is
frequently reverse-transcribed incorrectly.
Analysis of the (+) Strand DNA Transfer Mechanism--
The (+)
strand DNA transfer event was studied using an in vitro
system schematized in Fig. 1B.
The RNA template (HIV-1 5' RNA, 311 nt) for ss-cDNA synthesis
contained R, U5, and part of the encapsidation/dimerization signal.
ss-cDNA synthesis was primed by natural tRNALys,3,
synthetic tRNALys,3, an RNA PBS primer, or a DNA PBS
primer. RNA template was preincubated separately with these four
primers in the presence of NCp7 and RT and dNTP added to synthesize
ss-cDNA. Simultaneous addition of a DNA primer
(5'-32P-primer R5') complementary to the 3' end of the
ss-cDNA allowed for (+) strand DNA synthesis while the
oligodeoxynucleotide ODN 2 (50 nt) was available as the template for
strand transfer. Transfer resulted in the synthesis of a 228-nt
5'-32P product. Since all polymerization reactions were
performed in a single tube, ODN 2 was 3' blocked by a dideoxy adenosine
(ddA) to prevent its use as primer for ss-cDNA synthesis.
Experiments were first performed without acceptor template ODN 2 to
analyze events preceding (+) strand DNA transfer. (+) strand DNA
synthesis with natural tRNALys,3 revealed several
intermediates (Fig. 3A, lanes 1-3).
The 180- and 192-nt products are generated by termination at the 2nd
and 14th nt of the PBS (Fig. 3B), respectively, and are
probably due to the incomplete unwinding of the tRNA-PBS RNA complex.
The three last termination sites (196-, 200-, and 217-nt products)
correspond exactly to the previously observed stops of RT during
reverse transcription of natural tRNALys,3 (Fig.
2A). Thus, the 196-, 200-, and
217-nt products are due to the presence, in the natural
tRNALys,3, of m1A-58, Tm-54, and
ms2t6A-37, respectively (Fig.
3B). The same assay performed
using the synthetic tRNALys,3 reveals a different pattern
(Fig. 3A, lanes 8-10). While 180- and
192-nt products are observed as with natural tRNALys,3,
products of 196, 200, and 217 nt are absent, as expected. Instead, a
254-nt product is generated by complete reverse transcription of the
synthetic tRNALys,3. Results obtained with the RNA PBS
primer (Fig. 3A, lanes 15-17) are
similar to those for natural tRNALys,3 except that no
product was larger than 196 nt representing complete reverse
transcription of the RNA PBS primer. Finally a single product is
observed when the DNA PBS primer is used (Fig. 3A, lanes 22-24), reverse transcription terminating
at the end of the DNA PBS primer. These data support conclusions made
from Fig. 2 concerning the influence of modified nucleosides on reverse transcription of tRNA and show that RT is held up by RNA-RNA duplexes (tRNA-PBS) as opposed to DNA-RNA duplexes (DNA PBS primer-PBS) in
agreement with the results of Ben-Artzi et al. (6).
To analyze (+) strand DNA transfer in vitro, the acceptor
template ODN 2 was added to the reaction mixtures. (+) strand DNA transfer occurred efficiently only with natural tRNALys,3
(Fig. 3A, lanes 4-6) and RNA PBS
primer (Fig. 3A, lanes 19 and 20) reaching 15% and 13% after 60 min of incubation time,
respectively. Using synthetic tRNALys,3, (+) strand DNA
transfer was hardly detectable (Fig. 3A, lanes 11-13), whereas no transfer product was observed using the
DNA PBS primer (Fig. 3A, lanes
25-27). These results clearly indicate a role for modified
nucleosides of tRNALys,3 in the (+) strand DNA transfer
process. Since (+) strand DNA transfer may be facilitated by the RNase
H-catalyzed removal of the tRNA primer (26, 27), we propose that the
arrest of RT at m1A-58 of the tRNA is probably required for
tRNA cleavage. Even when RT was completely halted at the end of the PBS
by use of the DNA PBS primer, (+) strand DNA transfer was not detected
while it did occur efficiently with the RNA PBS primer. The above data suggest that removal of the primer for ss-cDNA synthesis by RNase H
is an obligatory step for (+) strand DNA transfer and cannot occur if
this primer is DNA. Parallel experiments carried out in the absence of
NCp7, indicate that NCp7 is an essential component in this in
vitro system (Fig. 3A, lanes 7,
14, 21, and 28). All assays shown in
Fig. 3A were repeated with an alternative acceptor template
(SA-5 MOD, see "Experimental Procedures") composed of the ( RNase H Activity Enhances (+) Strand DNA Transfer in
Vitro--
The absence of (+) strand DNA transfer with a DNA PBS
primer strongly suggested a role for the RNase H activity of RT in
strand transfer. To further investigate the requirement of RNase H for the (+) strand DNA transfer, we used an RNase H-defective RT mutant (mutation E478Q) (13). ss-cDNA was synthesized with natural tRNALys,3 using wild type RT, purified to eliminate the
enzyme, and used as template for (+) strand DNA synthesis and (+)
strand DNA transfer catalyzed by either RNase H( Sequence Analysis of (+) Strand DNA Transfer Products--
As
shown above with natural tRNALys,3, m1A-58 is
not an absolute stop site for RT before strand transfer, and in the
case of synthetic tRNALys,3 RT transcribes the complete
tRNALys,3 (Fig. 3A). We thus sequenced the (+)
strand DNA transfer products obtained from natural
tRNALys,3, synthetic tRNALys,3, and DNA PBS
primer in order to precisely determine the transfer site and eventual
nucleotide misincorporations at the transfer site. With acceptor
template SA-2 MOD (sequence 179-228) and natural tRNALys,3, there were no errors among 14 clones (Table
III). With synthetic tRNALys,3, 2 sequences out of 13 had nucleotide
misincorporations located at the transfer site. These errors correspond
to the reverse transcription of 3 additional nucleotides of the tRNA
(A-58, A-57, and C-56) that replace 3 or 4 nucleotides of the sequence
downstream of the PBS. Although no strand transfer product was visible
using the DNA PBS primer (Fig. 3), PCR did allow amplification of
products. Sequencing of 17 clones revealed 12 wild type sequences and 5 mutated sequences. One of these mutated sequences had an insertion of 9 nucleotides after the PBS, corresponding to the 9 last nucleotides of
the PBS. This insertion may have been due to misalignment of annealed
PBS sequences. The four other sequences had insertions of one or two
complete PBS domains but these products could not have been due to a
transfer process. With the other acceptor template SA-5 MOD (( Effect of Deletions at the 5' End of the Acceptor Template ( Immediatly prior to (+) strand DNA transfer, the 3' end of
tRNALys,3, still attached to the 5' end of ( (+) strand DNA transfer was reconstituted in an in vitro
system using a 3' blocked acceptor template. The advantage of the present system (Fig. 1B) is that the PBS segment of the (+)
RNA template remains hybridized to tRNALys,3 as it should
be in vivo, while this was not the case in previously used
transfer systems (6, 7). Furthermore, this system contains NCp7, known
to coat the genomic RNA in virion (33) and to greatly enhance ( (+) ssDNA synthesis performed in the presence of acceptor template for
strand transfer revealed that stalling of RT at m1A-58 is a
prerequisite for efficient (+) strand DNA transfer, since efficiency of
transfer was low with synthetic tRNALys,3 relative to
natural tRNALys,3. In fact, reverse transcription is not
completely arrested by m1A-58, indicating that (+) strand
DNA transfer is not sufficiently rapid to prevent continued
transcription of the tRNA beyond m1A-58. The use of an RNA
PBS primer confirmed this observation since transfer was as efficient
as transfer with natural tRNALys,3. In contrast, a DNA PBS
primer generated only very low levels of transfer product, suggesting
that (+) strand DNA transfer strictly requires RNA as primer for
ss-cDNA synthesis, probably to allow removal by RNase H. This
hypothesis was confirmed by using an RT mutant deficient of RNase H
activity (mutation E478Q) (13). In the absence of RNase H activity, (+)
strand DNA transfer occurred but with markedly decreased efficiency.
Thus, RNase H activity greatly enhances strand transfer and RT seems to
be able to displace the 3' end of tRNALys,3 from the (+)
PBS, even without tRNA cleavage. It has been shown that
tRNALys,3 is cleaved near DNA/RNA junction, leaving one
ribonucleotide A at the 3' end of ( The efficiency of (+) strand DNA transfer is equally dependent on base
pairing between the 3' end of (+) ssDNA and acceptor template. Indeed,
a two-nucleotide deletion at the 5' end of ( Another aim of this work was to analyze mutagenesis at the (+) strand
DNA transfer site. Sequencing of (+) strand DNA transfer products
revealed that m1A-58 is also important for the fidelity of
transfer. Transfer with natural tRNALys,3 showed only two
distinct nucleotide misincorporations immediately downstream of the
PBS. These mutations may not result from transfer errors but could
instead be due to nontemplated blunt-end nucleotide addition by RT.
Such RT terminal transferase activity has been incriminated in
nucleotide misincorporation events during ( Taken together, the data herein suggest that the absence of methylation
on A-58 of natural tRNALys,3 would allow mutations
contributing effectively to variability of the HIV genome. The
interesting observation by Morin et
al.2 that
tRNALys,3 of chronically HIV infected cells is drastically
hypomodified lends support to this hypothesis.
) PBS of the acceptor template and the RNase H activity of RT also influence the
efficacy of (+) strand DNA transfer.
INTRODUCTION
Top
Abstract
Introduction
References
) strand DNA
synthesis. (+) strand synthesis is primed by an RNase H-resistant
sequence, the polypurine tract (PPT), and continues as far as a
double-stranded RNA region corresponding to the 18-nt tRNA-viral PBS
RNA complex. RT needs to unwind this tRNA-PBS RNA to reverse-transcribe
the 3' end of primer tRNA. The stop signal for this (+) strand
synthesis has been proposed to be the methylated adenosine at position
58 (m1A-58) of primer tRNA (4, 5) but it is not the
absolute termination site of RT in the case of HIV (6, 7). Termination
at m1A-58 results in the (+) strong stop DNA species
carrying a complete (+) PBS sequence at its 3' terminus. Before (+)
strand DNA transfer, the tRNA primer is thought to be removed by the
RT-associated RNase H activity (7-11); thus, the (+) PBS at the 3' end
of (+) ssDNA becomes available for hybridization to the (
) PBS
synthesized at the 3' end of the (
) strand DNA, and synthesis of both
strands resumes with strands copying each other (Fig.
1A).
) PBS of acceptor template. We also
examined the fidelity of (+) strand DNA transfer by comparing transfer
products obtained with an in vitro system using the
following primers: natural tRNALys,3 containing
post-transcriptional modifications, synthetic tRNALys,3
lacking modifications, or oligonucleotides (RNA and DNA) complementary to the PBS.
EXPERIMENTAL PROCEDURES
-32P]ATP and purified by 12% PAGE in 7 M
urea.
DNA and RNA oligonucleotides used in the present study
) reverse
transcriptase (RT p66/p51; E478Q) (13) were purified from
Escherichia coli (14).
) Assay--
ss-cDNA was first synthesized by
wild type RT as above, in the absence of 5'-32P primer
SA-18 and acceptor template, using a 30-min incubation time.
ss-cDNA product was phenol/chloroform-extracted,
ethanol-precipitated, and resuspended in 10 µl of a mixture
containing 5'-32P primer SA-18 (3 pmol) and NCp7 (44 pmol)
and incubated 10 min at 37 °C for annealing. (+) ssDNA synthesis and
(+) strand DNA transfer were then initiated by addition of wild type RT
or RNase H(
) RT as follows: wild type RT or RNase H(
) RT (8 pmol),
dNTP (0.25 mM each), and SA-2 MOD were added, and samples
were incubated 30 min at 37 °C in a total volume of 25 µl.
RESULTS
Errors upon reverse transcription of the natural tRNALys,3
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Fig. 1.
A, scheme of retroviral reverse
transcription including (+) strand DNA transfer. Steps are explained in
more detail in the introduction. 1, strong stop cDNA
(ss-cDNA) synthesis is initiated by a specific cellular tRNA at the
primer binding site (PBS). 2, ( ) strand DNA transfer and
(
) strand DNA synthesis. 3, before (+) strand DNA
transfer: (+) strong stop DNA ((+) ssDNA) synthesis is initiated by the
polypurine tract (PPT) primer. 4, after (+) strand DNA
transfer: (+) strand DNA synthesis. B, summary of HIV-1 (+)
strand DNA transfer system. 1, the HIV RNA template (R, U5,
PBS, and part of encapsidation/dimerization signal) is preincubated in
the presence of tRNALys,3 and NCp7. 2,
tRNALys,3 is annealed to the PBS of HIV RNA template.
3, ss-cDNA synthesis occurs in the presence of HIV-1 RT
and dNTP. 4, the concomitant degradation of the RNA template
by the RNase H activity of RT allows annealing of the
5'-32P primer R5' (complementary to the 3' end of
ss-cDNA). 5, (+) strand DNA synthesis is initiated by
the 5'-32P primer R5'. 6, A second
oligodeoxyribonucleotide (ODN 2) acts as the acceptor template for
strand transfer by RT. The acceptor template possesses a
dideoxyadenosine at its 3' end to prevent its use as primer for
ss-cDNA synthesis.
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Fig. 2.
Primer tRNALys,3 as template for
reverse transcription by HIV-1 RT. A, natural bovine
tRNALys,3 (lanes 1 and 2)
or synthetic bovine tRNALys,3 (lanes
3 and 4) were hybridized individually to a 10-mer
DNA primer (SA-12, complementary to tRNA positions 67-76) at 37 °C
in the presence of NCp7. 5'-32P-labeled primer-tRNA
complexes were incubated with HIV-1 RT for 15 and 45 min as described
under "Experimental Procedures." Size markers were obtained by
tRNALys,3 gene sequencing using 5'-32P-labeled
primer SA-12 (see "Experimental Procedures"). B, bovine
tRNALys,3 cloverleaf structure.
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Fig. 3.
Analysis of (+) strand DNA transfer by HIV-1
RT. A, (+) strand DNA transfer was performed as
described for Fig. 2A. The ss-cDNA primer was natural
tRNALys,3 (lanes 1-7), synthetic
tRNALys,3 (lanes 8-14), RNA PBS
primer (lanes 15-21), or DNA PBS primer
complementary to (+) PBS (lanes 22-28). Only the
(+) strand DNA primer (primer R5') was 32P-labeled;
consequently, only (+) strand DNA synthesis and (+) strand DNA
transfer products are labeled. Reactions were performed with
(lanes 4-7, 11-14,
18-21, and 25-28) or without acceptor template
ODN 2 (lanes 1-3, 8-10,
15-17, and 22-24) and in the presence
(lanes 1-6, 8-13, 15-20,
and 22-27) or in the absence of NCp7 (lanes
7, 14, 21, and 28).
Expected products are (+) strong stop DNA ((+) ssDNA; 196 nucleotides)
and the strand transfer product (228 nucleotides). B, scheme
of products obtained with natural tRNALys,3 in the absence
of acceptor template. The ss-cDNA, including the primary structure
of tRNALys,3, is also schematized.
) PBS
sequence followed by a random sequence, with identical results (data
not shown). Thus, the (+) strand DNA transfer mechanism is independent
of acceptor template sequence between positions 197 and 228.
) RT or wild type RT
(Fig. 4). The amount of (+) strand DNA
transfer product was clearly decreased using RNase H(
) RT (Fig. 4,
lane 4) compared with that with wild type RNase
H(+) RT (Fig. 4, lane 2), thus indicating that
RNase H activity of RT is necessary for efficient (+) strand DNA
transfer. These results are in agreement with the model proposed above
in which tRNALys,3 is released by RNase H activity to
facilitate (+) strand DNA transfer. In addition, when RNase H(
) RT
was used, the amount of the 200-nt product, generated by RT arrested by
Tm at position 54 of primer tRNALys,3 (see Figs. 2 and 3),
was remarkably increased. This can be due to an extensive copying of
primer tRNALys,3 in the absence of release of
tRNALys,3 by RNase H activity of RT.
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Fig. 4.
(+) strand DNA transfer requires RNase H
activity. Reactions were carried out with natural
tRNALys,3, wild type RT, or RNase H( ) mutant RT. For all
assays, ss-cDNA was first synthesized by wild type RT and then
purified prior to the (+) ssDNA synthesis and (+) strand DNA
transfer.
) PBS
followed by a random sequence), sequencing gave generally similar data
(Table III). Among 17 clones using natural tRNALys,3 as
primer, 2 sequences had one nucleotide misincorporation located at the
transfer site, where the A immediately downstream of the PBS had been
replaced by G or T. These mutations are probably due to the 3' terminal
transferase activity of RT (19-21). With synthetic
tRNALys,3, 1 sequence out of 5 was mutated by insertion of
24 nucleotides resulting from the reverse transcription of 11 additional nucleotides of tRNA (A-58 to G-48), followed by partial
annealing to the (
) PBS of the acceptor template. Finally, with the
DNA PBS primer, 2 sequences out of 6 had one nucleotide deletion at the
transfer site and 3 sequences had an insertion of the whole PBS
sequence. Taken together, these data confirm that (+) strand DNA
transfer is independent of acceptor template sequence, except for the
PBS segment.
Sequencing data of (+) strand DNA transfer products obtained with three
distinct () ssDNA synthesis primers and two distinct acceptor
templates (ODN 2): underlined sequences are mutated
)
PBS--
Liang et al. (29) showed that HIV-1 containing
deletions at the 3' end of the (+) PBS of the viral genomic RNA,
exhibited an attenuated replication phenotype. This was shown to be
caused by the lower efficiency of tRNALys,3 placement onto
viral genomic RNA and the possibility of an effect on (+) strand DNA
transfer was also evoked, but not testable in vivo. In order
to investigate this possibility in our in vitro system, we
used acceptor templates containing 5' (
) PBS deletions (Table I and
Fig. 5A). Interestingly, (+)
strand DNA transfer was markedly affected by a 2-nt 5' (
) PBS
deletion (Fig. 5B, lane 3) or a 4-nt
5' (
) PBS deletion of the acceptor template (Fig. 5B,
lane 4) in support of the hypothesis of Liang
et al. (29). To determine whether the complete PBS sequence
can be restored by the appearance of compensatory deletions downstream of the PBS as observed by Liang et al. (29), we sequenced
transfer products. In contrast to the results of Liang et
al. (29), we did not find products possessing wild type PBS
sequence followed by a deletion (Table
IV). Furthermore, we observe a
predominance of wild type sequences lacking any deletions. The
discrepancy between these in vitro data and the in
vivo results of Liang et al. (29) suggests that
additional factors may be involved in (+) strand DNA transfer
in vivo.
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Fig. 5.
Effect of deletions at the 5' end of the ( )
PBS of acceptor template. A, schematic representation
of the transfer step of in vitro system and templates with
deletion at the 5' end of the PBS. B, (+) strand DNA
transfer assays were performed with or without wild type acceptor
template (lanes 2 and 1, respectively)
or two mutated acceptor templates containing 2- or 4-nt 5' (
) PBS
deletions (lanes 3 and 4,
respectively) as described in Fig. 1B.
Sequencing data of (+) strand DNA transfer products obtained with two
distinct acceptor templates
DISCUSSION
) strand DNA,
is reverse-transcribed. To shed light on this specific aspect of (+)
strand DNA transfer, we investigated the ability of HIV RT to
reverse-transcribe tRNALys,3 alone. The reverse
transcription of natural tRNALys,3 shows three RT
termination sites due to modified nucleosides m1A-58,
Tm-54, and ms2t6A-37, while these stops are not
observed with synthetic tRNALys,3. Sequencing of cDNA
products revealed that m1A is always transcribed
incorrectly into A. In contrast, other modified nucleosides between
m1A-58 and ms2t6A-37, comprising
Tm,
, m5C, D, and m7G are transcribed
correctly except for occasional transcription of m7G-46
into T. This indicates that post-transcriptional modifications in
natural tRNALys,3 not interfering with Waston-Crick base
pairing, as is the case for these five last modified nucleosides, do
not affect faithful reverse transcription. For m7G-46, the
positive charge due to methylation on its N-7 (30) may occasionally
interfere with reverse transcription, giving rise to a T. It should be
pointed out that although Tm-54 partially stops RT, it does not cause
nucleoside misincorporation. Tm-54 possesses two methylations (one on
the base, the other on the ribose) that have no effect on atoms
implicated in Waston-Crick base pairing. However stabilization of the
C3' endo rather than the usual C2' endo conformer by the
2'-O-methylation on ribose (31) may explain the difficulty
of RT to pass through this modified nucleoside. These results are
consistent with previous work (32), in which reverse transcription of
yeast tRNAPhe by the Klenow fragment of E. coli
DNA polymerase I demonstrated that m1A-58 partially
prevents elongation and that Y-base (wybutosine), a highly modified
nucleoside as ms2t6A, was able to block DNA
polymerase. However, the Klenow polymerase nonspecifically transcribed
m1A-58 into the four bases while, in our case, HIV RT
incorporates only A. Polymerases thus do not read m1A-58 in
a universal manner; the positive charge of m1A-58 (30) may
interact differently with the active sites of these two enzymes.
)
strand DNA transfer (2, 22, 34, 35). ss-cDNA was synthesized by
using four distinct primers: natural tRNALys,3, synthetic
tRNALys,3, an RNA PBS primer, and a DNA PBS primer. The
nature of (+) ssDNA synthesis products was dependent upon the primer
used. With natural tRNALys,3, (+) strand DNA synthesis was
partially terminated at nucleosides m1A-58 and Tm-54 and
was totally halted by ms2t6A-37. As expected,
these stops did not occur with synthetic tRNALys,3 where
(+) strand DNA synthesis continued as far as the 5' end of the tRNA.
With RNA PBS or DNA PBS as primer, full-length (+) ssDNA was obtained.
Two common products were synthesized, from all primers except DNA PBS
primer, generated by stops occurring at bases 2 and 14 of the PBS.
These results indicate that RT stalls, even in the presence of NCp7, at
the double-stranded 3' tRNALys,3-PBS RNA complex, but not
at the RNA/DNA heteroduplex composed of the DNA PBS primer and viral
RNA PBS. The difficulty of RT to catalyze strand displacement synthesis
through RNA/RNA duplexes has also been shown by Ben-Artzi et
al. (6) in a test performed without NCp7. Thus, while NCp7 is not
solely responsible for this phenomenon, its stabilization of
intermolecular nucleic acid duplexes (36) might exacerbate it. In
contrast, it is clear that NCp7 is essential for synthesis of (+) ssDNA
in our in vitro system. This can be explained by the well
established ability of NCp7 to promote annealing of the
tRNALys,3 to the PBS (reviewed in Ref. 3) as well as to
augment efficiency and processivity of RT (23, 34, 37-40).
) strand DNA (9) but the mechanism
is still unclear. Since polymerase and RNase H active sites are
separated by 18 nt, a single RT molecule could simultaneously
transcribe the first 18 nt of tRNA and cleave the tRNA. Alternatively,
one RT molecule might transcribe tRNA and another might rebind the RNA-DNA duplex to cleave the tRNA, as suggested for PPT release (41).
Consistent with data herein for (+) strand DNA transfer, Blain et
al. (42) suggested that (
) strand DNA transfer may require not
only the RNase H activity of RT to degrade the RNA template but also an
unwinding activity of RT to dissociate the small residual RNA fragment
remaining annealed to the 3' end of the ss-cDNA that cannot be
degraded because of the distance between polymerase and RNase H active sites.
) PBS in the acceptor
template that allows mispairing between the last two bases of (+) ssDNA
and acceptor template is sufficient to drastically decrease the amount
of transfer product. This result is not surprising since a mismatch at
the 3' end of a primer affects the kinetics of its extension by RT (23,
43). These observations are consistent with the hypothesis proposed to
explain low replication of HIV-1 containing deletions at the 3' end of
the PBS (29). However, in contrast to these in vivo results,
we did not observe reversion to a wild type PBS sequence in our system
in the form of concomitant deletion of nucleotides downstream of the
PBS. The efficiency of in vitro (+) strand DNA transfer in
our hands (10-15%) is also lower than that observed in infected cells
(80-85%) (44). These differences may indicate that other factors
influence (+) strand DNA transfer in vivo.
) strand DNA transfer (19,
20), as well as during internal strand transfers necessary for
recombination by a forced copy choice mechanism (21). In contrast,
transfer with synthetic tRNALys,3 or a DNA PBS primer
revealed more frequent mutations. With synthetic tRNALys,3,
we observed insertions of several nucleotides complementary to
tRNALys,3 downstream of G-59 that can be
reverse-transcribed because of the absence of methylation on A-58. With
the DNA PBS primer, we noted single nucleotide deletions downstream of
the PBS, possibly due to incomplete displacement of the DNA PBS primer
from (+) ssDNA allowing a misalignment between the (+) PBS and acceptor (
) PBS.
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ACKNOWLEDGEMENTS |
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We thank Damien Ficheux (IBCP, Lyon, France) for providing nucleocapsid protein NCp7. We thank Michael Rau and Henri Grosjean for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Agence Nationale de Recherches sur le SIDA, the Association pour la Recherche sur le Cancer, and Sidaction.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. Tel.:
33-4-72-72-81-69; Fax: 33-4-72-72-86-86; E-mail:
Jean-Luc.Darlix{at}ens-lyon.fr.
The abbreviations used are:
RT, reverse
transcriptase; PBS, primer binding site; HIV-1, human immunodeficiency
virus type 1; PPT, polypurine tract; NCp7, nucleocapsid protein; ODN, oligodeoxyribonucleotide; m1A, 1-methyladenosine; Tm, 5,
2'-O-dimethyluridine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; , pseudouridine; m5C, 5-methylcytidine; D, dihydrouridine; m7G, 7-methylguanosine; Y, wybutosine; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; ss, strong
stop; MOD, 3' modified.
2 A. Morin, C. Simon, F. Subra, and H. Grosjean, personal communication.
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REFERENCES |
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