From the Eccles Institute of Human Genetics and the
Department of Biochemistry, University of Utah, Salt Lake City, Utah
84112 and the
Center for Comparative Medicine, University of
California, Davis, California 95616
Received for publication, July 18, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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Many retroviruses either encode dUTP
pyrophosphatase (dUTPase) or package host-derived uracil DNA
glycosylase as a means to limit the accumulation of uracil in DNA
strands, suggesting that uracil is detrimental to one or more steps in
the viral life cycle. In the present study, the effects of DNA
uracilation on ( Human immunodeficiency virus type 1 (HIV-1),1 like other
retroviruses, is a plus-stranded RNA virus that replicates through a
DNA intermediate. Viral reverse transcriptase (RT), a DNA polymerase that uses RNA and DNA templates, converts the viral RNA to
double-stranded DNA after host cell infection (Fig.
1). This DNA is then integrated into a
host cell chromosome, where it serves as a template for synthesis of
new viral RNA. Full-length viral RNA and some viral gene products are
subsequently packaged into progeny virions (reviewed in Refs. 1 and
2)
) strand DNA synthesis, RNase H activity, and (+)
strand DNA synthesis were investigated in a cell-free system. This
system uses the activities of purified human immunodeficiency virus
type 1 (HIV-1) reverse transcriptase to convert single-stranded
RNA to double-stranded DNA in a single reaction mixture. Substitution
of dUTP for dTTP had no effect on (
) strand synthesis but
significantly decreased yields of (+) strand DNA. Mapping of nascent
(+) strand 5' ends revealed that this was due to decreased initiation
from polypurine tracts with a concomitant increase in initiation at
non-polypurine tract sites. Aberrant initiation correlated with a
change in RNase H cleavage specificity when assayed on preformed
RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate
"selection" of the (+) strand primer is affected. Collectively,
these data suggest that accumulation of uracil in retroviral DNA may
disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RNase H activity is
required for retroviral DNA synthesis. RNA (gray lines)
is converted to DNA (black lines) by RT in the accepted
model of retroviral DNA synthesis (1). Arrowheads indicate
the 3' direction of DNA polymerization. RNase H cleavage events
are indicated by the scissors. The precise cleavage event
required to liberate the 3' end of the 3'-ppt to serve as a (+) strand
synthesis primer is shown by the black scissors. DNA
synthesis from the ppt and the subsequent ppt primer removal must be
precise to faithfully generate the U3 end of the 5' long terminal
repeat (gray ovals). Genomic R, U5, PBS, PPT, and U3 regions
are indicated, with lowercase and uppercase
letters corresponding to (+) and ( ) strand, respectively.
For simplicity, the central ppt is omitted. Steps recapitulated in the
cell-free assay described in the legend for Fig. 2 are shown in the
gray box. The (
) strand DNA product of step 2 is shown as an intermediate having a 3' end in U3. This approximates
the positions of primers HIV 9091 and FIV 9133 used in the cell-free
assay (Fig. 2).
In HIV-1, both minus () and plus (+) strand DNA synthesis initiate
from RNA primers (Fig. 1). The former requires a cellular tRNA annealed
near the 5' end of the genomic RNA (Fig. 1, step 1), whereas
primers for the latter are generated by specific RNase H-mediated
cleavage of viral RNA (indicated in Fig. 1 with scissors). Following (
) strand DNA synthesis, selective degradation of viral RNA
primers leaves short (+) strand RNA fragments annealed to the (
)
strand DNA (3). One of these fragments, a highly conserved, purine-rich
sequence referred to as the 3' polypurine tract (3'-PPT), is located
near the 3' end of the genome and is used as an obligatory primer for
(+) strand synthesis in all retroviruses (Fig. 1, step 4).
Precise cleavage at (Fig. 1, black scissors) and initiation from the 3'-PPT is required to define the 5' end of the "left" long
terminal repeat and maintain the viral integrase (IN) attachment site
(att; (2)) (Fig. 1, gray oval). A second copy of
the PPT is located near the center of the genome (cPPT) and is also
used to prime (+) strand DNA synthesis in HIV-1 and other lentiviruses (4-7).
The pol gene in all retroviruses universally encodes RT and IN. However, in some non-primate lentiviruses such as feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV), the pol gene also encodes a dUTP pyrophosphatase (dUTPase) (8). This enzyme hydrolyzes dUTP to dUMP and pyrophosphate and thus provides dUMP as a precursor for de novo synthesis of dTTP in host cells (9). dUTPase also ensures that the dUTP/dTTP ratio is low to minimize uracil incorporation into DNA (9). Previous studies revealed that retroviruses encoding defective dUTPase exhibit delayed replication kinetics in macrophages (10-13) and accumulate G-to-A base substitutions (14, 15). Delayed replication in macrophages resulted from a block in the replication cycle at one or more steps after viral DNA synthesis (10). Thus, it was proposed that dUTPase functions primarily to increase replication efficiency and fidelity. It was further speculated the absence of dUTPase in some retroviruses may contribute to genetic diversity (15).
Interestingly, although primate lentiviruses lack dUTPase, they recruit and package cellular uracil DNA glycosylase (UDG) in the virion through a Vpr- or IN-dependent mechanism (16, 17). UDG removes uracil bases from DNA as part of the base excision repair pathway (9). Although UDG and dUTPase are mechanistically different enzymes, they both function to exclude uracil from DNA strands. This further suggests that uracil accumulation in retroviral DNA is detrimental to some critical step or steps in the viral life cycle.
In this work, the effects of uracilated DNA on () strand DNA
synthesis, RNase H activity, and (+) strand DNA synthesis initiation were evaluated in a cell-free system. This system uses purified RT and
its associated RNase H to convert regions of single-strand RNA to
double-strand DNA in a single incubation (18). In dUTP-containing reactions, DNA synthesis catalyzed by HIV-1 RT proceeded normally, but
the creation and extension of PPT primers were significantly compromised. Failure to correctly "select" and extend the PPT primers was largely explained by additional experiments in which the
effects of uracil on RNase H cleavage specificity were evaluated. The
results showed that cleavage specificity is altered by the presence of
uracil in the DNA strand of a PPT-containing RNA-DNA hybrid. A model is
proposed to explain why retroviruses have mechanisms to avoid uracil
incorporation into viral DNA.
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EXPERIMENTAL PROCEDURES |
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Materials--
The following reagents were from Amersham
Biosciences: Hybond N+ nylon membrane, Sephadex G-50, RNaseT1, RNaseU2,
Ultrapure dNTPs, [-32P]ATP (3000 Ci/mmol),
[
-32P]dCTP (6000 Ci/mmol), Redivue sequencing
terminator mix [
-33P]ddNTPs (1500 Ci/mmol), and
thermosequenase radiolabeled terminator cycle sequencing kit. T7 RNA
polymerase in vitro transcription kits, rNTPs, RNasin, and
pGEM-3Zf(+) were from Promega. T4 polynucleotide kinase and shrimp
alkaline phosphatase were purchased from U. S. Biochemical Corp. Quick
Spin Sephadex G-25 columns were from Roche Molecular Biochemicals.
Plasmids pHIV-pol and pHIV-nef and oligonucleotide 9091 were described previously (18, 19).
Oligonucleotide FIV 9133 is complementary to nucleotides (nt)
9133-9152 of the FIV molecular clone 34TF10 (20). Oligonucleotides
were synthesized by Operon Technologies. Restriction enzymes and
general reagents were from Invitrogen or Fisher.
Recombinant wild-type and RNase H-minus (D443N) HIV-1 RTs were
prepared according to published procedures (18, 19) and were >95%
homogeneous as determined from Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis. The wild-type RT had a specific
activity of 2.8 units/µg on poly(rC)·oligo(dG) (units defined in
Ref. 19) and was ~33% active as determined by titration with
a synthetic U5 long terminal repeat DNA·DNA primer·template (21,
22). FIV RT (1 unit/µl) was purified from the Petaluma virus strain
using the procedure and unit definition of North et al.
(23). All RT preparations were stored in multiple aliquots at
70 °C. Shortly before conducting experiments, their activities were reassessed in dilution and time course assays on each of the
substrates described below. RT concentrations and incubation times were
chosen that spanned the linear range of product formation in each
experiment. Thus, all experiments were conducted under "steady-state" conditions of synthesis in which the products
assayed arose in direct proportion to RT concentration and time.
Preparation of RNA Templates--
pFIV-PPT was created by
subcloning a NheI/SpeI fragment of p34TF10 (nt
8287-9229) into the XbaI site of pGEM-3Zf(+). FIV 3'-PPT RNA (1 kb) was generated by in vitro transcription of
HindIII-linearized pFIV-PPT as recommended by Promega. HIV-1
nef RNA (1.5 kb), containing the 3'-PPT, was
described previously (18). The 73-nt HIV-1 3'-PPT and 61-nt cPPT RNAs
were generated by in vitro transcription of PCR products.
The 5' and 3' primers for the cPPT PCR product were complementary to nt
4772-4789 and 4807-4826, respectively (based on NL4-3 HIV-1
sequence). The 5' and 3' primers for the 3'-PPT PCR product were
complementary to nt 9042-9059 and 9091-9110, respectively. PCR
conditions and product purification were as described (24), except the
annealing temperature was 55 °C. The PCR primers added a T7 RNA
polymerase promoter sequence (5'-TGTAATACGACTCACTATAGGGCGA-3') to the 5' end of each product amplified from pHIV-pol or
pHIV-nef. In some cases, the 73-nt 3'-PPT and 61-nt cPPT
RNAs were precipitated with ethanol and 10 mM
MgCl2 (25), dephosphorylated with alkaline phosphatase, and
5'-end-labeled with [-32P]ATP as described (26), and
free nt were removed by G-25 spin columns. In all cases, RNAs
were purified by 7 M urea/8% PAGE and eluted by the
crush and soak method (25).
Cell-free DNA Synthesis Reactions--
Annealing and extension
steps for processivity reactions were performed as described
(18, 19). See the figure legends for details. The assay for (+) strand
synthesis initiation is described in detail elsewhere (18). Reaction
products were treated with 0.3 N NaOH, neutralized and
passed through Sephadex G-50 spin columns (18), or treated with 45 mM EDTA followed by G-50 spin columns. Products were
resolved by 7 M urea/8% PAGE and transferred to Hybond N+
nylon membranes. Nascent (+) strand DNAs are visualized by
PhosphorImager analysis after hybridization to a radioactively labeled
(+) strand specific probe. The probe was the same oligonucleotide used
as primer for initial () strand synthesis. Thus, the probe will only
detect (+) strand DNAs that have been extended by RT to the 5' end of
the (
) strand DNA initiated from the same primer-probe sequence. Plus
strand DNA 5' ends are identified by their electrophoretic mobility
relative to a sequencing ladder created with the same primer. See the
figure legends for specific conditions.
HIV-1 RT/RNase H Cleavage Assay--
5'-end-labeled
(+) strand RNA (40 nM) was hybridized to a 35-mer ()
strand DNA oligonucleotide (400 nM) in 100 mM
KCl and 40 mM Tris-HCl, pH 8, at 65 °C for 10 min,
37 °C for 15 min, 25 °C for 15 min, and 4 °C for 15 min.
RNA-DNA duplexes were diluted 5-fold and incubated at 37 °C with 0.7 units of HIV-1 RT/ml in 10-µl reactions containing 25 mM
Tris-HCl, pH 8.0, 30 mM KCl, 10 mM
MgCl2, 0 or 100 µM each of dNTP, RNasin (1 unit/µl), and 2 mM dithiothreitol. Reactions were halted
by the addition of an equal volume of 90% formamide, 10 mM EDTA, pH 8.0, 0.1% bromphenol blue, and 0.1% xylene
cyanol. Samples were loaded on prerun 7 M urea/8%
polyacrylamide gels, and bands were visualized by PhosphorImager. RNA
sequencing ladders were generated with RNaseT1 and RNaseU2 or alkaline
hydrolysis according to a protocol kindly provided by Amersham Biosciences.
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RESULTS |
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dUTP Minimally Affects () Strand DNA Synthesis--
To determine
how dUMP incorporation affects reverse transcription, (
) strand DNA
synthesis reactions were examined in a cell-free system (Fig.
2, step 1). A 1.5-kb RNA
derived from the 3' end of the HIV-1 genome was hybridized to a
[5'-32P]-labeled 20-mer DNA primer and incubated for
varying lengths of time with a fixed amount of purified, recombinant
HIV-1 RT (Fig. 3). Two different reaction
sets were performed: one containing all four normal dNTPs (Fig. 3,
labeled dTTP) and a second set in which dUTP was substituted
for dTTP (Fig. 3, labeled dUTP). Synthesis of (
) stand DNA
was performed with an excess of primer-template relative to HIV-1 RT to
ensure steady-state synthesis and facilitate comparison with previous
work (18, 19).
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Resolution of primer extension products by sequencing gels revealed that primer extension terminated at distinct sites called "pause sites," where RT frequently dissociates from the primer-template (19). The pausing pattern in each reaction set remained essentially unchanged as the incubation time increased, and total extension products increased linearly with time up to about 15 min (Fig. 3). Thus, RT and unextended primer-template are in the steady state for ~15 min, and the products result from multiple turnovers initiated at the excess of primer-template. The HIV-1 RT pause pattern was largely unaffected by dUMP incorporation with the exception of a single site where pausing was significantly reduced ~90 nt from the primer terminus (indicated by the filled circle). In addition, the steady-state rates of primer extension (1-3 nt/sec) were comparable in the dTTP and dUTP reaction sets. Similar results were obtained with FIV RT (data not shown). Taken together, these data show that dUMP is incorporated efficiently in place of dTMP by HIV-1 and FIV RTs.
dUMP Decreases Initiation of (+) Strand DNA Synthesis from
PPTs--
The sequence of the central and 3'-PPTs in HIV-1 (+) strand
RNA is 5'-AAAAGAAAAGGGGGG-3' (27). This A-rich sequence is a good
template for dUMP incorporation by RT (Fig. 3), and the uracil in the
complementary () strand DNA may alter the utilization of the PPT for
(+) strand DNA synthesis. This was tested using a cell-free assay that
converts regions of single-strand RNA to double-strand DNA in a single
incubation (Fig. 2, steps 1-3). This
RT-dependent system recapitulates several of the required intermediate steps of retroviral DNA synthesis: RNA-templated (
)
strand DNA synthesis, RNase H cleavage and preferential (+) strand
initiation at PPTs, and subsequent DNA-templated (+) strand DNA
synthesis (18). Briefly, HIV-1 or FIV RNAs containing PPTs are
hybridized to oligonucleotide primers and incubated with RT in the
presence of either a dTTP- or a dUTP-containing dNTP set. More RT is
required in these reactions to increase yields of (
) strand DNA
intermediates and in turn achieve steady-state accumulation of nascent
(+) strand DNAs. Half of the products of each reaction are subjected to
alkaline hydrolysis to degrade the RNA, and all reaction products are
resolved by denaturing PAGE and transferred to a nylon membrane.
Nascent (+) strand DNA products are detected by probing with
32P-labeled strand-specific oligonucleotides. The 5' ends
of nascent DNA or RNA-DNA chimeras (nascent DNAs still attached to RNA
primers) are identified by mapping against sequence ladders.
Formation of (+) strand DNA requires () strand DNA synthesis and
yields three groups of reaction products (Fig. 2, step 3 products, and Fig. 4, P1,
P2, and P3). P1 bands are nascent (+) strand DNAs
liberated from the PPT primer by RNase H. P2 bands are (+) strand DNAs
covalently linked to the PPT RNA primer. The bands labeled
P3 reflect various (+) strand DNAs initiated from non-PPT
primers upstream (5') of the PPT. The P2 bands and several of the P3
bands disappeared and/or showed an increased gel mobility after NaOH
treatment (Fig. 4A, +NaOH lanes), demonstrating
that RNA primers were still linked to the nascent (+) strand DNAs and not yet removed by RT/RNase H. Specific (+) strand DNA initiation products were not detected in reactions where an RNase H-minus HIV-1 RT
mutant was used (D443N; (18)) or in reactions lacking the (
) strand
DNA primer (data not shown).
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Initiation of (+) strand DNA synthesis from the HIV-1 3'-PPT required
RT and dNTPs (Fig. 4A, () lanes and data not
shown). In reactions containing dTTP in the absence of dUTP, two PPT
initiation products of similar intensity were detected with 5' ends
mapping to the last G residue and the adjacent A residue at the 3' end of the PPT (Fig. 4A,
NaOH lanes, bands labeled
P1). NaOH treatment yielded two additional P1 bands (Fig.
4A, +NaOH lanes, dTTP-containing reactions),
revealing the four-band pattern observed previously (18). Substitution
of dUTP for dTTP in these reactions significantly decreased the amount
of P1 and caused a slight increase in the mobility of the P1 bands,
presumably due to the absence of the C5 methyl group in uracil (Fig.
4A,
NaOH lanes, dUTP reactions). Reactions containing dUTP yielded shorter P2 products (Fig.
4A). In addition, the intensity of non-PPT initiation
products (P3) increased in reactions containing dUTP relative to the
dTTP-containing reactions (Fig. 4A, right
inset).
Using an FIV RNA-DNA substrate, HIV-1 and FIV RTs recognized and initiated (+) strand DNA synthesis from the FIV PPT (Fig. 4B). The amount of PPT initiation decreased in dUTP-containing reactions, and a decrease in heterogeneity at the PPT 5' end was observed with HIV-1 RT (Fig. 4B, bands labeled P1 and P2, respectively). These data demonstrate that the PPT itself, not the specific RT or flanking sequences (HIV-1 versus FIV), directs (+) strand synthesis initiation in these experiments (see also Refs. 18 and 28-30). Thus, the effect of dUMP on primer formation and recognition by RT is directed predominately by the double-strand PPT region.
PPT-initiated (+) strand DNAs (P1) were detected as early as 10 min
(Figs. 4A and 5A) and accumulated linearly with
time for up to 60 min, whereas the amount of P2 was essentially
constant over the 90-min incubation (Fig.
5A and data not shown).
Presumably P2 never accumulated due to rapid removal of the PPT by
RNase H (31). The rate of P1 accumulation decreased ~3-fold in the dUTP reaction set (Fig. 5A). This decrease must result from
an effect on either RNase H cleavage and/or subsequent PPT primer selection and extension because () strand synthesis is unaffected by
uracil (Fig. 3). Interestingly, the quantity of non-PPT initiated products increased significantly when dUTP was included in the reactions, and their rate of formation increased 3-fold (Fig. 5B). Taken together, the data in Fig. 4 show that uracilated
DNA diminished specific PPT initiation and concomitantly increased the
rate of non-PPT initiation.
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dUMP in () Strand DNA Decreases RNase H Cutting at the
PPT--
The change in (+) strand DNA initiation specificity imparted
by dUMP incorporation may be the result of altered RNase H activity. An
RT/RNase H activity assay was used to directly investigate the
influence of dUMP on RNase H cleavage of the HIV-1 PPTs (Fig. 6). [5'-32P]-HIV-1 RNAs
(61-mer containing the cPPT or 73-mer containing the 3'-PPT) were
hybridized to 35-mer (
) strand DNAs containing either dTMP or dUMP
(Fig. 6A). These substrates position the polymerase active
site at the 3' end of the (
) strand DNA and the RNase H active site
18 nt behind (i.e. toward the 5' end of the (
) strand) to
allow precise RNA cleavage between the last G residue of the PPT and
the adjacent A residue. The substrate design suppresses cleavage
directed by the 5' end of RNA (26). RNA-DNA hybrids are incubated with
RT and dNTPs for various time points, and the resultant RNase H
cleavage products are resolved by urea-PAGE and visualized by
PhosphorImager. Unlike the concerted assay (Fig. 4), HIV-1 RT is
present in limiting quantities for this experiment to study RNase H
activity under steady-state conditions. Thus, the observed cleavage
products result primarily from hydrolysis of the initial substrate.
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The RNA-DNA duplex containing dTMP was cleaved by RT/RNase H between
the A and G residue precisely at the 3' end of both the 3' and cPPTs,
similar to the positive control lane (+) (Fig. 6, B and
C, reaction set labeled DNA/T and indicated with
an arrow). Note that RNA fragments generated by RNase H
activity have a 3'-hydroxyl, causing them to migrate ~1 nt slower
than the corresponding fragments of the RNA sequence marker that have
3'-phosphates left by RNases U2 and T1 (32). Cleavage also occurred 1 nt inside the 3' end of each PPT. These two RNA fragments are
consistent with the two major (+) strand synthesis initiation sites
observed in Fig. 4. The remainder of the PPT sequence was resistant to
RNase H in these reaction conditions. RT extended the () strand DNA
oligonucleotide, resulting in significant RNA cleavage 14-18 nt from
the 5' end of each RNA substrate (Fig. 6, B and
C, bottom left). The amount of each of these
cleavage products increased with time on the 3' and cPPT substrates. No
cleavage products were detected in the absence of RT (Fig. 6, (
)
lanes) nor in control reactions incubated with HIV-1 D443N
RT lacking RNase H activity (data not shown).
The amount of precise PPT cleavage product decreased on duplexes
containing dUMP in place of dTMP (Fig. 6, reaction sets labeled DNA/U). Analysis of the data indicates that the precise PPT
cut represents only 17 ± 6% of the total cleavage products
observed at and within the 3' and cPPTs. In contrast, on the DNA/T
substrate, the precise cut accounted for 44 ± 8% of the cleavage
products at the PPT (10-min time points). The cleavage pattern 14-18
nt from the 5' end of the RNA was unaffected by uracil in the () strand DNA. Interestingly, the total amount of RNA-DNA/U duplex that
was cleaved by RNase H was equal to or slightly increased relative to
the RNA-DNA/T duplex. Thus, RNase H activity is specifically decreased
only at the 3' end of the PPT in the presence of dUMP and not at other
sites on the RNA/DNA duplex. Additional imprecise RNA cleavage occurred
within the PPT (Fig. 6, B and C) as observed in
Fig. 4A (
NaOH reaction set). However, these shorter PPT
RNA fragments were incapable of serving as (+) strand primers based on
the absence of (+) strand DNA with 5' ends mapping to this sequence
(Fig. 4A, +NaOH reactions). In the absence of DNA synthesis, the total RNase H activity on all PPT substrates increased (data not
shown). In addition, the cleavage specificity decreased, and the PPT
sequence itself was significantly more susceptible to cleavage by RNase
H when the (
) strand DNA contained uracil. In summary, uracilated DNA
causes a decrease in precise PPT cleavage while increasing nonspecific
cleavage within the PPT, demonstrating that the activity of RNase H is
greatly affected by uracil in the DNA/RNA duplex.
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DISCUSSION |
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Lentiviral virions contain enzymes that are predicted to limit the
amount of uracil present in viral DNA, suggesting that incorporated
dUMP may disrupt an essential step in the viral life cycle. In this
work, we investigated whether incorporated dUMP alters primer-template
recognition by RT in biochemical assays of () strand DNA synthesis,
RNase H activity, and (+) strand DNA synthesis initiation.
RTs from both HIV-1 and FIV efficiently utilized dUTP to synthesize
() strand DNA (Fig. 3 and data not shown); neither the steady-state
polymerization rates nor the processivity were altered. Likewise, the
3'-5' RNase H activity of HIV-1 RT (33) was similar on substrates
containing either thymine or uracil bases (data not shown). Thus, dUMP
residues in the primer strand do not impede movement of RT along the
nucleic acid substrate. Polymerization of dUMP is not unique to RT as
other DNA polymerases also readily incorporate dUMP (9). Therefore,
efficient incorporation and subsequent extension of dUMP are general
properties of DNA polymerases.
The effect of uracilated () strand DNA on RT/RNA-DNA substrate
recognition was investigated. When dTTP was replaced by dUTP in the
concerted reaction, there was a significant reduction in (+) strand DNA
synthesis initiation at the PPT and an increase in the number and
intensity of non-PPT initiation sites (Figs. 4 and 5). Analysis of
RNase H activity revealed that the decrease in PPT initiation was the
result of diminished PPT-specific RNase H activity and increased
nonspecific cutting (Fig. 6). Interestingly, the number of (+) strand
DNA products mapping to the HIV-1 PPT 3' periphery increased from two
bands to four bands after NaOH treatment (Fig. 4A). There
are two reasons for this. Primers removed by RNase H activity yield DNA
with 5' phosphates, whereas DNA liberated by NaOH treatment has 5'
hydroxyls (28), and the loss of a phosphate reduces apparent gel
mobility of short DNAs by ~1 nt (32). In addition, imprecise RNase H
activity yields PPT primers shortened at the 3' end by 2-3 nt (Fig.
6B). These primers are extended by RT during (+) strand
synthesis, but unlike the full-length PPT primer, they are not
efficiently removed by RNase H (Fig. 4A) (34). Hence, the
resultant (+) DNA (2-3 nt longer than precisely initiated (+) DNA) is
visualized only after NaOH removal of the primer.
Minor groove-contacting amino acid residues are important for the interaction of HIV-1 RT with nucleic acid substrates (35, 36). These residues also influence PPT primer recognition and cleavage and affect interactions with DNA containing minor groove adducts (37, 38). Because the RNase H active site contacts the RNA backbone near the minor groove of the nucleic acid (36, 39), alteration of the minor groove width and/or depth may modulate RNase H specificity. A recent chemical footprinting analysis of the HIV-1 PPT structure revealed that the PPT duplex contains a pre-existing structural distortion that correlates with precise PPT cleavage (40). Nuclease accessibility data indicate that removal of the thymine C5 methyl group (to form uracil) widens the minor groove in duplex DNA (41). Thus, uracil presumably alters the PPT structure and changes the specificity of RT/RNase H activity. RNase H activity appears to be more sensitive to the structural changes imparted by uracil than polymerase activity as processive DNA synthesis is not impaired. Biophysical analysis of RT complexed with T- or U-containing PPT substrates could test these ideas.
Our data show that initiation of (+) strand synthesis at the HIV-1 and FIV PPTs is less precise under these cell-free conditions as compared with the virus (18) and that DNA containing uracil decreases specific PPT cleavage and initiation and increases aberrant non-PPT initiation. If uracil incorporation in replicating HIV-1 has the same effects, the number of non-PPT (+) strand synthesis initiation sites should increase. This may enhance the overall rate of (+) strand DNA synthesis completion as (+) strand initiation from a second site, the cPPT, increases HIV-1 replication in culture (4, 7). Indeed, double-strand viral DNA is synthesized more efficiently in macrophages by dUTPase-minus EIAV than by wild-type EIAV (10). The effects of uracil incorporation on HIV-1 replication in the cell are not known but can be tested (18, 42).
Another perhaps more significant effect of uracil-mediated aberrant initiation of (+) strand synthesis would be decreased viral DNA integration frequency. Plus-strand DNA originating downstream of the 3'-PPT (i.e. within u3, r, u5, or pbs; Fig. 1, steps 4 and 5) would compromise and/or eradicate the critical att site, thereby preventing integration (43). In addition, IN binds less efficiently to viral DNAs with minor or major groove perturbations imparted by base analogs (44). Thus, dUMP incorporation is deleterious for retroviruses because it affects specific protein/DNA interactions and consequently alters critical protein functions. This hypothesis is supported by the biochemical data presented in Figs. 4-6. Furthermore, the observations in macrophages that uracilated EIAV DNA forms proviruses 2.5 times less efficiently than viral DNA lacking uracil, and the subsequent decreased transcription of uracilated EIAV DNA, provide additional evidence for this hypothesis (10). Moreover, interactions of several other viral and cellular proteins with DNA are also altered by the presence of uracil (45-47). Thus, we propose that retroviruses require dUTPase and/or UDG to exclude dUMP from viral DNA, ensuring that critical protein/DNA interactions occur with the highest possible efficiency and fidelity, thereby enhancing viral fitness.
The substrates used in our studies contained exclusively dTMP or dUMP to maximize the effects in the cell-free system. The replicating virus has access to small amounts of dUTP (48); therefore, the effects on initiation or integration may be less pronounced, particularly if, as expected, the effects are proportional to the amount of uracil in the DNA. Nevertheless, even subtle impairment of integration efficiency and the resultant decreased viral fitness will have significant effects on overall virus replication after many replication cycles in a selective environment (49, 50).
A previous study concluded that the function of dUTPase is to maintain
genetic stability by preventing the misincorporation of dUMP opposite
template G residues (15). The work reported here suggests an
alternative reason for the necessity of UDG and dUTPase. The models are
not mutually exclusive, and additional studies are needed to explore
both in more detail. Neither of the models can rule out the effects
that dUTPase, UDG, or uracilated DNA might have on the nuclear import
of viral DNAs. In addition, the effect of UDG on nascent HIV-1 DNA has
not been directly tested. The presence of UDG in HIV-1 virions (16, 17)
suggests that retroviral DNA requires repair mechanisms in the host
cell to patch the apyrimidinic sites formed by UDG (51). The fact that several virus families have enzymatic means to minimize uracilated DNA
accumulation (52-54) suggests that these enzymes are important to
virus survival and are thus targets for antiviral therapy.
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ACKNOWLEDGEMENTS |
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We thank Jason Rausch and Robert Smith for critical reading of this manuscript and C. Palaniappan for technical advice with the RNA cleavage assay.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grants R01 AI34834, R01 AI38755, and P30 CA42014 (to B. D. P.) and R01 AI28189 (to T. W. N.) from the National Institutes of Health.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 authors contributed equally to this work.
¶ To whom correspondence should be addressed: HIV Drug Resistance Program National Cancer Institute-Frederick, P. O. Box B, Frederick, MD 21702. Tel.: 301-846-5395; Fax: 301-846-6013; E-mail: gklarmann@ncifcrf.gov.
** Present address: University of Washington Dept. of Pathology K-072 Health Sciences Bldg., Box 357705, 1959 NE Pacific St., Seattle, WA 98195-7705.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M207223200
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; PPT, polypurine tract; cPPT, PPT located near the center of the genome; IN, integrase protein; att, IN attachment site; FIV, feline immunodeficiency virus; EIAV, equine infectious anemia virus; dUTPase, dUTP pyrophosphatase; ddNTP, dideoxynucleotide triphosphate; UDG, uracil DNA glycosylase; nt, nucleotide.
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