Current HIV Research, 2006, 4, 31-42
31
Uracils as a Cellular Weapon Against Viruses and Mechanisms of Viral
Escape
Stéphane Priet, Joséphine Sire and Gilles Quérat*
Unité des Virus Emergents. Faculté de Médecine. 27 Boulevard Jean Moulin. 13385 Marseille Cedex 5. France
A b s t r a c t : Uracil in DNA is a deleterious event that may arise either by cytosine deamination or
misincorporation of dUTP. Consequently, cells from all free-living organisms have developed strategies to
protect their genome against the presence of uracils, by using uracil DNA glycosylase (UNG) and deoxyuridine
triphosphatase (dUTPase) enzymatic activities. In the viral kingdom, some (namely poxviruses and
herpesviruses) but not all of the DNA viruses encode their own UNG and dUTPase to control uracilation of their
genome. Some retroviruses, which are RNA viruses using DNA as an intermediate of replication, also encode
dUTPase. Surprisingly, though most of nonprimate lentiviruses encode dUTPase, primate lentiviruses such as
HIV-1, HIV-2 or SIV do not. Because these latter viruses also replicate in nondividing cells where the
dUTP/dTTP ratio is high, it is probable that they have found other ways to fight against the emergence of
uracilated-viral transcripts. Indeed, recent studies showed that HIV-1 efficiently controls both the cytosine
deamination and the dUTP misincorporation. The viral Vif protein acts in preventing the packaging into viral
particles of the host-derived cytosine deaminase APOBEC3G enzyme, while the viral integrase domain of the
Gag-Pol precursor mediates the packaging of the host-derived uracil DNA glycosylase UNG2 enzyme. In the
absence of Vif or UNG2, HIV-1 viral transcripts are heavily charged in uracil bases leading to inactivation of the
virus.
Keywords: HIV-1; uracil DNA glycosylase; integrase domain; reverse transcriptase; dUTP misincorporation; uracil base
excision repair.
The integrity of cellular DNA is constantly jeopardized
by a large number of exogenous or endogenous agents as
well as by some cellular processes [49]. The incorporation
and retention of uracil bases in DNA strands represent a
permanent danger as it triggers mutations, but cell developed
several mechanisms to highly control uracilation of its
genome. Viruses that replicate their genome in a cellular
environment favoring the incorporation of uracils in DNA
also evolved strategies to counteract the uracilation of viral
DNA transcripts. In this review, we describe what are the
consequences of uracils in DNA for the replication of some
viruses, and what are the molecular mechanisms used by
HIV-1 to avoid uracilation of viral transcripts.
URACILS IN DNA: ORIGIN AND CONSEQUENCES
Uracil is a natural base present in RNA that is replaced
by thymine in DNA. However, uracils may be abnormally
found in DNA via two distinct mechanisms including either
spontaneous or enzymatically-induced cytosine deamination
to uracils and dUTP misincorporation in place of dTTP.
Deamination of cytosine residue in DNA is a genotoxic
event in cell due to the creation of G: U mismatched pairs
that leads, if unrepaired before the next round of replication,
to G: A mismatch generating G-to-A transition mutations.
Spontaneous deamination of cytosines is a frequent event
that has been estimated between 60 to 500 times per day and
*Address correspondence to this author at the Unité des Virus Emergents.
Faculté de Médecine. 27 Boulevard Jean Moulin. 13385 Marseille Cedex
5. France; Tel: 33 (4) 91 32 45 53; Fax: 33 (4) 91 32 44 95; E-mail:
[email protected]
1570-162X/06 $50.00+.00
per genome [49]. The modified form of cytosine, namely the
5-methylcytosine, is subjected to spontaneous deamination 3
to 4 times more rapidly than its unmodified form [30].
Some chemical agents, like nitric oxide (NO) [105]) or
bisulfites [13] may enhance the frequency of cytosine
deamination. Two kinds of cellular enzymes are able to
convert cytosine to uracil, the (cytosine-5)-methyltransferase
and the cytidine deaminase. In mammalian cells, 2 to 7% of
cytosines are methylated and located in areas involved in
gene expression, the CpG islets. Cytosines within CpG
islets are essentially converted to 5-methylcytosines by the
(cytosine-5)-methyltransferase enzyme. The first step of the
conversion starts by the formation of a covalent bond
between enzyme and cytosine that leads to transient
dihydropyrimidinic intermediate products that are highly
subjected to spontaneous deamination. Then, the enzyme
catalyzes the transfer on the cytosine base of a methyl group
provided by the methyl donor S-adenosylmethionine. Thus,
a cytosine residue can be converted to uracil residue upon the
abortive action of (cytosine-5)-methyltransferase [84] or in
cellular conditions where the concentration of Sadenosylmethionine is low [86].
The cytidine deaminase APOBEC family includes
members that share similar catalytic activity. Among these
members, are found APOBEC1 (apolipoprotein B mRNA
editing catalytic subunit 1), AID (activation-induced cytidine
deaminase), APOBEC2, also referred as APOBEC1-related
cytidine deaminase 1 (ARCD-1) and proteins APOBEC3A
to APOBEC3G. At this time, no function has been
attributed to APOBEC3A, APOBEC3D and APOBEC3E.
APOBEC1 is responsible for the tissue-specific deamination
of the cytosine residue C6666 of the apolipoprotein B
mRNA leading to a premature stop codon and the expression
© 2006 Bentham Science Publishers Ltd.
32 Current HIV Research, 2006, Vol. 4, No. 1
of a truncated form of apolipoprotein B [68, 93]. APOBEC
1 acts exclusively in deaminating apolipoprotein B mRNA
in cells, although when expressed in bacteria, it is also able
to deaminate cytosines present in DNA [3, 34]. The
expression of AID is restricted to activated B lymphocytes
[64] and is required to generate antibody diversity in
allowing the class-switch recombination and the somatic
hypermutation processes [63, 79]. AID is specific of singlestranded DNA and acts in deaminating cytosine present in
the immunoglobulin gene locus [9, 19]. Concerning
APOBEC3 proteins, the seminal publication from Sheehy
and collaborators [85] identifying the APOBEC3G enzyme
as a host cell restriction factor that impedes HIV-1
replication, led to a spectacular interest as revealed by the
high number of reports published during the last three years
(see below).
Incorporation of dUTP into DNA leads to A: U
mismatched pairs that are not mutagenic. Indeed, uracil that
replaces thymine will be paired with adenine to generate A:
T matched pairs in the next round of replication. In
Priet et al.
eukaryotic cells, dUTP is synthesized either from the
conversion by a dCTP deaminase of dCMP to dUMP or
from the phosphorylation of dUDP arising from UDP under
the action of rNDP reductase (Fig. (1)). dUMP is an
essential intermediate for the synthesis of intracellular dTTP
pool. Thus, dUMP is always present in cell and represents a
permanent source of dUTP. DNA polymerases, including
reverse transcriptases, but excluding vent and pfu DNA
polymerases from hyperthermophilic archea [33], are not able
to discriminate dUTP from dTTP rendering the
incorporation of dUTP as the major source of uracils in
DNA. In physiological conditions, there is a low
concentration of dUTP in cell (∼ 0.2 µM) in comparison to
that of dTTP (37±30 µM) [95]. The intracellular ratio
dUTP/dTTP has been determined between 0.3 and 3%.
However, in some cell types such as HT29 cell line, primary
spleen cells, macrophages or quiescent lymphocytes, dUTP
levels are high and even exceed those of dTTP [2, 17, 38].
One main consequence of uracils in DNA is the
enhancement of recombination frequency in eukaryotic cells
Fig (1). Depiction of the biosynthesis pathway of nucleotides in mammalian cells. De novo synthesis of AMP, GMP and UMP leads to
the generation of dATP, dGTP, dCTP, dTTP and dUTP nucleotides that are substrates for cellular polymerases and therefore are
incorporated into DNA.
Uracilation of Viral DNA
that leads to DNA fragmentation [22, 32]. Moreover,
accumulation of uracils in promoter sequences may alter
gene expression due to the failure of some transcription
factors to recognize their target sequence [27, 101].
CELLULAR STRATEGIES TO COUNTERACT
URACILS IN DNA
To impede deleterious effects of uracils in DNA, cells
have evolved in setting up two mechanisms to prevent
incorporation and retention of uracils in DNA. The first
mechanism [22, 36, 100] involves dUTPase that acts
directly on the intracellular dUTP pool level by hydrolyzing
dUTP to dUMP and inorganic pyrophosphate. The role of
dUTPase is to maintain a low level of intracellular dUTP
but also to contribute to the biosynthesis of nucleotides
derived from thymidine. In prokaryotes, the total deficiency
in dUTPase is lethal, and in conditions where the deficiency
is only partial, it is associated with an increased time of
replication and an increased frequency of spontaneous
mutations and recombinations [22, 36, 100]. In S. cerevisiae
cells, the total deficiency in dUTPase is lethal except if
dTMP is exogenously added, indicating that lethality was
probably the consequence of a blockage in DNA synthesis
due to the absence of dTTP [32]. The human dUTPase gene
is composed of four exons that encode two isoforms
generated by alternative splicing, one expressed in nucleus
and the other one expressed constitutively in mitochondria
[44]. The expression of nuclear form is dependent on cell
cycle with a high expression level in S phase that becomes
undetectable in G0 and G1 phases.
The second mechanism involves uracil DNA
glycosylases (UNG) that recognize and excise uracil residues
that are already present in DNA using two distinct base
excision repair (BER) pathways, i. e. the short and the long
patch repair pathway (Fig. (3)). UNG excises the uracil base
leading to an abasic site that is further cleaved on its 5’ side
by an apurinic/apyrimidinic (AP)-endonuclease, leaving a
free 3’-OH end. In the short patch repair pathway, the
removal of the baseless nucleotide and the insertion of
correct nucleotide are performed by the polymerase ß via its
lyase and polymerase activities, respectively. The sealing of
the remaining nick is performed by the XRCC1-DNA ligase
3 complex. In the long patch repair pathway, polymerases
such a pol ε and/or pol δ associated with proliferating cell
nuclear antigen (PCNA) and replication factor C (RFC)
allow the polymerization of a 2-10 nucleotides flap from the
AP-endonuclease-induced free 3’-OH end through stranddisplacement polymerization, which is then cut by flap
endonuclease 1 (FEN1). A patch of the same size is then
synthesized by DNA polymerase and PCNA and ligated by
DNA ligase I. Other mechanisms of DNA repair that
included nucleotide excision repair and homologous- or
nonhomologous recombination also exist in cells [37].
Sequence comparisons between UNGs expressed from
mammals, bacteria, yeast, or herpesvirus show a high degree
of homology. In human cells, several enzymes with uracil
DNA glycosylase activity have been reported, namely TDG,
MBD4, SMUG, UNG1 and UNG2 [43]. The human ung
gene encodes two UNG isoforms generated by alternative
splicing, the UNG2 form (314 aminoacids) displaying a
nuclear localization signal and the UNG1 form (305
Current HIV Research, 2006, Vol. 4, No. 1
33
aminoacids) displaying a mitochondrial localization signal
[67]. These forms differ in their N-terminal sequence.
Sequences downstream the position 44 of UNG2 and
sequences downstream the position 35 of UNG1 are
identical. The UNG1 mitochondrial form is constitutively
expressed in cells, while the expression of the UNG2 nuclear
form depends on cell cycle with high levels in S phase and
low levels in G0 phase [26].
VIRAL STRATEGIES TO COUNTERACT URACILS
IN DNA
Viruses belonging to the herpesviridae, poxviridae and
retroviridae family have evolved in encoding their own
dUTPase and/or UNG, arguing that these viruses seem to be
sensitive to the presence of uracils in their genome.
Members of the Herpesviridae family replicate their
genome in the nucleus of infected cells. They possess a
linear double-stranded DNA genome that rapidly circularizes
in nucleus. These viruses infect distinct cell types and some
have the ability to infect nondividing cells, like neurons.
Alpha-herpes viruses, like Herpes simplex (HSV) or
Varicella zooster (VZV), and gamma-herpes viruses, like
Epstein-Barr virus (EBV), encode enzymes of
deoxynucleotides metabolism, including dUTPase [69] and
thymidine kinase, and DNA repair enzymes such as UNGs.
Studies have demonstrated that HSV-1 or VZV deficient for
dUTPase replicated well in cultured dividing cells [11, 28]
where dUTP is low and cellular dUTPase is high. dUTPaseminus HSV-1 was severely impaired in replication,
neuroinvasiveness and reactivation from latency in mice
nervous system where the levels of dUTP are high and those
of cellular dUTPase and UNG are low [77]. In addition,
dUTPase or thymidine kinase deficient HSV-1 displayed
enhanced frequency of mutations [75]. Mutations of the
UNG gene of HSV-1 or VZV did not impaired replication in
dividing cells in culture [78] but led to alterations of viral
propagation in a in vivo murine system [76]. The situation is
somewhat different for beta-herpes viruses, such as
cytomegalovirus (CMV), which do not encode a functional
dUTPase nor any other dNTP biosynthesis enzymes and
might only rely on enzymes expressed in cellular targets
[10]. However, CMV encodes a viral UNG and this virallyencoded enzyme was somewhat dispensable for viral
replication in highly dividing cells, although DNA
replication was slightly delayed and the replicative cycle was
longer. CMV-encoded UNG was much more required for
viral propagation in slowly or nondividing cells, as UNGdeficient CMV exhibited markedly delayed DNA synthesis
in these cells [16, 70]. Further studies have suggested that
UNG is a crucial determinant in the transition from early- to
late-phase CMV DNA replication that plays a role in
excising uracils incorporated during early phase replication
to create sites that serve as substrates for initiation of
recombination-dependent replication late in infection [16].
These data support the notion that CMV UNG is required to
compensate for low levels of cellular UNG in nondividing
cells.
Members of the Poxviridae family replicate their linear
double-stranded DNA genome exclusively in the cytoplasm
of infected cells. Although deletion of the dUTPase gene of
34 Current HIV Research, 2006, Vol. 4, No. 1
Priet et al.
the vaccinia virus prototypic member had no effect on viral
propagation in actively dividing cells [29], deletion of the
UNG gene altered viral replication in the same cell type [24,
62]. Vaccinia virus-encoded UNG had also an essential
function in the viral DNA synthesis that was independent of
its glycosylase activity [18]. It is probable that the lack of
replication of UNG-deficient vaccinia virus could be
attributed to the fact that cellular UNG enzymes which are
mainly localized in nucleus could not gained access into the
cytoplasmic viral capsid, where viral replication occurs,
suggesting that these viruses have to encode their own UNG
to target it to the site of the viral DNA synthesis.
promoted by the cellular DNA repair pathway. In
macrophages infected by dUTPase deficient EIAV there was
a reduction of integrated provirus and a strong (up to 300
fold) reduction in transcription [88] probably because of
faulty recognition of transcriptional signals.
Members of the Retroviridae family replicate their RNA
genome via a double-stranded DNA intermediate generated
by the virally-encoded reverse transcriptase, which is then
integrated in the genome of host cell. Retroviruses have
captured a dUTPase gene on at least three different
occasions, highly suggesting that dUTP hydrolysis must
have conferred a strong selective advantage for these viruses.
Retroviral dUTPase is a part of the polymerase precursor
protein, expressed either downstream of the integrase domain
for human or murine endogenous retroviruses ERV-L [4, 15]
or between the reverse transcriptase and the integrase
domains for some lentiviruses [23, 58, 59]. Retroviral
dUTPase is expressed as a fusion protein with the
nucleocapsid domain for ß-retroviruses, such as MazonPfizer monkey virus (MPMV) or murine mammary tumor
virus (MMTV) [5]. As a general rule, the dUTPase protein is
predicted to be encapsidated into budding particles and to be
in close spatial proximity of the reverse transcription process
[23]. The exact role of dUTPase in the replication of ßretroviruses has not yet been established. In contrast, studies
on the role of dUTPase of nonprimate lentiviruses, like
visna-maedi virus (MMV), caprine arthritis-encephalitis
virus (CAEV), feline immunodeficiency virus (FIV) and
equine infectious anemia virus (EIAV), revealed that virallyencoded dUTPase is dispensable for viral replication in
dividing cells, but is required for fully efficient replication
in nondividing cells, such as primary macrophages that are
their natural target cells [47, 88, 94, 99, 102]. The proviral
DNA of dUTPase-negative EIAV or CAEV was shown to be
uracilated suggesting that in the absence of viral dUTPase,
the reverse transcriptase was highly efficient at dUTP
incorporation in place of TTP [88, 99]. It must be noted that
this uracilation is not mutagenic per se. Experimental
infection of goats or cats with dUTPase-deficient CAEV or
FIV showed reduced viral load associated with only a slight
increase of mutation rates [46, 97]. G-to-A transitions were
observed in dUTPase-deficient proviruses, but they were
very different, both in terms of frequency and of sequencecontext, from the bona fide G-to-A hypermutations observed
in Vif-negative HIV-1 DNA. This small excess of G-to-A
subsitutions had been hypothesized to result from dUTP
incorporation in place of dCTP and opposite to guanine in
the first strand DNA. If this happens, this must be a very
rare event, as revealed by in vitro assays with recombinant
RT, owing to the actual ratio of dCTP and dUTP in a cell
[57, 74].
PRIMATE HIV-1 LENTIVIRUS STRATEGY TO
COUNTERACT CYTOSINE DEAMINATION IN
VIRAL DNA
Uracilation of DNA by dUTP incorporation, although
generally not mutagenic, may nevertheless lead to a strong
impairment of replication. High levels of uracils in both
DNA strands could lead to excessive strands breaks
Collectively, these reports highlight the fact that high
levels of dUTP are present in lentivirus target cells, that this
could lead to uracilation of proviral DNA and reduced viral
replication and explain why a captured dUTPase gene may
have been selected for.
Primate lentiviruses, such as human immunodeficiency
virus type 1 (HIV-1), human immunodeficiency virus type 2
(HIV-2) and simian immunodeficiency virus (SIV), are
phylogenetically closely related to nonprimate lentiviruses
and replicate their genome in nondividing cells where
dUTPase and UNG expression levels are low. The
intracellular dUTP pool level in macrophages and
lymphocytes has been estimated to be 30% of that of dTTP
[2, 17, 38], highly enhancing the probability to
misincorporate dUTP during the HIV-1 life cycle. Because
these viruses do not encode dUTPase or UNG, they need to
acquire enzymatic activities to counteract the uracilation of
their genome. Recent studies have unveiled strategies used
by HIV-1 to fight uracils arising in viral DNA either by
dUTP misincorporation during the reverse transcription
process or by the recently described cytosine deamination of
reverse transcripts.
HIV-1 viral particles have the property to incorporate the
host-derived cytidine deaminases APOBEC3G [85] and
APOBEC3F [103, 112]. APOBEC3G incorporation occurs
via either a specific association with the RNA-recruiting
nucleocapsid (NC) domain of Gag [1, 12, 51, 110], or a
nonspecific association with viral or cellular RNA [40, 92].
The virion-associated APOBEC3G triggers G-to-A
hypermutations due to the deamination of cytosines in the
minus-strand viral cDNA in a CC context [35, 45, 53, 111],
while APOBEC3F deaminates cytosines in a TC context.
Providing that these uracils were not recognized and
eliminated by virion-associated UNG (see below), these
mutations will be fixed in the plus-strand DNA following
second strand cDNA synthesis. Together, these two cytidine
deaminase specificities account for the majority of observed
G-to-A hypermutations.
The retention of uracils in neosynthesized viral DNA is
incompatible with virus survival as shown by the
degradation of uracilated viral transcripts prior to integration
[53, 55]. To protect its genome from the cytosine
deamination process, HIV-1 encodes the Vif protein that has
the unique property to bind and to induce polyubiquitination
of APOBEC3G followed by its proteosomal degradation,
leading to a substantial reduction of the packaging of
APOBEC3G. The Vif-mediated degradation of APOBEC3G
required an interaction between Vif and a Skp-1-cullin-F box
(SCF)-like complex including the cellular Cul5, elongins B
and C, and Rbx1 [56, 60, 109]. Moreover, HIV-1 Vif was
polyubiquitinated and degraded in the presence of
Uracilation of Viral DNA
APOBEC3G through a Vif-APOBEC3G complex [61]. In
addition, it has been reported that Vif can trigger the
depletion of cellular APOBEC3G through a pathway that
involved the inhibition of the translation of APOBEC3G
mRNA [39, 89]. The protective role of HIV-1 Vif was
efficient to restrict the packaging of APOBEC3G expressed
in human cells but not that of APOBEC3G expressed in
African green monkey (AGM) or rhesus monkey cells or that
of APOBEC3 expressed in murine cells [31, 55]. This
species-specific restriction was attributed to the presence in
the APOBEC3G protein of a charged amino acid at position
128 that is a critical determinant for its interaction with Vif
[7, 52, 82, 106]. APOBEC3B also played a role in
deaminating cytosines of HIV-1 genome, while APOBEC1,
APOBEC3A and APOBEC3C did not [6, 48, 103, 113].
APOBEC3B and APOBEC3C were reported to be potent
inhibitors of SIV [107]. Remarkably, a recent study revealed
that the function of human APOBEC3G as a restriction
factor for HIV-1 in human resting T lymphocytes or in
monocytes could be alleviated by subsequent mitogen
stimulation of T cells or by induction of differentiation of
monocytes into macrophages [14]. In mitogen-stimulated
cells and differentiated-induced monocytes, APOBEC3G
was recruited in an enzymatically incompetent, highmolecular-mass ribonucleoprotein complex creating a
favorable environment for productive HIV-1 infection. In
unstimulated cells and monocytes, APOBEC3G was part of
an enzymatically competent, low-molecular-mass
ribonucleoprotein complex acting in blocking HIV-1
infection, explaining why these cells were refractory to HIV.
This antiviral activity did not trigger massive G-to-A
hypermutations, indicating that APOBEC3G-mediated DNA
editing was not involved, but rather involved a nonenzymatic mechanism that is Vif insensitive. Consistently,
studies have reported that some deaminase-defective
APOBEC3G variants still exhibited anti-HIV activity [66,
87]. Altogether, these findings indicate that the antiviral
function of APOBEC3G may be the result of two separable
properties, one involving uracilation of the DNA and an
other one that is not associated to G-to-A hypermutation.
The precise mechanism of the deamination-independent
antiviral activity of APOBEC3G remains elusive, however.
Infectivity of simple retroviruses like murine leukemia
virus (MLV) was strongly inhibited by the expression of
human APOBEC3G and APOBEC3B, but was unaffected
by the expression of murine APOBEC3 [6, 20, 42]. This
difference correlated with the ability of MLV to package
human APOBEC3G and APOBEC3B, but not murine
APOBEC3, into MLV virion particles, explaining the
resistance of MLV to its cognate APOBEC3 [20]. Human Tcell leukemia virus-1 (HTLV-1) oncoretroviruses infectivity
was profoundly affected by the packaging of human
APOBEC3G, although no significant accumulation of G-toA mutations was reported [65, 81], suggesting that
APOBEC3G exerted its antiviral activity through a
deaminase-independent mechanism. Feline foamy virus
(FFV), a member of the foamy retrovirus subfamily
spumaretrovirinae, was susceptible to feline APOBEC3mediated viral DNA editing that was counteracted by the
expression of the FFV-encoded Bet protein [50]. Hepatitis B
viruses (HBV), the replication of which requires a reverse
transcription step, was susceptible to APOBEC3G and
Current HIV Research, 2006, Vol. 4, No. 1
35
APOBEC3F-mediated antiviral activity. Depending on the
cellular type infected by HBV, it has been observed either no
G-to-A mutations [80, 98] or G-to-A mutations on both
plus- and minus-DNA strand [90].
Beside its role as antiviral factor, APOBEC3G plays a
physiological role in markedly inhibiting retrotransposition
of endogenous retroviruses, namely mammalian longterminal-repeat (LTR) retroposons such as murine IAP and
MusD copies [25]. These retroelements share with infectious
retroviruses a reverse transcription process during which
APOBEC3G functions to edit retroviral DNA preventing
further movement. In contrast, a non-LTR retrotransposon,
such as human LINE-1 (long interspersed nucleotide
element-1), evaded human APOBEC3G inhibition by
replicating in the nucleus, thus avoiding its genome to be
targeted by APOBEC3G that is a cytoplasmic protein [96].
Although the physiological relevance was unclear, it has
been reported that overexpression of human APOBEC3C can
inhibit yeast retrotransposon Ty1 [21].
In conclusion, some proteins of the APOBEC family
exert an antiviral effect against various viruses which
replicate through a reverse transcription step, HBV included.
Part of this antiviral effect can be due to cytosine
deamination of the nascent reverse transcripts, leading to
uracilated cDNAs which are subsequently degraded by, still
uncharacterized, enzymatic activities in target cell. To
counteract this cellular defense mechanism, HIV have
evolved by using the Vif protein which acts by preventing
the packaging of the negative factor into the viral capsid
where reverse transcription takes place. It must be noted that
Vif has no direct effects on uracils per se.
HIV-1 LENTIVIRUS STRATEGY TO COUNTERACT
dUTP MISINCORPORATION IN VIRAL DNA
Beside the emergence of uracils in viral DNA by cytosine
deamination that was counteracted by HIV-1 Vif, there exists
the possibility to uracilate viral DNA v i a dUTP
misincorporation. As noted before, the intracellular dUTP
pool level in macrophages and lymphocytes is quite high [2,
17, 38], and so is the probability to misincorporate dUTP in
place of TTP during reverse transcription. It must be stated
again that this kind of uracilation is not supposed to be
mutagenic, but could nevertheless lead to the degradation of
cDNA and abortion of the viral life cycle. Therefore, it is
probable that HIV-1 is endowed with specific enzymatic
activities to counteract dUTP misincorporation.
In a yeast double-hybrid assay, HIV-1 Vpr protein was
found to bind a human clone encoding a UNG polypeptide
deleted from 52 aminoacid residues corresponding to UNG2
or UNG1 [8]. Because HIV-1 Vpr is packaged into HIV-1
virions particles, the question arises to check whether UNG
was packaged into virions via a specific association with
Vpr. Using sucrose gradient purified virions, we have
identified that the UNG2 isoform was a virion-associated
protein that was recovered in both HIV-1 wild-type and Vprdeficient particles, indicating that Vpr was not required to
package UNG2 [104]. Further studies using GST pull-down
assays demonstrated that UNG2 had the ability to associate
with the integrase (IN) domain of the HIV-1 Gag-Pol
precursor, in addition to its association with HIV-1 Vpr.
36 Current HIV Research, 2006, Vol. 4, No. 1
Priet et al.
This interaction was confirmed by demonstrating that viral
particles proficient for Vpr but deficient for IN or containing
point mutations in IN that inhibited the IN-UNG2
interaction were unable to package endogenous UNG2,
clearly indicating that IN is the sole determinant that
allowed the packaging of UNG2 [71, 104]. Note that one
other group found that overexpressed HIV-1 Vpr directed the
packaging of overexpressed UNG2 into pseudoparticles [54],
while one another group found that overexpressed Vpr
directed the degradation of UNG2 and prevented its
packaging [83]. The opposite conclusions in those studies
could result from either aberrant behavior of overexpressed
and tagged Vpr and UNG2 or by the lack of gradient
purification of bona fide virions.
that the presence of UNG2 within viral particle is
indispensable to obtain infectious virus [74]. Viral
infectivity could be rescued by transfection in UNG2deficient producer cells of a UNG2 gene engineered to escape
siRNA silencing. Interestingly, viral infectivity was also
restored by overexpression in UNG2-deficient producer cells
of a siRNA-insensitive UNG2 protein containing point
mutations that abrogate its binding to HIV-1 Vpr, thus
confirming that the association of UNG2 with HIV-1 Vpr
has no influence on HIV-1 propagation. The blockage of
UNG2 catalytic activity by overexpression in producer cells
of the UNG inhibitor Ugi led to the production of
noninfectious virus, revealing the importance of the uracil
removal capability of UNG2 in the viral life cycle.
HIV-1 virus produced from cells depleted of UNG2 by
RNA interference-mediated knock-down were unable to
propagate either in dividing or nondividing cells, indicating
The impaired propagation of UNG2-depleted virions was
due to the failure of accumulation of viral transcripts. This
impairment was reminiscent of that observed for ∆Vif
Fig. (2). Prevention of uracil incorporation in newly synthesized reverse transcripts in HIV-1 infected cells. UNG2 is packaged into
virions via a specific interaction with the integrase (IN) domain of the Gag-Pol precursor and acts to repair dUTP misincorporated
during reverse transcription. The uracil-free proviral DNA is then imported into the nucleus and expressed. Virus produced from
UNG2-deficient cells is devoid of UNG2. Reverse transcription allows uracil incorporation from dUTP into the first strand cDNA.
Unrepaired uracil lead to abnormal second strand synthesis. The damaged and uracilated proviral DNA recruits UNG2 and other repair
activities from the nucleus and is degraded.
Uracilation of Viral DNA
viruses that were unable to counteract cellular APOBEC3G
and thus generated uracilated transcripts that are degraded in
the cytoplasm of infected cells [53, 55]. The viral infectivity
of UNG2-deficient virus could be rescued by targeting
overexpressed dUTPase into UNG2-deficient viral particles,
demonstrating that the failure of UNG2-deficient virus to
replicate was due to uracils in viral DNA arising from
misincorporated dUTP. Despite the presence of uracils in
transcripts from UNG2-deficient virus, no G-to-A mutations
Current HIV Research, 2006, Vol. 4, No. 1
37
were found nor were there any excess mutations with the
Vpr-deficient virus. These findings were not unexpected as
HIV-1 RT readily incorporated dUTP opposite to dAMP,
but strongly resisted at incorporating dUTP opposite to
dGMP.
Although not mutagenic, the presence of uracils as dU-rA
pairs in transcripts led to the degradation of the proviral
DNA in target cell, thus blocking HIV-1 replication. The
Fig. (3). Cellular and HIV-1 pathways utilized in uracil base excision repair (BER). The cellular short-patch BER pathway is depicted
in left panel. The cellular long-patch BER pathway is depicted in middle panel. The HIV-1 specific uracil BER pathway is depicted in
right panel. Left panel: cellular uracil DNA glycosylase-induced abasic site is cleaved on the 5’ side by AP-endonuclease APE 1and
on the 3’ side by the AP-lyase activity of ß-polymerase. The resulting gap is filled-in by the polymerase activity of ß-polymerase and
the remaining nick is sealed by the XRCC- DNA ligase 3 complex. Middle panel: upon the action of APE1, the concerted action of
DNA Pol∂/ε, RFC, PCNA and FEN1 displaces the strand 3’ to the nick to produce a flap of 2-10 nucleotides, which is cut by FEN1
endonuclease. Pol∂/ε and PCNA then synthesized a new 2-10 nucleotides patch that is ligated by DNA ligase 1. Right panel: HIV-1associated uracil DNA glycosylase excises the uracil base. The resulting abasic site is then incised on its 5’ side by the dNTPdependent AP-endonuclease activity of HIV-1 reverse transcriptase. The remaining free 3’-OH end serves to prime polymerization via
the long strand displacement synthesis property of HIV-1 reverse transcriptase. The uracil base is indicated by a hatched box. Grey
boxes indicate newly inserted nucleotides.
38 Current HIV Research, 2006, Vol. 4, No. 1
mechanism(s) by which this degradation occured is not yet
characterized. For UNG2-deficient viruses, one could
envisage that uracils will be efficiently incorporated in the
viral minus strand cDNA. Uracils in minus strand are known
to alter the RNase H activity and could disrupt the second
strand synthesis [41], leaving breaks and/or RNA primers.
Once in the nucleus of target cell, the heavily uracilated and
damaged proviral DNA would induce catastrophic DNA
repair on both strands leading to degradation of the provirus
(Fig. 2).
The main difference in the role of UNG2, either
protective from uracilation when associated to the reverse
transcription/preintegration complexes or destructive of
uracilated proviruses in the nucleus of target cells would
therefore result from difference in both timing and
environment. During reverse transcription in the capsid and
or in the cytoplasm of target cell, the UNG2 would exert a
protective role because the UNG2-containing HIV-1
replication complex contains all the activities needed to fully
repair an uracil in the minus strand cDNA. We will see
below what we already know about the mechanisms of uracil
repair by this replication complex.
It is noteworthy that both UNG2 and APOBEC3G exert
their effects very early in the viral life cycle, as soon as the
minus strong stop cDNA begins. Accorddingly, both Vif
and UNG2 defects must be transcomplemented in producing
cells and not in target cells.
URACIL REPAIR MECHANISM BY HIV-1 VIRION
HIV-1 viral lysate contained all enzymatic activities to
convert G: U mispair to G: C pair [72]. Although the
cellular BER process required the intervention of an APendonuclease, we [72] and other [54] failed to detect the
presence within HIV-1 particles of human AP-endonucleases
APE1 or APE2 suggesting that the HIV-1 specific uracil
repair process may use a virally-encoded unknown enzymatic
activity that may play a role similar to that of an APendonuclease enzyme. To understand how HIV-1 repaired
dUTP misincorporated in viral transcripts, we have
developed a cell-free system and we demonstrated that the
concerted action of UNG2 and HIV-1 reverse transcriptase
(RT) can replace rG: dU mispairs to rG: dC pairs. This
uracil repair was successfully accomplished because UNG2
was active on RNA/DNA heteroduplexes, and because HIV-1
RT displayed an unexpected dNTP-dependent APendonuclease activity [74] on either DNA-DNA homoduplex
or RNA/DNA heteroduplex. The HIV-1 specific BER
mechanism was similar to that of the cellular long-patch
excision repair mechanism, in that it used a DNA strand
displacement step. However, these mechanisms diverged in
that cellular BER utilized a combination of multiple
enzymatic activities including AP-endonuclease, DNA
polymerase, PCNA, RFC, FEN1 and DNA ligase 1 to
replace a stretch of 2-10 nucleotides containing the baseless
nucleotide, while HIV-1 BER utilized only HIV-1 RT to
cleave 5’ to the baseless nucleotide leading a free 3’-OH end
that was used to polymerize longer stretch of nucleotides
(Fig. (3)). It is worth noting that HIV-1 repair does not
require FEN1 nor ligase because the strand displacement
activity of the reverse transcriptase will reconstitute the
Priet et al.
strand ahead of the lesion. We have found that the repair was
most efficient when uracil was not more than 25 to 30 nt
from the 3’end of the cDNA. This may be because the
reverse transcriptase needs a 3’OH-end to attach to the cDNA
and this could reflect the size of the RT-UNG2 complexes
onto the cDNA.
The composition of this replication complex is presently
unknnown. Individuallly, UNG2 interacts with Vpr,
integrase and RT. RT interacts also with integrase and with
NC. Is there a large holo-enzyme? Could it be that these
interactions result in unmasking new enzymatic activities in
either the reverse transcriptase and/or integrase?
VIRALLY-ASSOCIATED UNG2 WILL NOT REPAIR
DEAMINATED CYTOSINE.
Typically, the mechanism of uracil repair requires a
strand template allowing polymerase to incorporate the
correct nucleotide in place of the baseless nucleotide. It has
been demonstrated that the single-stranded minus DNA was
the substrate for APOBEC3G and that the removal of viral
RNA template by the RNase H activity of RT was a
prerequisite for the cytosine deamination process [91, 108].
This means that the UNG2-RT complex might be unable to
repair uracils arising from cytosine deamination because of
the absence of RNA template to guide repair. Indeed, the
recovery of G-to-A mutations in Vif-deficient viral
transcripts confirmed that uracils arising from cytosine
deamination were fixed in minus-strand DNA and therefore
escaped the viral UNG2-mediated uracil repair pathway.
CONCLUDING REMARKS
Viruses have evolved in acquiring genes that allow them
to replicate in an adverse cellular environment, like those
promoting the synthesis of uracil-substituted viral transcripts
that impairs replication. Viruses that replicate in
nondividing cells, such as neurons or macrophages, are
equipped with their own UNG and/or dUTPase enzymes to
compensate the low levels of cellular UNG and dUTPase
enzymes. Viruses that replicate in the cytoplasm of cells also
encode their own UNG and/or dUTPase to compensate the
fact that UNG and dUTPase enzymes are essentially
localized in nucleus. Since HIV-1 infects nondividing cells
and since reverse transcription takes place mainly in the
cytoplasm, the ability to package host UNG2 and to encode
Vif are crucial events for virus survival. HIV-2 and SIV
replicate, like HIV-1, in the cytoplasm of nondividing cells,
but no UNG2 was recovered in viral particles [73]. To cope
with the danger of dUTP misincorporation, HIV-2 may have
evolved in packaging another member of the human UNG
family or in packaging the human dUTPase enzyme, and
SIV may have evolved in packaging the simian UNG2
counterpart. Whether other retroviruses such as MLV or
HTLV-1, or HBV, were sensitive to dUTP misincorporation
and how they resist to uracilation of their proviral DNA
remains to be established.
We have showed that forcing the retention of uracils in
viral transcripts represented an innate immune response
against viruses. Note that dUTP levels in infected versus
noninfected cells have not been evaluated, raising the
Uracilation of Viral DNA
possibility that dUTP levels may be increased in cells in
response to a viral infection, thus representing a second
barrier against viral invasion. Therefore, any strategies
aiming to enhance uracil levels in transcripts of a variety of
viruses may reinforce the innate response contributing to the
potent inhibition of viral replication. It is expected that such
a strategy will not be easily hurdled by viral mutations, in
contrast to the frequency of multi-drug resistant HIV-1
mutants found in patients under highly active antiretroviral
therapy (HAART). We can thus envisage new therapeutic
roads targeting dUTP misincorporation to control HIV-1
infection by manipulating interactions between host cell
factors and viruses. These may include drugs that act in
impeding the interaction between HIV-1 integrase and UNG2
to block the packaging of UNG2 during virus budding, or
drugs that impede the interaction between HIV-1 reverse
transcriptase and UNG2 to inhibit the viral uracil repair
mechanism. However, to rule out any effect on host genomic
DNA, these drugs will have to target exclusively the viral
partner involved in the interaction. Also, it would be
important to target the AP-endonuclease activity of HIV-1
reverse transcriptase, when the molecular mechanism(s) will
be understood.
Current HIV Research, 2006, Vol. 4, No. 1
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
ACKNOWLEDGMENTS
We thank Houssam Attoui for carefully reading of the
manuscript. This work was supported by INSERM, by
Ensemble Contre le SIDA (Sidaction), and by the French
Agency against AIDS (ANRS).
[18]
[19]
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Alce TM and Popik W. (2004). APOBEC3G is incorporated into
virus-like particles by a direct interaction with HIV-1 Gag
nucleocapsid protein. The Journal of Biological Chemistry. 279:
34083-34086.
Aquaro S, Calio R, Balzarini J, Bellocchi MC, Garaci E, and
Perno CF. (2002). Macrophages and HIV infection: therapeutical
approaches toward this strategic virus reservoir. Antiviral
Research. 55: 209-225.
Beale RC, Petersen-Mahrt SK, Watt IN, Harris RS, Rada C, and
Neuberger MS. (2004). Comparison of the differential contextdependence of DNA deamination by APOBEC enzymes:
correlation with mutation spectra in vivo. Journal of Molecular
Biology. 337: 585-596.
Benit L, De Parseval N, Casella JF, Callebaut I, Cordonnier A,
and Heidmann T. (1997). Cloning of a new murine endogenous
retrovirus, MuERV-L, with strong similarity to the human HERVL element and with a gag coding sequence closely related to the
Fv1 restriction gene. Journal of Virology. 71: 5652-5657.
Bergman AC, Bjornberg O, Nord J, Nyman PO, and Rosengren
AM. (1994). The protein p30, encoded at the gag-pro junction of
mouse mammary tumor virus, is a dUTPase fused with a
nucleocapsid protein. Virology. 204: 420-424.
Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, and
Malim MH. (2004). Cytidine deamination of retroviral DNA by
diverse APOBEC proteins. Current Biology. 14: 1392-1396.
Bogerd HP, Doehle BP, Wiegand HL, and Cullen BR. (2004). A
single amino acid difference in the host APOBEC3G protein
controls the primate species specificity of HIV type 1 virion
infectivity factor. Proceedings of the National Academy of
Sciences of the United States of America. 101: 3770-3774.
Bouhamdan M, Benichou S, Rey F, Navarro JM, Agostini I, Spire
B, Camonis J, Slupphaug G, Vigne R, Benarous R, and Sire J.
(1996). Human immunodeficiency virus type 1 Vpr protein binds
to the uracil DNA glycosylase DNA repair enzyme. Journal of
Virology. 70: 697-704.
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
39
Bransteitter R, Pham P, Scharff MD, and Goodman MF. (2003).
Activation-induced cytidine deaminase deaminates deoxycytidine
on single-stranded DNA but requires the action of RNase.
Proceedings of the National Academy of Sciences of the United
States of America. 100: 4102-4107.
Caposio P, Riera L, Hahn G, Landolfo S, and Gribaudo G. (2004).
Evidence that the human cytomegalovirus 46-kDa UL72 protein
is not an active dUTPase but a late protein dispensable for
replication in fibroblasts. Virology. 325: 264-276.
Caradonna SJ and Cheng YC. (1981). Induction of uracil-DNA
glycosylase and dUTP nucleotidohydrolase activity in herpes
simplex virus-infected human cells. The Journal of Biological
Chemistry. 256: 9834-9837.
Cen S, Guo F, Niu M, Saadatmand J, Deflassieux J, and Kleiman
L. (2004). The interaction between HIV-1 Gag and APOBEC3G.
The Journal of Biological Chemistry. 279: 33177-33184.
Chen H and Shaw BR. (1993). Kinetics of bisulfite-induced
cytosine deamination in single-stranded DNA. Biochemistry. 32:
3535-3539.
Chiu YL, Soros VB, Kreisberg JF, Stopak K, Yonemoto W, and
Greene WC. (2005). Cellular APOBEC3G restricts HIV-1
infection in resting CD4+ T cells. Nature. 435: 108-114.
Cordonnier A, Casella JF, and Heidmann T. (1995). Isolation of
novel human endogenous retrovirus-like elements with foamy
virus-related pol sequence. Journal of Virology. 69: 5890-5897.
Courcelle CT, Courcelle J, Prichard MN, and Mocarski ES.
(2001). Requirement for uracil-DNA glycosylase during the
transition to late- phase cytomegalovirus DNA replication.
Journal of Virology. 75: 7592-7601.
Cross DR, Miller BJ, and James SJ. (1993). A simplified HPLC
method for simultaneously quantifying ribonucleotides and
deoxyribonucleotides in cell extracts or frozen tissues. Cell
Proliferation. 26: 327-336.
De Silva FS and Moss B. (2003). Vaccinia virus uracil DNA
glycosylase has an essential role in DNA synthesis that is
independent of its glycosylase activity: catalytic site mutations
reduce virulence but not virus replication in cultured cells.
Journal of Virology. 77: 159-166.
Dickerson SK, Market E, Besmer E, and Papavasiliou FN. (2003).
AID mediates hypermutation by deaminating single stranded
DNA. Journal of Experimental Medecine. 197: 1291-1296.
Doehle BP, Schafer A, Wiegand HL, Bogerd HP, and Cullen BR.
(2005). Differential Sensitivity of Murine Leukemia Virus to
APOBEC3-Mediated Inhibition Is Governed by Virion Exclusion.
Journal of Virology. 79: 8201-8207.
Dutko JA, Schafer A, Kenny AE, Cullen BR, and Curcio MJ.
(2005). Inhibition of a yeast LTR retrotransposon by human
APOBEC3 cytidine deaminases. Current Biology. 15: 661-666.
el-Hajj HH, Zhang H, and Weiss B. (1988). Lethality of a dut
(deoxyuridine triphosphatase) mutation in Escherichia coli.
Journal of Bacteriology. 170: 1069-1075.
Elder JH, Lerner DL, Hasselkus-Light CS, Fontenot DJ, Hunter E,
Luciw PA, Montelaro RC, and Phillips TR. (1992). Distinct subsets
of retroviruses encode dUTPase. Journal of Virology. 66: 17911794.
Ellison KS, Peng W, and McFadden G. (1996). Mutations in
active-site residues of the uracil-DNA glycosylase encoded by
vaccinia virus are incompatible with virus viability. Journal of
Virology. 70: 7965-7973.
Esnault C, Heidmann O, Delebecque F, Dewannieux M, Ribet D,
Hance AJ, Heidmann T, and Schwartz O. (2005). APOBEC3G
cytidine deaminase inhibits retrotransposition of endogenous
retroviruses. Nature. 433: 430-433.
Fischer JA, Muller-Weeks S, and Caradonna S. (2004).
Proteolytic degradation of the nuclear isoform of uracil-DNA
glycosylase occurs during the S phase of the cell cycle. DNA
Repair (Amst). 3: 505-513.
Fisher EF and Caruthers MH. (1979). Studies on gene control
regions XII. The functional significance of a lac operator
constitutive mutation. Nucleic Acids Research. 7: 401-416.
Fisher FB and Preston VG. (1986). Isolation and characterisation
of herpes simplex virus type 1 mutants which fail to induce
dUTPase activity. Virology. 148: 190-197.
Fleming SB, Lyttle DJ, Sullivan JT, Mercer AA, and Robinson AJ.
(1995). Genomic analysis of a transposition-deletion variant of
orf virus reveals a 3.3 kbp region of non-essential DNA. Journal
of General Virology. 76 (Pt 12): 2969-2978.
40 Current HIV Research, 2006, Vol. 4, No. 1
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
Frederico LA, Kunkel TA, and Shaw BR. (1990). A sensitive
genetic assay for the detection of cytosine deamination:
determination of rate constants and the activation energy.
Biochemistry. 29: 2532-2537.
Gaddis NC, Sheehy AM, Ahmad KM, Swanson CM, Bishop KN,
Beer BE, Marx PA, Gao F, Bibollet-Ruche F, Hahn BH, and
Malim MH. (2004). Further investigation of simian
immunodeficiency virus Vif function in human cells. Journal of
Virology. 78: 12041-12046.
Gadsden MH, McIntosh EM, Game JC, Wilson PJ, and Haynes
RH. (1993). dUTP pyrophosphatase is an essential enzyme in
Saccharomyces cerevisiae. The EMBO Journal. 12: 4425-4431.
Greagg MA, Fogg MJ, Panayotou G, Evans SJ, Connolly BA, and
Pearl LH. (1999). A read-ahead function in archaeal DNA
polymerases detects promutagenic template-strand uracil.
Proceedings of the National Academy of Sciences of the United
States of America. 96: 9045-9050.
Harris RS, Petersen-Mahrt SK, and Neuberger MS. (2002). RNA
editing enzyme APOBEC1 and some of its homologs can act as
DNA mutators. Molecular Cell. 10: 1247-1253.
Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt
SK, Watt IN, Neuberger MS, and Malim MH. (2003). DNA
deamination mediates innate immunity to retroviral infection. Cell.
113: 803-809.
Hochhauser SJ and Weiss B. (1978). Escherichia coli mutants
deficient in deoxyuridine triphosphatase. Journal of Bacteriology.
134: 157-166.
Hoeijmakers JH. (2001). Genome maintenance mechanisms for
preventing cancer. Nature. 411: 366-374.
Horowitz RW, Zhang H, Schwartz EL, Ladner RD, and Wadler
S. (1997). Measurement of deoxyuridine triphosphate and
thymidine triphosphate in the extracts of thymidylate synthaseinhibited cells using a modified DNA polymerase assay.
Biochemical Pharmacology. 54: 635-638.
Kao S, Miyagi E, Khan MA, Takeuchi H, Opi S, Goila-Gaur R,
and Strebel K. (2004). Production of infectious human
immunodeficiency virus type 1 does not require depletion of
APOBEC3G from virus-producing cells. Retrovirology. 1: 27.
Khan MA, Kao S, Miyagi E, Takeuchi H, Goila-Gaur R, Opi S,
Gipson CL, Parslow TG, Ly H, and Strebel K. (2005). Viral RNA
is required for the association of APOBEC3G with human
immunodeficiency virus type 1 nucleoprotein complexes. Journal
of Virology 79: 5870-5874.
Klarmann GJ, Chen X, North TW, and Preston BD. (2003).
Incorporation of uracil into minus strand DNA affects the
specificity of plus strand synthesis initiation during lentiviral
reverse transcription. The Journal of Biological Chemistry. 278:
7902-7909.
Kobayashi M, Takaori-Kondo A, Shindo K, Abudu A, Fukunaga
K, and Uchiyama T. (2004). APOBEC3G targets specific virus
species. Journal of Virology. 78: 8238-8244.
Krokan HE, Otterlei M, Nilsen H, Kavli B, Skorpen F, Andersen
S, Skjelbred C, Akbari M, Aas PA, and Slupphaug G. (2001).
Properties and functions of human uracil-DNA glycosylase from
the UNG gene. Progress in Nucleic Acid Research and
Molecular Biology. 68: 365-386.
Ladner RD and Caradonna SJ. (1997). The human dUTPase gene
encodes both nuclear and mitochondrial isoforms. Differential
expression of the isoforms and characterization of a cDNA
encoding the mitochondrial species. The Journal of Biological
Chemistry. 272: 19072-19080.
Lecossier D, Bouchonnet F, Clavel F, and Hance AJ. (2003).
Hypermutation of HIV-1 DNA in the absence of the Vif protein.
Science. 300: 1112.
Lerner DL, Wagaman PC, Phillips TR, Prospero-Garcia O,
Henriksen SJ, Fox HS, Bloom FE, and Elder JH. (1995).
Increased mutation frequency of feline immunodeficiency virus
lacking functional deoxyuridine-triphosphatase. Proceedings of
the National Academy of Sciences of the United States of
America. 92: 7480-7484.
Lichtenstein DL, Rushlow KE, Cook RF, Raabe ML, Swardson
CJ, Kociba GJ, Issel CJ, and Montelaro RC. (1995). Replication in
vitro and in vivo of an equine infectious anemia virus mutant
deficient in dUTPase activity. Journal of Virology. 69: 2881-2888.
Liddament MT, Brown WL, Schumacher AJ, and Harris RS.
(2004). APOBEC3F properties and hypermutation preferences
Priet et al.
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
indicate activity against HIV-1 in vivo. Current Biology. 14: 13851391.
Lindahl T. (1993). Instability and decay of the primary structure
of DNA. Nature. 362: 709-715.
Lochelt M, Romen F, Bastone P, Muckenfuss H, Kirchner N, Kim
YB, Truyen U, Rosler U, Battenberg M, Saib A, Flory E, Cichutek
K, and Munk C. (2005). The antiretroviral activity of APOBEC3
is inhibited by the foamy virus accessory Bet protein. Proceedings
of the National Academy of Sciences of the United States of
America. 102: 7982-7987.
Luo K, Liu B, Xiao Z, Yu Y, Yu X, Gorelick R, and Yu XF.
(2004). Amino-terminal region of the human immunodeficiency
virus type 1 nucleocapsid is required for human APOBEC3G
packaging. Journal of Virology. 78: 11841-11852.
Mangeat B, Turelli P, Liao S, and Trono D. (2004). A single
amino acid determinant governs the species-specific sensitivity of
APOBEC3G to Vif action. The Journal of Biological Chemistry.
279: 14481-14483.
Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, and Trono D.
(2003). Broad antiretroviral defence by human APOBEC3G
through lethal editing of nascent reverse transcripts. Nature. 424:
99-103.
Mansky LM, Preveral S, Selig L, Benarous R, and Benichou S.
(2000). The interaction of vpr with uracil DNA glycosylase
modulates the human immunodeficiency virus type 1 In vivo
mutation rate. Journal of Virology. 74: 7039-7047.
Mariani R, Chen D, Schrofelbauer B, Navarro F, Konig R,
Bollman B, Munk C, Nymark-McMahon H, and Landau NR.
(2003). Species-specific exclusion of APOBEC3G from HIV-1
virions by Vif. Cell. 114: 21-31.
Marin M, Rose KM, Kozak SL, and Kabat D. (2003). HIV-1 Vif
protein binds the editing enzyme APOBEC3G and induces its
degradation. Nature Medicine. 9: 1398-1403.
Martinez MA, Sala M, Vartanian JP, and Wain-Hobson S. (1995).
Reverse transcriptase and substrate dependence of the RNA
hypermutagenesis reaction. Nucleic Acids Research. 23: 25732578.
McClure MA, Johnson MS, and Doolittle RF. (1987). Relocation
of a protease-like gene segment between two retroviruses.
Proceedings of the National Academy of Sciences of the United
States of America. 84: 2693-2697.
McGeoch DJ. (1990). Protein sequence comparisons show that
the 'pseudoproteases' encoded by poxviruses and certain
retroviruses belong to the deoxyuridine triphosphatase family.
Nucleic Acids Research. 18: 4105-4110.
Mehle A, Goncalves J, Santa-Marta M, McPike M, and Gabuzda
D. (2004). Phosphorylation of a novel SOCS-box regulates
assembly of the HIV-1 Vif-Cul5 complex that promotes
APOBEC3G degradation. Genes and Development. 18: 28612866.
Mehle A, Strack B, Ancuta P, Zhang C, McPike M, and Gabuzda
D. (2004). Vif overcomes the innate antiviral activity of
APOBEC3G by promoting its degradation in the ubiquitinproteasome pathway. The Journal of Biological Chemistry. 279:
7792-7798.
Millns AK, Carpenter MS, and DeLange AM. (1994). The
vaccinia virus-encoded uracil DNA glycosylase has an essential
role in viral DNA replication. Virology. 198: 504-513.
Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y,
and Honjo T. (2000). Class switch recombination and
hypermutation require activation-induced cytidine deaminase
(AID), a potential RNA editing enzyme. Cell. 102: 553-563.
Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K,
Davidson NO, and Honjo T. (1999). Specific expression of
activation-induced cytidine deaminase (AID), a novel member of
the RNA-editing deaminase family in germinal center B cells.
The Journal of Biological Chemistry. 274: 18470-18476.
Navarro F, Bollman B, Chen H, Konig R, Yu Q, Chiles K, and
Landau NR. (2005). Complementary function of the two catalytic
domains of APOBEC3G. Virology. 333: 374-386.
Newman EN, Holmes RK, Craig HM, Klein KC, Lingappa JR,
Malim MH, and Sheehy AM. (2005). Antiviral function of
APOBEC3G can be dissociated from cytidine deaminase activity.
Current Biology. 15: 166-170.
Nilsen H, Otterlei M, Haug T, Solum K, Nagelhus TA, Skorpen F,
and Krokan HE. (1997). Nuclear and mitochondrial uracil-DNA
glycosylases are generated by alternative splicing and
Uracilation of Viral DNA
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
transcription from different positions in the UNG gene. Nucleic
Acids Research. 25: 750-755.
Powell LM, Wallis SC, Pease RJ, Edwards YH, Knott TJ, and
Scott J. (1987). A novel form of tissue-specific RNA processing
produces apolipoprotein-B48 in intestine. Cell. 50: 831-840.
Preston VG, and Fisher FB. (1984). Identification of the herpes
simplex virus type 1 gene encoding the dUTPase. Virology. 138:
58-68.
Prichard MN, Duke GM, and Mocarski ES. (1996). Human
cytomegalovirus uracil DNA glycosylase is required for the
normal temporal regulation of both DNA synthesis and viral
replication. Journal of Virology. 70: 3018-3025.
Priet S, Navarro JM, Querat G, and Sire J. (2003). Reversion of
the Lethal Phenotype of an HIV-1 Integrase Mutant Virus by
Overexpression of the Same Integrase Mutant Protein. The
Journal of Biological Chemistry. 278: 20724-20730.
Priet S, Navarro JM, Gros N, Querat G, and Sire J. (2003).
Functional Role of HIV-1 Virion-associated Uracil DNA
Glycosylase 2 in the Correction of G:U Mispairs to G:C Pairs. The
Journal of Biological Chemistry. 278: 4566-4571.
Priet S, Navarro JM, Gros N, Querat G, and Sire J. (2003).
Differential incorporation of uracil DNA glycosylase UNG2 into
HIV-1, HIV-2, and SIV(MAC) viral particles. Virology. 307:
283-289.
Priet S, Gros N, Navarro JM, Boretto J, Canard B, Querat G, and
Sire J. (2005). HIV-1-associated uracil DNA glycosylase activity
controls dUTP misincorporation in viral DNA and is essential to
the HIV-1 life cycle. Molecular Cell. 17: 479-490.
Pyles RB, and Thompson RL. (1994). Mutations in accessory
DNA replicating functions alter the relative mutation frequency
of herpes simplex virus type 1 strains in cultured murine cells.
Journal of Virology. 68: 4514-4524.
Pyles RB, and Thompson RL. (1994). Evidence that the herpes
simplex virus type 1 uracil DNA glycosylase is required for
efficient viral replication and latency in the murine nervous
system. Journal of Virology. 68: 4963-4972.
Pyles RB, Sawtell NM, and Thompson RL. (1992). Herpes
simplex virus type 1 dUTPase mutants are attenuated for
neurovirulence, neuroinvasiveness, and reactivation from
latency. Journal of Virology. 66: 6706-6713.
Reddy SM, Williams M, and Cohen JI. (1998). Expression of a
uracil DNA glycosylase (UNG) inhibitor in mammalian cells:
varicella-zoster virus can replicate in vitro in the absence of
detectable UNG activity. Virology. 251: 393-401.
Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O,
Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A,
Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N,
Muramatsu M, Notarangelo LD, Kinoshita K, Honjo T, Fischer A,
and Durandy A. (2000). Activation-induced cytidine deaminase
(AID) deficiency causes the autosomal recessive form of the
Hyper-IgM syndrome (HIGM2). Cell. 102: 565-575.
Rosler C, Kock J, Malim MH, Blum HE, and von Weizsacker F.
(2004). Comment on "Inhibition of hepatitis B virus replication by
APOBEC3G". Science. 305: 1403; author reply 1403.
Sasada A, Takaori-Kondo A, Shirakawa K, Kobayashi M, Abudu
A, and Uchiyama T. (2005). APOBEC3G targets human T-cell
leukemia virus type 1. Retrovirology. 2.
Schrofelbauer B, Chen D, and Landau NR. (2004). A single
amino acid of APOBEC3G controls its species-specific
interaction with virion infectivity factor (Vif). Proceedings of the
National Academy of Sciences of the United States of America.
101: 3927-3932.
Schrofelbauer B, Yu Q, Zeitlin SG, and Landau NR. (2005).
Human Immunodeficiency Virus Type 1 Vpr Induces the
Degradation of the UNG and SMUG Uracil-DNA Glycosylases.
Journal of Virology 79: 10978-10987.
Selker EU. (1990). DNA methylation and chromatin structure: a
view from below. Trends in Biochemical Sciences. 15: 103-107.
Sheehy AM, Gaddis NC, Choi JD, and Malim MH. (2002).
Isolation of a human gene that inhibits HIV-1 infection and is
suppressed by the viral Vif protein. Nature. 418: 646-650.
Shen JC, Rideout WM, 3rd, and Jones PA. (1992). High
frequency mutagenesis by a DNA methyltransferase. Cell. 71:
1073-1080.
Shindo K, Takaori-Kondo A, Kobayashi M, Abudu A, Fukunaga
K, and Uchiyama T. (2003). The enzymatic activity of
CEM15/Apobec-3G is essential for the regulation of the
Current HIV Research, 2006, Vol. 4, No. 1
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
[99]
[100]
[101]
[102]
[103]
[104]
[105]
41
infectivity of HIV-1 virion but not a sole determinant of its
antiviral activity. The Journal of Biological Chemistry. 278:
44412-44416.
Steagall WK, Robek MD, Perry ST, Fuller FJ, and Payne SL.
(1995). Incorporation of uracil into viral DNA correlates with
reduced replication of EIAV in macrophages. Virology. 210:
302-313.
Stopak K, de Noronha C, Yonemoto W, and Greene WC. (2003).
HIV-1 Vif blocks the antiviral activity of APOBEC3G by
impairing both its translation and intracellular stability. Molecular
Cell. 12: 591-601.
Suspene R, Guetard D, Henry M, Sommer P, Wain-Hobson S, and
Vartanian JP. (2005). Extensive editing of both hepatitis B virus
DNA strands by APOBEC3 cytidine deaminases in vitro and in
vivo. Proceedings of the National Academy of Sciences of the
United States of America. 102: 8321-8326.
Suspene R, Sommer P, Henry M, Ferris S, Guetard D, Pochet S,
Chester A, Navaratnam N, Wain-Hobson S, and Vartanian JP.
(2004). APOBEC3G is a single-stranded DNA cytidine
deaminase and functions independently of HIV reverse
transcriptase. Nucleic Acids Research. 32: 2421-2429.
Svarovskaia ES, Xu H, Mbisa JL, Barr R, Gorelick RJ, Ono A,
Freed EO, Hu WS, and Pathak VK. (2004). Human
apolipoprotein B mRNA-editing enzyme-catalytic polypeptidelike 3G (APOBEC3G) is incorporated into HIV-1 virions through
interactions with viral and nonviral RNAs. The Journal of
Biological Chemistry. 279: 35822-35828.
Teng B, Burant CF, and Davidson NO. (1993). Molecular cloning
of an apolipoprotein B messenger RNA editing protein. Science.
260: 1816-1819.
Threadgill DS, Steagall WK, Flaherty MT, Fuller FJ, Perry ST,
Rushlow KE, Le Grice SF, and Payne SL. (1993).
Characterization of equine infectious anemia virus dUTPase:
growth properties of a dUTPase-deficient mutant. Journal of
Virology. 67: 2592-2600.
Traut TW. (1994). Physiological concentrations of purines and
pyrimidines. Molecular and Cellular Biochemestry. 140: 1-22.
Turelli P, Vianin S, and Trono D. (2004). The innate antiretroviral
factor APOBEC3G does not affect human LINE-1
retrotransposition in a cell culture assay. The Journal of
Biological Chemistry. 279: 43371-43373.
Turelli P, Guiguen F, Mornex JF, Vigne R, and Querat G. (1997).
dUTPase-minus caprine arthritis-encephalitis virus is attenuated
for pathogenesis and accumulates G-to-A substitutions. Journal of
Virology. 71: 4522-4530.
Turelli P, Mangeat B, Jost S, Vianin S, and Trono D. (2004).
Inhibition of hepatitis B virus replication by APOBEC3G. Science.
303: 1829.
Turelli P, Petursson G, Guiguen F, Mornex JF, Vigne R, and
Querat G. (1996). Replication properties of dUTPase-deficient
mutants of caprine and ovine lentiviruses. Journal of Virology. 70:
1213-1217.
Tye BK, Nyman PO, Lehman IR, Hochhauser S, and Weiss B.
(1977). Transient accumulation of Okazaki fragments as a result
of uracil incorporation into nascent DNA. Proceedings of the
National Academy of Sciences of the United States of America.
74: 154-157.
Verri A, Mazzarello P, Biamonti G, Spadari S, and Focher F.
(1990). The specific binding of nuclear protein(s) to the cAMP
responsive element (CRE) sequence (TGACGTCA) is reduced
by the misincorporation of U and increased by the deamination of
C. Nucleic Acids Research. 18: 5775-5780.
Wagaman PC, Hasselkus-Light CS, Henson M, Lerner DL,
Phillips TR, and Elder JH. (1993). Molecular cloning and
characterization of deoxyuridine triphosphatase from feline
immunodeficiency virus (FIV). Virology. 196: 451-457.
Wiegand HL, Doehle BP, Bogerd HP, and Cullen BR. (2004). A
second human antiretroviral factor, APOBEC3F, is suppressed by
the HIV-1 and HIV-2 Vif proteins. The EMBO Journal. 23: 24512458.
Willetts KE, Rey F, Agostini I, Navarro JM, Baudat Y, Vigne R,
and Sire J. (1999). DNA repair enzyme uracil DNA glycosylase
is specifically incorporated into human immunodeficiency virus
type 1 viral particles through a Vpr-independent mechanism.
Journal of Virology 73: 1682-1688.
Wink DA, Kasprzak KS, Maragos CM, Elespuru RK, Misra M,
Dunams TM, Cebula TA, Koch WH, Andrews AW, Allen JS, and
42 Current HIV Research, 2006, Vol. 4, No. 1
[106]
[107]
[108]
[109]
Priet et al.
et al. (1991). DNA deaminating ability and genotoxicity of nitric
oxide and its progenitors. Science. 254: 1001-1003.
Xu H, Svarovskaia ES, Barr R, Zhang Y, Khan MA, Strebel K,
and Pathak VK. (2004). A single amino acid substitution in human
APOBEC3G antiretroviral enzyme confers resistance to HIV-1
virion infectivity factor-induced depletion. Proceedings of the
National Academy of Sciences of the United States of America.
101: 5652-5657.
Yu Q, Chen D, Konig R, Mariani R, Unutmaz D, and Landau NR.
(2004). APOBEC3B and APOBEC3C are potent inhibitors of
simian immunodeficiency virus replication. The Journal of
Biological Chemistry. 279: 53379-53386.
Yu Q, Konig R, Pillai S, Chiles K, Kearney M, Palmer S, Richman
D, Coffin JM, and Landau NR. (2004). Single-strand specificity
of APOBEC3G accounts for minus-strand deamination of the
HIV genome. Nature Structural & Molecular Biology. 11: 435442.
Yu Y, Xiao Z, Ehrlich ES, Yu X, and Yu XF. (2004). Selective
assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin
Received: June 21, 2005
[110]
[111]
[112]
[113]
ligase complex through a novel SOCS box and upstream
cysteines. Genes and Development. 18: 2867-2872.
Zennou V, Perez-Caballero D, Gottlinger H, and Bieniasz PD.
(2004). APOBEC3G incorporation into human immunodeficiency
virus type 1 particles. Journal of Virology. 78: 12058-12061.
Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, and
Gao L. (2003). The cytidine deaminase CEM15 induces
hypermutation in newly synthesized HIV-1 DNA. Nature. 424:
94-98.
Zheng R, Jenkins TM, and Craigie R. (1996). Zinc folds the Nterminal domain of HIV-1 integrase, promotes multimerization,
and enhances catalytic activity. Preceedings of the National
Academy of Sciences of the United States of America. 93:
13659-13664.
Zheng YH, Irwin D, Kurosu T, Tokunaga K, Sata T, and Peterlin
BM. (2004). Human APOBEC3F is another host factor that blocks
human immunodeficiency virus type 1 replication. Journal of
Virology. 78: 6073-6076.
Revised: August 29, 2005
Accepted: August 30, 2005