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INTRODUCTION |
The control of uracil residue levels in cellular DNA is ensured by
a family of uracil DNA glycosylase
(UNG)1 DNA repair enzymes
involved in the base excision repair (BER) pathway (for a review see
Ref. 1). Uracil in DNA may result from misincorporation of dUTP instead
of dTTP or from spontaneous deamination of cytosine. Incorporation into
DNA of uracil opposite to adenine is not directly mutagenic. In
contrast, incorporation of uracil opposite to guanine is promutagenic
and will result, if not corrected, in a G-to-A mutation in the next
round of DNA replication.
The uracil DNA glycosylase enzyme recognizes and excises the uracil
base from DNA, thus initiating the BER pathway by creating an abasic
site. The sugar-phosphate backbone 5' of the abasic site is then
cleaved by an AP-endonuclease, leaving a 3'-OH end. The removal of the
baseless sugar residue and insertion of the correct nucleotide are
performed by the polymerase
via its lyase and polymerase
activities, respectively. Finally, the remaining nick is sealed by the
XRCC1-ligase3 complex (2). The nuclei of human cells contain at least
five distinct enzymes to excise uracil from DNA, namely UNG2, UDG2,
TDG, MBD4, and SMUG1 (3-7). This redundancy of enzymatic
activities required to process uracil residues from DNA argues for the
importance of this process in the survival of the cell. In addition to
UNGs, all free-living organisms also express the deoxyuridine
triphosphatase (dUTPase) enzyme that prevents misincorporation of
uracil residues into DNA but through a mechanistically different
pathway by acting on the pool of intracellular nucleotides to maintain
a low ratio of dUTP to dTTP.
In the viral kingdom, genomes of some DNA viruses, namely pox and
herpes viruses, encode both UNG and dUTPase enzymatic activities (8,
9). dUTPase and/or UNG minus the mutants of herpes viruses replicate
well in cultured dividing cells but are severely impaired in
replication, neuroinvasiveness, and reactivation from latency in
non-dividing neurons (10-12). Genomes of
-retroviruses such as
Mazon Pfizer monkey virus and murine mammary tumor virus and non-primate lentiviruses such as Visna-maedi virus, caprine
arthritis-encephalitis virus, feline immunodeficiency virus, and equine
infectious anemia virus encode only dUTPase (13). The presence of these
virally encoded enzymes in viral particles suggests that they might
play an important role in the viral life cycle. Indeed,
dUTPase-deficient non-primate lentiviruses replicate well in dividing
cells but are impaired in replication in non-dividing macrophages or
resting T-cells and display a high level of G-to-A mutations and a loss of invasiveness and pathogenicity in vivo (14-18). These
data strongly argue for the necessity to control the incorporation of
uracil residues into proviral DNA in the course of infection of
non-dividing cells.
Interestingly, genomes of primate lentiviruses such as human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses encode neither UNG nor dUTPase. Because primate lentiviruses infect non-dividing macrophage cells that are
characterized by low intracellular dNTP pool levels with a low
dCTP/dTTP ratio and a high level of dUTP pool (19), they have a
non-negligible probability to misincorporate uracil residues into their
genome during viral DNA synthesis. Therefore, it is likely that
they have evolved to control uracil misincorporation into viral
DNA. Indeed, we have previously demonstrated that HIV-1 viral particles
have the ability to package one of the members of the cellular uracil
DNA glycosylase enzyme family, the UNG2 enzyme (20). This packaging
occurred via a specific association with the integrase (IN) domain of
the Gag-Pol precursor. We have proposed that host UNG2 localized inside
HIV-1 viral particles might have a role similar to that played by viral
dUTPase encoded by non-primate lentiviruses (i.e. to prevent
the fixation of uracil residues in DNA), avoiding the accumulation of
G-to-A mutations in the viral genome. The presence of UNG2 in HIV-1
viral particles highly suggests that HIV-1 might have the ability to
control the level of uracil residues misincorporated during viral DNA synthesis.
In this study, we investigated the functional role of virion-associated
UNG2 by examining the ability of wild-type or UNG2-deficient HIV-1
viral lysate to process uracil residues from a primer-template DNA
substrate containing G:U mismatched pairs. For this purpose, we first
demonstrated that leucine residue 172 of IN was critical for UNG2
binding and that recombinant HIV-1 viruses carrying the L172A mutation
were impaired for UNG2 packaging into viral particles. We then
demonstrated that (i) HIV-1 wild-type viral lysate is competent to
correct G:U mispairs to G:C pairs from a uracil-containing primer-template DNA substrate, (ii) virion-associated UNG2 is a
critical component for this process, and (iii) UNG2 and RT can be
physically associated. Our data support the notion that HIV-1 virion-associated UNG2 initiates the processing of uracil residues from
DNA and that HIV-1 viruses are able to counteract the mutagenic threat
of uracil misincorporation into DNA during viral DNA synthesis.
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EXPERIMENTAL PROCEDURES |
GST Fusion Plasmid Constructions and GST Pull-down
Assays--
The IN open reading frame was amplified by PCR from
the HIV-1AD8 molecular clone and subcloned in-frame into
the pGEX-5X-2 plasmid vector (Amersham Biosciences) to obtain a
resulting plasmid encoding a glutathione S-transferase
(GST)-wild-type IN fusion protein. Site-directed mutagenesis was
performed by PCR amplification procedures to obtain IN point mutants.
The cDNA encoding the UNG2 sequence (21) was amplified by PCR and
cloned in-frame with the GST sequence. The construction of the GST-Gag
fusion protein has been described elsewhere (22). All of the constructs
were checked by DNA sequencing. GST fusion proteins and His-tagged UNG2
were expressed in Epicurean Coli BL21-codonPlus RIL (Stratagene). The
purification of bacterially expressed GST derivatives was performed as
reported previously (23). Experimental procedures for GST pull-down
assays were done as described previously (20). GST derivatives and
recombinant proteins were allowed to interact on a rotating wheel for
2 h at 4 °C in 20 mM Hepes, pH 7.6, 250 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, and 0.5% Nonidet P-40 and then were
washed four times with 1 ml of incubation buffer. Anti-UNG2 and anti-IN
antibody were kind gifts from G. Slupphaug and D. Trono, respectively.
Anti-RT and anti-CA antibodies were purchased from Intracel and
Aalto BioReagents, respectively. Primary antibody binding was revealed
by horseradish peroxidase-conjugated secondary antibody (DAKO). The
horseradish peroxidase activity was detected with ECL Western blotting
detection reagents (Amersham Biosciences). Recombinant p66/p51 RT
heterodimer, p66/p66 RT homodimer, and p51/p51 RT homodimer were
purified until near homogeneity as reported previously (24).
Recombinant His6-tagged UNG2 was purified until near
homogeneity according to Slupphaug et al. (21).
Viral Molecular Clones and Purification of Viruses--
Viral
molecular clones used in this study were those from
HIV-1NL43 and HIV-1AD8. The 3743-bp
ApaI-EcoRI fragment of pHIV-1AD8 was
subcloned in pBluescript. An artificial unique ClaI
restriction site was then introduced by PCR amplification to modify the
fifteenth nucleotide (A versus C) of the IN open reading
frame without affecting the codon. To introduce point mutations within
the IN sequence, the 708-bp ClaI-HindIII fragment
of IN was used as a template, and site-directed mutagenesis was
performed using PCR procedures with internal primers containing each of
the point mutations and external primers containing ClaI and
HindIII restriction sites, respectively. Finally, the
ApaI-EcoRI fragment with IN point mutations was
cloned back into pNL43 plasmid to obtain HIV-1-mutated IN molecular
clones. All of the mutants were checked by sequencing. To obtain the
HIV-1
IN construct, the ClaI restriction site located in
HIV-1 IN was filled in and the resulting construct encoded only the
five first residues of IN. 293T cells were transfected with proviral
DNA in the presence of FuGENE 6 transfectant reagent (Roche Molecular
Biochemicals), and 48 h post-transfection, viral particles
released in the cell-free supernatant were filtered on 0.45-µm
pore-size filters and highly purified by ultracentrifugation through
8-18% Optiprep density gradient as described previously (25).
Gradient fractions coinciding with the peak of the reverse transcriptase activity were pooled and normalized for equivalent amounts of CA p24 antigen. Viral lysate was obtained by lysis of
purified virions in TNE buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA) in the presence of 0.2% of
Triton X-100.
Processing of Uracil-containing Substrate--
The 32/70-mer
primer-template DNA substrate was composed of a 5'-end labeled 32-mer
primer oligonucleotide with a uracil residue at position 32 annealed
with a 3-fold molar excess of the unlabeled complementary 70-mer
template oligonucleotide. The complete sequence of the 70-mer
oligonucleotide is as follows:
5'-CACTCTCCATACTTCCAATCTCCACTCCACCCTCCTCCGTCGACTTCCTGCGCGCTGCGGCTGCGCCTTC-3'. In the duplex, the uracil residue is included opposite to guanine within the palindromic sequence recognized by the SalI
restriction enzyme. We also used a 32/70-mer primer-template DNA
substrate but with a thymine at position 32 so that a G:T mispair was
created. The mismatch-containing substrate processing was assayed as
reported previously (26). Viral lysate was incubated with 2 nM of labeled uracil-containing DNA substrate in a final
volume of 20 µl of a buffer containing 20 mM Hepes, pH
7.6, 60 mM KCl, 10 mM MgCl2, 10 µM ZnCl2, 0.05% Triton X-100, 1 mM dithiothreitol, 5% glycerol, and 100 µg/ml bovine
serum albumin in the presence of 200 µM of each of the
four dNTPs. After incubation at 37 °C for 30 min and extraction with
phenol/chloroform, DNA was recovered by ethanol precipitation in the
presence of 3 µg of tRNA carrier and subjected to SalI
digestion (2 units/point) for 60 min at 37 °C in a final volume of 4 µl. Four microliters of formamide loading dye was then added, and
samples were heated at 75 °C for 3 min and resolved by
electrophoresis on a 8 M urea-containing 15%
polyacrylamide gel in 1× TBE buffer. Radiolabeled DNA products were
revealed by autoradiography.
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RESULTS |
Mapping of the UNG2 Epitope within IN--
In a previous study
(20), we reported that HIV-1 viruses deficient for the expression of IN
(HIV-1
IN) failed to incorporate host UNG2, indicating that IN acts
as a carrier to package UNG2 into viral particles. We proposed that
virion-associated UNG2 in a manner similar to its cellular counterpart
might have a role in controlling misincorporation of uracil residues
into DNA during viral DNA synthesis. Because it has been reported that
HIV-1
IN was impaired for efficient viral DNA synthesis (27), we
decided to analyze the role of virion-associated UNG2 in the processing of uracil residues by using viruses containing point mutations into IN
so that the host UNG2 packaging was altered but not the reverse
transcriptase activity.
To map residues of IN required for association with UNG2, we generated
an affinity matrix consisting of a glutathione S-transferase (GST) fused to HIV-1 IN with N-terminal deletions (Fig.
1A) or C-terminal deletions
(Fig. 1B) and immobilized on glutathione-agarose beads.
Recombinant purified His6-tagged UNG2 was incubated with matrix, and matrix-bound UNG2 was analyzed by SDS-PAGE followed by
Western blotting. Similar amounts of GST derivatives were used as
judged by Coomassie Blue staining. Results showed that all of the
deleted INs still retained their ability to bind UNG2 with the
exception of when the region encompassing residues 170-180 was
missing. These data indicate that residues 170-180 are required for
UNG2 binding.

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Fig. 1.
Domain of HIV-1 IN involved in the
association with UNG2. GST pull-down assays using recombinant
His6-tagged UNG2 and bacterially expressed GST-IN mutants
containing C-terminal deletions (panel A) or N-terminal
deletions (panel B). Interacting UNG2 was visualized by
Western blot analysis with anti-UNG antibody. Similar amounts of
GST derivatives were analyzed as revealed by Coomassie Blue staining of
the gel. Lane marked input contains one-fifth of
His6-tagged UNG2 before binding to GST. Molecular mass
markers are shown in kilodaltons.
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To determine what residues within the region 170-180 of IN were
critical for the UNG2 binding, we generated IN point mutants by
alanine-scanning mutagenesis of residues 170-181. Each of the mutants
was expressed as a GST fusion protein, and GST pull-down assays were
carried out using recombinant His6-tagged UNG2. Results from Fig. 2, left panel,
revealed that the mutation of both leucine 172 and lysine 173 residues
of IN failed to retain UNG2. All other mutations did not affect the
binding of UNG2. To map precisely the residue of IN critical for UNG2
binding, we introduced single point mutations on residues belonging to
the region 170-173 of IN. As shown in Fig. 2, right panel,
the mutation of residues 170, 171, and 173 has no effect on UNG2
binding. In contrast, the L172A mutation impaired the binding of UNG2.
These data indicate that leucine residue 172 is important for the
interaction of IN with UNG2, although it cannot be ascertained that
leucine 172 is the binding residue of UNG2.

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Fig. 2.
Leucine residue 172 of IN is important for
IN-UNG2 association. GST pull-down assays using recombinant
His6-tagged UNG2 and bacterially expressed GST-IN
containing alanine substitution of residues located in the 170-181
region of IN (panel A) or containing single alanine
substitution of residues 170-173 (panel B). Interacting
His6-tagged UNG2 was visualized by Western blot analysis
with anti-UNG antibody. Lane marked input
contains one-fifth of UNG2 before binding to GST. Molecular mass
markers are shown in kilodaltons.
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Incorporation of Host UNG2 into IN Point Mutants--
We then
analyzed whether L172A IN mutation inserted in the context of the
HIV-1NL4.3 molecular clone impaired UNG2 packaging into
viral particles. Recombinant viral molecular clones were engineered to
express a IN gene with point mutations in the 170-181 region and used
to transfect 293T cells. Viruses produced in the cell-free supernatant
were highly purified by Optiprep gradient velocity centrifugation. Each
of the viral lysates was then assayed by Western blotting for the
packaging of host UNG2. As shown in Fig. 3,
left panel, UNG2 was
undetectable in viral lysate from viruses either deficient for IN
(
IN) or containing L172A/K173A IN mutations. In contrast, all other
point mutant viruses exhibited levels of virion-associated UNG2 similar
to that of wild-type viruses. The mutation of leucine residue 172 of IN
abrogated the packaging of UNG2 (Fig. 3, right panel).
Similar amounts of each of the viral lysates were used as judged by
amounts of CA p24 antigen revealed by Coomassie Blue staining of the
gel. In addition, Western blotting with anti-CA and anti-IN antibody
revealed that Gag and Gag-Pol precursors in viruses carrying IN
mutations were processed as well as in wild-type viruses, indicating
that mutations did not induce obvious viral protein alterations. These
in vivo data are consistent with in vitro data
and demonstrate that leucine residue 172 of IN is required to fully
package host UNG2 into viral particles.

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Fig. 3.
Leucine residue 172 of IN is important for
host UNG2 packaging into viral particles. 293T cells were
transfected with HIV-1 molecular clones containing point mutations
within the 170-181 IN region, and viruses produced in the cell-free
supernatant were purified by Optiprep gradient velocity centrifugation.
Purified viruses were solubilized, and viral lysate was analyzed by
Western blot for the presence of host UNG2 (upper panel).
Expression of mutated IN in viral lysate was visualized by using
anti-IN antibody (middle panel). Amounts of viral lysate
were estimated by amounts of CA p24 antigen revealed with anti-p24
antibody (lower panel). Molecular mass markers are shown in
kilodaltons.
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Correction of G:U Mispairs to G:C Pairs by Wild-type but Not
172/173 IN HIV-1 Viral Lysate--
The role of the cellular
UNG2 DNA repair enzyme is to initiate the BER process by excising
misincorporated uracil residues from DNA. We designed experiments to
analyze whether virion-associated UNG2 plays a role similar to that of
its cellular counterpart. The uracil-containing DNA substrate
(32/70-mer) was composed of a 5' end-labeled 32-mer primer
oligonucleotide with a uracil residue at position 32 annealed with the
unlabeled complementary 70-mer template oligonucleotide (Fig.
4A). In the primer-template
DNA substrate, the uracil residue is included opposite to guanine residue within the palindromic sequence recognized by the
SalI restriction enzyme. The presence of uracil residue
opposite to guanine residue in the SalI sequence has been
reported to prevent DNA cleavage by SalI (26). The
restoration of the SalI digest implies that the uracil
residue in G:U mispairs was corrected to G:C pairs.

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Fig. 4.
Processing of uracil-containing DNA substrate
by HIV-1 viral lysate. A, the 32/70-mer
uracil-containing substrate used is depicted. Asterisk
indicates the 5' end-labeling. The SalI restriction site is
boxed. B, the DNA substrate containing G:U
mispairs was incubated with increasing amounts (0.3, 0.6, and 1.2 µg
of CA p24 antigen) of wild-type or 172/173 IN viral lysate and
subjected to SalI restriction enzyme digestion. Resulting
DNA products were resolved on a 15% denaturing gel and revealed by
autoradiography. As a control (right panel), viral lysate
was incubated with a DNA substrate containing G:T mispairs instead of
G:U pairs. Arrow indicates the position of the
SalI digest product.
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The 32/70-mer DNA substrate was incubated with increasing amounts (0.3, 0.6, and 1.2 µg of CA p24 antigen) of highly purified HIV-1 wild type
or L172A/K173A IN mutant viral lysate in the presence of each of the
four dNTPs. The substrate was then recovered by ethanol precipitation
and subjected to SalI digestion. DNA products were resolved
on urea-denaturing gel and analyzed by autoradiography (Fig.
4B). Efficient DNA extension of the primer by increased levels of wild-type or L172A/K173A IN viral lysate was evidenced by the
appearance of increased levels of the labeled 70-mer product concomitant with decreased levels of the labeled 32-mer primer. A
proportion of the DNA substrate upon incubation with increasing amounts
of wild-type viral lysate became susceptible to cleavage by
SalI as judged by the appearance of a
dose-dependent labeled DNA product with the expected size
(27-mer) for the SalI digestion product. This results
presumably from a specific repair of G:U mismatched pairs to G:C
matched pairs. In contrast, incubation of the DNA substrate with
similar amounts of L172A/K173A IN viral lysate failed to generate the
27-mer SalI digestion product. The failure to observe uracil
processing upon incubation of the DNA substrate with
UNG2-deficient viruses serves as an internal control to certify that
cellular proteins in microvesicles did not significantly contaminate
our purified virion preparations (28) and to eliminate the possibility
that the G:U versus G:C correction was the result of a
virion-independent endonucleolytic processing of the substrate. As a
control, we incubated a primer-template DNA substrate containing a G:T
mismatch instead of a G:U mismatch with increased amounts (0.3, 0.6, and 1.2 µg of CA p24 antigen) of wild-type viral lysate, and we
subjected it to SalI digestion. As shown in Fig.
4B, right panel, no SalI digest
product was observed, indicating the specificity of the repair process.
Altogether, our data indicate that uracil residues are corrected from
G:U mispairs to G:C pairs in the presence of HIV-1 viral lysate and
that virion-associated UNG2 plays a key role in initiating the repair process.
Physical Association of UNG2 with the Viral Reverse Transcriptase
Enzyme--
In a previous study, we reported that a G:U mispair buried
in the central part of a double-stranded DNA substrate was not corrected to a G:C pair by HIV-1 viral lysate (20). In contrast, we
observed here the correction of G:U mispairs located near the free
3'-OH end of the primer in the primer-template DNA substrate. The
presence of a free 3'-OH end in close proximity to the G:U mispair to
anchor the viral RT enzyme on the DNA primer seems to be important for
uracil processing. We hypothesized that UNG2 and RT might be spatially
close to act in a concerted manner to process uracil.
Therefore, we examined whether UNG2 and RT have the ability to
associate. GST affinity matrix containing HIV-1 IN, HIV-1
Pr55gag, or UNG2 was incubated with recombinant purified
p66/p51 RT heterodimer, and matrix-bound RT was analyzed by SDS-PAGE
followed by Western blotting (Fig. 5,
left panel). RT binds to HIV-1 integrase as reported
previously (29) as well as to UNG2 but not to GST or GST-Pr55gag. The possibility that nucleic acids interfered
in the interaction RT-UNG2 was ruled out by pretreating the mixture of
RT-GST derivatives with micrococcal nuclease and RNase A, which did not
decrease amounts of the RT pull-down assay (data not shown). We also
tested the ability of p66/p66 RT homodimer (Fig. 5, middle
panel) or p51/p51 RT homodimer (Fig. 5, right panel) to
associate with UNG2. The results indicated that each of the homodimers
still retained UNG2 association, suggesting that the RNase H domain of
RT was probably not involved in the interaction with UNG2. These data indicate that UNG2 and RT physically associated.

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Fig. 5.
Physical association of UNG2 with RT.
Left panels, recombinant purified p66/p51 RT heterodimer
incubated with equivalent amounts of GST, GST-HIV-1 IN, GST-UNG2, or
GST-Pr55gag fusion proteins affinity-purified on
glutathione-agarose beads. After washes, bound proteins were
analyzed by Western blot with anti-RT antibody. Middle
panels, GST pull-down assays performed with GST derivatives and
the p66/p66 RT homodimer. Right panels, GST pull-down assays
performed with GST derivatives and the p51/p51 RT homodimer.
Lane marked input contains one-fifth of RT before
binding to GST derivatives. Similar amounts of GST derivatives were
revealed by Coomassie Blue staining of the gel. Molecular mass markers
are shown in kilodaltons.
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DISCUSSION |
We have previously reported that HIV-1 viral particles have the
unique property of packaging the host uracil DNA glycosylase DNA repair
enzyme UNG2 into viral particles (20). In this paper, we investigated
the functional role of virion-associated UNG2 in uracil processing by
studying the ability of HIV-1 viral lysate to correct G:U mismatched
pairs incorporated into a primer-template DNA substrate. The repair of
G:U mispairs to G:C pairs was revealed by the restoration of the
cytidine residue instead of the uridine residue in the nucleotidic
sequence recognized and cleaved by the SalI restriction
enzyme. We showed that HIV-1 wild-type viral lysate, but not
UNG2-deficient viral lysate, can process uracil residues when they are
present at the free 3'-OH end of the primer in the primer-template DNA
substrate. In our early study (20), we showed that HIV-1 viral lysate
failed to process uracil residues present in a G:U mispair located in
the middle of a blunt double-stranded DNA substrate, probably because
of the absence of a free 3'-OH end to anchor RT on its primer. In
contrast, recombinant UNG2 alone efficiently processed uracil residues
in this case. These findings suggested that UNG2, in the context of
HIV-1 viral particles, might act in a manner distinct from that of
recombinant UNG2. Indeed, we demonstrated that (i) UNG2 is associated
and packaged with IN, (ii) UNG2 can associate with RT, and (iii) RT and
IN are associated. This latter association has been previously reported to be critical for the initiation of the retrotranscription of viral
genome (29). We hypothesized that RT, IN, and UNG2 are physically
associated and are part of the viral retrotranscription complex. As a
consequence of such a multienzymatic complex, UNG2 would probably not
be free in the viral particle and could act only on uracil residues in
close proximity of a free 3'-OH anchor for the retrotranscription
complex. On the basis of this hypothesis, we proposed that DNA
polymerization by RT and DNA repair initiated by virion-associated UNG2
are coupled events.
The viral retrotranscription process is a two-step process consisting
of the synthesis of the minus-strand DNA using viral RNA as a template
followed by the synthesis of the plus-strand DNA using the minus-strand
DNA as a template to obtain the proviral double-stranded DNA. One
mispairing mutation occurring in the minus-strand DNA of the
heteroduplex is mutagenic and is fixed in the plus-strand DNA of the
DNA/DNA homoduplex constituting the proviral genome. For example, dUMP
residues in the minus-strand DNA will be paired with dAMP residues
during the synthesis of the plus-strand DNA, contributing to the
accumulation of G-to-A substitutions. We demonstrated that
virion-associated UNG2 has the function of preventing accumulation of
uracil and, therefore, G-to-A transition in the context of a DNA/DNA
duplex. We have not looked at the repair process for RNA/DNA
heteroduplexes, which are intermediate products formed during the
retrotranscription process, but a previous study (30) have shown
that the repair process of a RNA/DNA duplex was effective, albeit with
a reduced efficiency, compared with that on a DNA/DNA duplex.
Moreover, it has been reported that HIV-1 viruses with an excess
of UNG2 packaged by Vpr displayed a more accurate reverse transcription during a unique cycle of infection (31), suggesting that UNG2 has the
function of lowering the mutation rate. Taken together, these data
support the notion that HIV-1 is able to control the amount of uracil
incorporated into its genome during reverse transcription and to
prevent the catastrophic G-to-A hypermutations, which have been
reported when the dNTP concentration is biased (32, 33).
The question arises whether the viral BER process occurred through a
pathway similar to that of cellular BER and thus required an
AP-endonuclease activity to cleave 5' to the abasic site resulting from
the action of the DNA glycosylase 2. Western blot analysis of HIV-1
viral lysate revealed no packaging of host AP-endonuclease 1 (34, 35) in experimental conditions where host UNG2 was clearly
detected in virions (data not shown). Similar negative results (data
not shown) were obtained by analyzing viral lysate of virions produced
from cells overexpressing either hemagglutinin-tagged AP-endonuclease
1 or hemagglutinin-tagged AP-endonuclease 2, a newly described
member of the human AP-endonuclease family (36). Therefore, we
speculate that HIV-1 viruses did not contain known AP-endonucleases,
suggesting that the processing of uracil residues by HIV-1 viral lysate
occurred independently of the presence of a AP-endonuclease activity.
An alternative hypothesis would be that the creation of an abasic site
by the virally associated UNG2 is followed by the elimination of the
baseless nucleotide by the pyrophosphorolytic activity of RT, leaving a
free 3'-OH end to be used by the polymerase activity of RT. Further
studies are in progress to understand the molecular mechanism(s)
involved in viral BER.
The fact that the HIV-1 reverse transcription process is mainly
accomplished in the cytoplasm of infected cells and the fact that
cellular enzymes participating in the DNA repair process are nuclear
proteins might explain why HIV-1 particles have to be equipped with
their own enzymatic DNA repair activities. In conclusion, we propose
that HIV-1 viruses in the manner of non-primate lentiviruses, pox
viruses, or herpes viruses which replicate in non-dividing cells, have
developed an original strategy to bypass the deleterious consequence of
uracil in viral DNA by using IN as a carrier to package host UNG2 into
viral particles and by using RT to target UNG2 at the site of nascent
DNA synthesis.