Recruitment of the nuclear form of uracil DNA glycosylase into virus particles participates in the full infectivity of HIV-1.
ABSTRACT The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: TRIM11 has been reported to be able to restrict HIV-1 replication, but the detailed aspects of the interfering mechanisms remain unclear. In this study, we demonstrated that TRIM11 mainly suppressed the early steps of HIV-1 transduction, resulting in decreased reverse transcripts. Additionally, we found that TRIM11 could inhibit HIV-1 long terminal repeat (LTR) activity, which may be related to its inhibitory effects on NF-κB. Deletion mutant experiments showed that the RING domain of TRIM11 was indispensable in inhibiting the early steps of HIV-1 transduction but was dispensable in decreasing NF-κB and LTR activities. Moreover, we found that low levels of Vpr decreased TRIM11 protein levels, while high levels increased them, and these regulations were independent of the VprBP-associated proteasome machinery. These results suggest that the antiviral factor TRIM11 is indirectly regulated by HIV-1 Vpr through unknown mechanisms and that the concentration of Vpr is essential to these processes. Thus, our work confirms TRIM11 as a host cellular factor that interferes with the early steps of HIV-1 replication and provides a connection between viral protein and host antiviral factors.PLoS ONE 08/2014; 9(8):e104269. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR.Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Like other HIV-1 auxiliary proteins, Vpr is conserved within all the human (HIV-1, HIV-2) and simian (SIV) immunodeficiency viruses. However, Vpr and homologous HIV-2, and SIV Vpx are the only viral auxiliary proteins specifically incorporated into virus particles through direct interaction with the Gag precursor, indicating that this presence in the core of the mature virions is mainly required for optimal establishment of the early steps of the virus life cycle in the newly infected cell. In spite of its small size, a plethora of effects and functions have been attributed to Vpr, including induction of cell cycle arrest and apoptosis, modulation of the fidelity of reverse transcription, nuclear import of viral DNA in macrophages and other non-dividing cells, and transcriptional modulation of viral and host cell genes. Even if some more recent studies identified a few cellular targets that HIV-1 Vpr may utilize in order to perform its different tasks, the real role and functions of Vpr during the course of natural infection are still enigmatic. In this review, we will summarize the main reported functions of HIV-1 Vpr and their significance in the context of the viral life cycle.Frontiers in Microbiology 01/2014; 5:127. · 3.94 Impact Factor
Recruitment of the Nuclear Form of Uracil DNA Glycosylase into
Virus Particles Participates in the Full Infectivity of HIV-1
Carolin A. Guenzel,a,b,cCécile Hérate,a,b,cErwann Le Rouzic,a,b,cPriscilla Maidou-Peindara,a,b,cHolly A. Sadler,d
Marie-Christine Rouyez,a,b,cLouis M. Mansky,dand Serge Benichoua,b,c
INSERM, U1016, Institut Cochin, Paris, Francea; CNRS, UMR8104, Paris, Franceb; Université Paris-Descartes, Paris, Francec; and Institute for Molecular Virology, University of
Minnesota, Minneapolis, Minnesota, USAd
interaction with the p6 C-terminal domain of the Pr55Gag pre-
subsequently required during the early steps of the virus life cycle
in the newly infected cell. After virus entry, the viral core is re-
leased into the cytoplasm, where the viral reverse transcriptase
catalyzes the synthesis of viral DNA from RNA. One reported
function of Vpr is to influence the accuracy of the reverse tran-
rate (24, 26). In addition, Vpr displays several other activities,
including a perturbation of the cell cycle progression resulting in
transcriptional modulation of host cell genes (23).
Initial studies showed that incorporation of Vpr into virions
ensued a significant reduction of mutations introduced by the
this activity was associated with its binding to the nuclear form of
the uracil DNA glycosylase (UNG2) (9, 26). UNG2 is a base exci-
the specific removal of uracil residues from DNA resulting from
nation (42). Therefore, the role of UNG2 in DNA repair at the
replication fork during chromosomal replication is well estab-
lished, since UNG2 contains determinants required for interac-
tions with proliferating cell nuclear antigen (PCNA) and the 32-
30). In addition, UNG2 plays a specific role in somatic hypermu-
tations and class-switch recombination (CSR) at the immuno-
on the CSR process when it is ectopically expressed in B cells (3).
Subsequently, we reported evidence indicating that the inter-
action between Vpr and UNG2 results in the incorporation of the
catalytically active form of this cellular enzyme into HIV-1 parti-
IV-1 Vpr is a small basic protein of 96 amino acids that is
specifically incorporated into virus particles through a direct
crucial role in this interaction (9, 26, 40, 41), and a Vpr mutant
recruit UNG2 into virus particles, even if it was itself correctly
incorporated into virions (25, 26). Although the VprW54R mu-
tant was not able to assure the accuracy of reverse transcription,
UNG2 expressed as a chimeric protein fused to the C-terminal
rate equivalent to that measured with wild-type Vpr (9). This
ipated in the maintenance of the integrity of the viral genome by
influencing the accuracy of reverse transcription. Other studies
ence the accuracy of the reverse transcription process and had a
positive influence on virus replication (9, 17, 37). Interestingly, it
has been recently reported that HIV-1 DNA generated in infected
macrophages and CD4-positive T cells is heavily uracilated (47).
However, the specific role of UNG2 incorporation into virions
was also challenged by other studies (18, 40, 48). While the spec-
ificity of the interaction between Vpr and UNG2 was not ques-
tioned, these studies reported data suggesting that UNG2 had ei-
ther a detrimental effect on virus replication (40, 48) or was
Received 19 May 2011 Accepted 2 December 2011
Published ahead of print 14 December 2011
Address correspondence to Serge Benichou, firstname.lastname@example.org, or Louis
M. Mansky, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0022-538X/12/$12.00Journal of Virologyp. 2533–2544jvi.asm.org
a detrimental effect on virus replication, the role of Vpr was
proposed to induce the proteasomal degradation of UNG2 in
particles (39, 40). However, other data have indicated that the
Vpr-induced reduction of endogenous UNG2 observed in HIV-
1-infected cells was not related to proteasomal degradation (21).
The goal of the present study was to further investigate the
contribution of UNG2 virion incorporation on viral mutation
and to reevaluate the role of UNG2 on viral infectivity. We report
tation through a nonenzymatic mechanism and that UNG2 has a
net positive impact on viral replication. Indeed, HIV-1 particles
produced in cells overexpressing UNG2 resulted in a net increase
MATERIALS AND METHODS
Vectors and expression plasmids. The HIV-1 vector used for analysis of
the HIV-1 mutation rate, as well as plasmids used for expression of the
hemagglutinin (HA)-tagged forms of the wild-type (wt) UNG2 protein
and UNG2 fused to the C terminus of the VprW54R mutant (VprW54R-
UNG2 fusion), have been described previously (9). Plasmids for expres-
tions within the UNG2 part of the fusion were constructed by
PCR-mediated site-directed mutagenesis using specific primers contain-
pAS1B plasmid as described previously (9). The plasmids for the expres-
of the murine UNG2 were kindly provided by Tasuku Honjo and Nasim
Begum (Kyoto, Japan) (3). The HIV-1-based packaging vectors
pCMVDR8.3 (lacking the env and vpr genes) and pCMVDR8.2 (lacking
only the env gene) were kindly provided by Didier Trono (Geneva, Swit-
zerland), while the HIV-1 vector encoding GFP (pHIvec2.GFP) and the
plasmids encoding the HIV-1 HXBc2 and YU-2 envelope glycoproteins
were described previously (20). The wt infectious clone of the NL4.3
HIV-1 isolate (pNL4.3) has been described (9). The pLKO.1 lentiviral
vectors harboring short hairpin RNA (shRNA) targeting either UNG2 or
the p32 subunit (RPA2) of RPA were purchased from Sigma.
Cell culture and transfection. 293T cells and HeLa-CD4 cells were
grown in Dulbecco minimal essential medium supplemented with 10%
fetal calf serum, 100 IU of penicillin/ml, and 100 ?g of streptomycin/ml
(Invitrogen); shRNA-transduced cells were cultivated with 1 ?g of puro-
mycin (Invitrogen/ml). Human monocytes were isolated from blood of
healthy donors (Hôpital Saint-Vincent-de-Paul, Paris) by density gradi-
ent sedimentation in Ficoll (GE Healthcare), followed by adhesion selec-
tion for 2 h at 37°C. After extensive washing, the monocytes were differ-
entiated into macrophages for 8 days in complete culture medium RPMI
1640 supplemented with 10% human serum (from total blood of same
blood donors), 100 IU of penicillin/ml, and 100 ?g of streptomycin/ml
(Invitrogen). All cells were grown at 37°C with 5% CO2. 293T cells were
transfected for virus productions and immunoprecipitation assay by the
(9, 21, 26). For UNG2 transient expression, HeLa-CD4 cells were trans-
according to the manufacturer’s instructions.
Analysis of HIV-1 mutant frequencies. The ability of wt or mutated
Vpr or VprW54R-UNG2 fusions to complement a vpr-defective HIV-1
was analyzed in a single-cycle replication assay for mutant frequencies, as
described previously (9, 26). Briefly, the plasmids for expression of HA-
transiently cotransfected with helper packaging plasmids into cells con-
as a mutation target. The viruses produced were then used to infect per-
frequency per round of replication. Proviral DNA was then purified with
ber of colonies observed provided the forward mutation rate for a single
retroviral replication cycle. A total of 421 colonies were screened, and the
Vpr transcomplementation was 0.15 (63/421) mutant/cycle.
UNG2- and RPA2-depleted cells. To stably knockdown UNG2 or
RPA2 endogenous expression, pLKO.1 lentiviral vectors harboring short
hairpin RNA (shRNA) targeting UNG2 or RPA2 were obtained from
oligonucleotide sequence targeting UNG2 was 5=-GCAGTTGTGTCCTG
GCTAAAT-3=, and that for RPA2 was 5=-CAATCAAGCAAGCTGTGGA
TT-3=. First, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudo-
or RPA2 were produced in 293T cells by cotransfecting pLKO1-shRNA,
a VSV-G expression plasmid. The pLKO.1 vector plasmid expressing
tor particles. At 48 h posttransfection, LVPs were pelleted from superna-
tants by ultracentrifugation (22,000 rpm for 1.5 h at 4°C) and used to
transduce 293T or HeLa-CD4 cells. After 24 h, transduced cells were cul-
immunofluorescence using specific antibodies.
Virus production and infection. Single-round-infection HIV-1 car-
rying the GFP gene was produced in 293T cells as follows. Cells were
seeded in T75 flasks at a density of ?2.5 ? 106cells/T75 flask and
transfected 16 h later by the calcium phosphate precipitation technique
with a DNA mix containing 8 ?g of the HIV-1-packaging plasmid
(pCMVDR8.3, pCMVDR8.2, or pNL4.3), 4 ?g of the HIV-1 vector en-
coding GFP (pHIvec2.GFP), 2 ?g of the plasmid encoding the HIV-1
envelope glycoproteins HXBc2 or YU-2, and 8 ?g of the additional plas-
mid for expression of UNG2 or VprW54R-UNG2 fusions. The cells were
washed 6 h later and then cultured in 8 ml of complete medium for 48 h.
filter, and ultracentrifuged to pellet viruses as described previously (20).
seeded into wells of a six-well plate at a density of 105or 106cells/well,
were then fixed in 1% paraformaldehyde (Sigma-Aldrich) and analyzed
on a Cytomix FC500 cytometer (Beckman-Coulter). The percentage of
GFP-positive cells was determined by analyzing the data with the RXP
analysis software. Viral infectivity was calculated by normalizing the per-
centage of GFP-positive cells to that obtained in cells infected with wt or
infected with 0.2 ?g of CAp24 of replication-competent virus, and cell
culture supernatant was collected 2, 4, 6, and 8 days after infection for
CAp24 determination by ELISA.
crude viral protein with the single-stranded DNA oligonucleotide sub-
Quantification of viral DNA. At 24 h prior to infection, HeLa-CD4
cells were seeded into a six-well plate at a density of 2 ? 105cells/well.
Before infection, replication-competent viruses were incubated with
DNase I (Roche) for 1 h at 37°C, and 0.5 ?g of CAp24 was then used for
infection. At 3 h after infection, the viruses were washed off, and the cells
were subsequently incubated at 37°C in complete medium supplemented
with 0.5 ?M saquinavir in order to restrict viral replication to a single
Guenzel et al.
jvi.asm.orgJournal of Virology
cycle. Then, 7 h later, cell samples were collected, and DNA was extracted
turer’s protocol. The total level of HIV-1 DNA was quantified via the
LightCycler 480 qPCR system (Roche Applied Science) using the follow-
ing protocols. Briefly, the quantitative PCR for total HIV-1 DNA was
carried out using primers targeting the gag region within the HIV-1
Tag polymerase (Roche) and 0.3 ?M concentrations of sense MH532
(5=-TGTGTGCCCGTCTGTTGTGT-3=) and antisense MH531 (5=-GAG
TCCTGCGTCGAGAGATC-3=) primers (TIB MolBiol). The fluorescent
probe primers 5=-LC640-TCTCTAGCAGTGGCGCCCGAACAG-PH
and 5=-CCCTCAGACCCTTTTAGTCAGTGTGGAA-FL were used at a
concentration of 0.2 ?M. Total DNA was expressed as copy numbers per
cell, with the DNA template normalized by ?-globin gene amplification
using a LightCycler control kit DNA (Roche).
Immunoprecipitation and immunoblot analyses. 293T cells were
with 10 ?g of the GFP-tagged murine or human UNG2 expression plas-
mid in combination with the HA-Vpr expression plasmid. After 24 h, the
cells were lysed in NP-40 lysis buffer supplemented with protease inhibi-
tor (Roche) for 30 min under gentle agitation at 4°C, as described previ-
carried out on 450 ?g of cell lysate proteins by incubation with 3 ?g of
beads (Sigma) for 2 h under gentle agitation at 4°C. Elution from beads
was carried out by incubation in 30 ?l of 1? Laemmli buffer containing
5% ?-mercaptoethanol for 10 min at 95°C. The concentrations of pro-
teins were quantified by Bradford analysis according to the manufactur-
er’s protocol (Bio-Rad), and 25-?g portions of the proteins were then
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 10% acrylamide NuPAGE Novex bis-Tris precast gels
(Invitrogen). Immunoprecipitated and cell lysate proteins were then an-
alyzed by Western blotting with anti-HA (3F10; Roche) and anti-GFP
ish peroxidase (HRP)-coupled antibodies (Sigma). For analysis of the
CAp24 contents of viral particles produced in cells overexpressing UNG2
or the VprW54R-UNG2 fusion, purified virions were resuspended in 80
cam). The incorporation into viral particles of the VprW54R-UNG2 fu-
sions and murine or human UNG2 proteins was analyzed as described
previously (9) using anti-Flag (Sigma), anti-HA (3F10; Roche), and anti-
p24 (NIH; Abcam) antibodies. For analysis of UNG2 or RPA2 expression
in shRNA-transduced or transfected 293T or HeLa-CD4 cells, cell lysate
proteins were similarly analyzed by SDS-PAGE and Western blotting us-
RPA2 (Abcam), and anti-?-actin antibodies (Sigma).
or 293T cells were seeded at low density into a six-well plate containing
coverslips. After 24 h, the cells were washed in phosphate-buffered saline
coverslips with PBS. Coverslips were then incubated for 1 h at room tem-
Triton X-100 (Sigma-Aldrich) and anti-HA antibody (Roche) for UNG2
transient-expression experiments and with anti-UNG2 (PU59 [kindly
provided by Geir Slupphaug, Trondheim, Norway]) or anti-RPA2
(Abcam) antibody for shRNA experiments. The cells were then washed
twice in PBS-BSA and incubated for 45 min in PBS-BSA supplemented
with Alexa 488-coupled anti-rat, Alexa 555-coupled anti-rabbit, or Alexa
were then washed twice in PBS-BSA and once in PBS and inverted in
mounting medium containing DAPI (4=,6=-diamidino-2-phenylindole;
Slow-Fade, Invitrogen). Samples were examined under an epifluores-
cence microscope (Leica), and acquisition of images was carried out as
described previously (21).
Enzymatic activity of UNG2 is not required for modulating
HIV-1 mutation. In order to further explore the specific contri-
bution of UNG2 incorporated into HIV-1 particles for modula-
as a chimeric protein fused to the C terminus of the VprW54R
mutant. This Vpr variant failed to recruit cellular UNG2 into vi-
rions and to influence the virus mutation rate, although it was
mentation of a vpr-defective HIV-1 (HIV-1?vpr) with the
VprW54R mutant, the VprW54R-UNG2 fusion gave rise to a vi-
tation with the wt Vpr protein (Fig. 1A). This demonstrates that
ing the direct contribution of UNG2 incorporation to the modu-
lation of the virus mutation rate.
Substitutions of residues specifically required for the catalytic
activity, recognition of uracil residues, and the binding to
of VprW54R-UNG2 to generate the mutated VprW54R-
UNG2D154N, -UNG2N181V, and -UNG2N213V fusions, re-
vector lacking the vpr gene in combination with the plasmids for
expression of either wt or one of the mutated VprW54R-UNG2
fusions. As shown in Fig. 1B (left part), all VprW54R-UNG2 fu-
of UNG activity could be recovered from virions produced from
by detection of the 12-bp product (Fig. 1B, right panel). In con-
trast, no UNG activity was detected from virions produced from
cells expressing the VprW54R-UNG2D154N and -UNG2N213V
mutated fusions. However, these three mutated fusions were still
able to complement the vpr-defective HIV-1 as efficiently as Vpr
or VprW54R-UNG2 in the mutation rate assay (Fig. 1A). Simi-
larly, fusions containing double- or even triple-point mutations
VprW54R to modulate HIV-1 mutant frequency (Fig. 1C) and
were efficiently incorporated into virions (Fig. 1D). These data
modulation of the HIV-1 mutation rate.
The N-terminal region of UNG2 is required for modulating
viral mutation. In order to determine the region within UNG2
being responsible for the modulation of viral mutant frequency,
we then generated several N-terminal and C-terminal deletion
mutants of UNG2 (Fig. 2A). Mutants deleted of 30, 90, and 140
amino acids (aa) from the N terminus of UNG2 (?30, ?90, and
?140), as well as truncation mutants spanning aa 1 to 90 and aa 1
virus-producing cells and tested for mutant frequencies as de-
scribed above. As shown in Fig. 2B, the VprW54R-UNG2?30 de-
letion mutant, lacking the first 30 aa of UNG2, was still able to
restore a mutation rate equivalent to that measured by comple-
the VprW54R-UNG2?90 fusion, lacking the first 90 aa, failed to
complement the vpr-defective virus despite the fact that it was
UNG2 and HIV-1 Infectivity
March 2012 Volume 86 Number 5jvi.asm.org 2535
correctly incorporated into virions (Fig. 2C), suggesting that the
restoration of a relative mutant frequency comparable to the wt
VprW54R-UNG2 fusion. Indeed, VprW54R-UNG2-1-90 and -1-
between residues 30 and 90 of the UNG2 protein is sufficient for
UNG2 contains the determinants of the protein required for in-
teraction with the p32 subunit of the RPA trimeric complex (12,
28, 30, 31).
Incorporation of UNG2 into virus particles positively influ-
ences HIV-1 infectivity. Since the results reported in Fig. 1 and 2
demonstrated that the recruitment of UNG2 into virions was di-
rectly responsible for influencing HIV-1 mutation, we then de-
reported in the literature about the impact of UNG2 on HIV-1
infectivity and replication (9, 17, 18, 22, 37, 40, 48) were further
challenged using a single-round infection assay for the investiga-
tion of virus infectivity when UNG2 was overexpressed in either
virus-producing cells or target cells. Wild-type reporter viruses
expressing the HA-tagged form of UNG2, and equivalent
amounts of virus, as measured by HIV-1 CAp24 in ELISA, were
used to assay virus infectivity on HeLa cells stably expressing the
CD4 receptor (HeLa-CD4). As shown in Fig. 3A, overexpression
of UNG2 in virus-producing cells had no detrimental effect on
virus infectivity, but rather resulted in a 3-fold increase of infec-
tivity of viruses containing UNG2. Moreover, an equivalent 2.5-
FIG 1 Analysis of catalytically inactive mutants of UNG2 for modulation of HIV-1 mutation rate. (A and C) The ability of wt or mutated Vpr or VprW54R-
of HA-tagged forms of Vpr, VprW54R, or VprW54R-UNG2 fusions were cotransfected with helper packaging plasmids into cells containing a single integrated
HIV-1 vector provirus containing the lacZ gene as a mutation target. The viruses produced were then used to infect permissive HeLa cells, which allowed for a
determination of the virus mutant frequency per round of replication. A total of 421 colonies were screened, and the average mutant frequency of the vpr-null
were cotransfected with a vpr-defective HIV-1-based vector in combination with plasmids for expression of HA-tagged VprW54R-UNG2 mutated fusions.
B (right side), UNG activity from the VprW54R-UNG2 fusions incorporated into virions was assayed with a 25-bp single-stranded DNA oligonucleotide
on a polyacrylamide denaturing gel. The gel was stained with SYBR Gold, and nucleic acids were visualized with an UV transilluminator. The control lane
contains untreated DNA substrate.
Guenzel et al.
jvi.asm.orgJournal of Virology
fold increase of infectivity was measured when viruses produced
from UNG2-overexpressing cells were used to infect primary
monocyte-derived macrophages (Fig. 3B). In contrast, no signif-
icant increase in infectivity was observed when HeLa-CD4 target
cells were transfected with HA-UNG2 expression plasmid 1 day
related to a low level of transfection since we confirmed that al-
HA-UNG2, as evidenced by immunofluorescence analysis (Fig.
3D). These results further clarify that UNG2 expression in both
virus producing and target cells does not have a negative but has
either a supportive effect (virus-producing cells) or no effect (tar-
get cells) on virus infectivity.
enced through expression of the VprW54R-UNG2 fusion in
virus-producing cells. ?vpr viruses containing VprW54R-UNG2
were produced in 293T cells by transfection with the vector for
expression of the fusion and then used as previously to infect
HeLa-CD4 target cells. As shown in Fig. 3C, ?vpr virus particles
produced in cells expressing the VprW54R-UNG2 fusion showed
a 2.5-fold increase of infectivity compared to ?vpr viruses. Simi-
primary monocyte-derived macrophages were used as target cells
(Fig. 3B). Again, overexpression of HA-UNG2 in target cells did
not significantly affect infectivity of the ?vpr viruses containing
the VprW54R-UNG2 fusion (Fig. 3C). Similarly, VprW54R-
UNG2 fusions with point mutations in the catalytic site
motif (VprW54R-UNG2WxxF/AxxG) of UNG2 were still able to
increase infectivity of ?vpr viruses, as efficiently as the wt
VprW54R-UNG2 fusion, when they were expressed in virus-
producing cells (Fig. 3C). Therefore, the enzymatic activity of
UNG2 and the WxxF motif are not necessary for modulation of
virus infectivity. In order to rule out any detrimental effect on
virus maturation and release related to UNG2 or VprW54R-
UNG2 overexpression in virus-producing cells, cell fractions and
purified virus particles were analyzed by Western blotting with
anti-CAp24 antibody. As shown in Fig. 3E, overexpression of
UNG2 or VprW54R-UNG2 affected neither the expression levels
of the Pr55Gag precursor in producer cells nor the release of viral
particles in the cell culture supernatant. Taken together, the data
fectivity and replication. We next sought to further confirm the
net positive influence of UNG2 on virus infectivity, and UNG2
fusions to the VprW54R mutant; amino acids are numbered according to the system of Haug et al. (15). (B) Viruses produced from cells expressing the deleted
VprW54R-UNG2 fusions were assayed for mutant frequency phenotype as indicated in Fig. 1A. A total of 356 colonies were screened. and the average mutant
experiments. Error bars represent one SD from the mean. (C) Virion incorporation of the VprW54R-UNG2 fusion proteins. 293T cells were cotransfected with
supernatants; proteins from cell and virion lysates were analyzed by Western blotting with anti-HA and anti-CAp24.
UNG2 and HIV-1 Infectivity
March 2012 Volume 86 Number 5 jvi.asm.org 2537
expression was then depleted not only in virus-producing 293T
cells but also in target HeLa-CD4 cells. In these experiments, we
used lentiviruses harboring specific shRNA against UNG2
(shUNG2) and luciferase as a control (shLuc). As evidenced by
Western blot analysis of cell lysates from transduced 293T and
HeLa-CD4 cells (Fig. 4A), the UNG2 protein band of 39 kDa was
efficiently reduced in lysates from shUNG2-transduced cells but
by immunofluorescence analysis using anti-UNG2 antibody (Fig.
4B). Wild-type HIV-1 was then produced in shUNG2- or shLuc-
transduced 293T cells and subsequently used for infection of
shUNG2- or shLuc-transduced HeLa-CD4 target cells (Fig. 4C).
Interestingly, when viruses were produced in UNG2-depleted
to viruses produced in shLuc-treated cells. In contrast, depletion
of UNG2 in target cells did not significantly decrease virus infec-
tivity (Fig. 4C), even though downregulation of nuclear UNG2
CD4 cells, as determined by immunofluorescence analysis (Fig.
were produced in 293T cells overexpressing HA-tagged forms of UNG2, or wt or mutated VprW54R-UNG2 fusions, and were used to infect HeLa-CD4 cells
overexpressing or not HA-UNG2. Viruses were normalized for CAp24 before infection. The percentages of GFP-positive infected cells were then measured by
or VprW54R-UNG2. Values are the means of at least four independent experiments. Error bars represent one SD from the mean. Statistical significance was
determined by using the Student t test (n.s., P ? 0.05; *, P ? 0.05; **, P ? 0.01). (B) Wild-type or ?vpr GFP reporter viruses carrying the HIV-1 YU-2 envelope
were produced in 293T cells overexpressing HA-tagged forms of either UNG2 or VprW54R-UNG2 and were used to infect primary macrophages derived from
blood monocytes from three healthy donors. Viral infectivity was normalized to that of wt viruses produced in cells that did not overexpress UNG2 or
VprW54R-UNG2. Values are the means of three independent experiments. Error bars represent one SD from the mean. Statistical significance was determined
Nuclei were stained with DAPI (left panels). Cells were analyzed by epifluorescence microscopy, and images were acquired by using a charge-coupled device
camera. (E) Virus maturation of wt and ?vpr HIV-1. 293T cells were cotransfected with wt or vpr-defective HIV-1-based vector in combination with plasmids
for expression of HA-tagged forms of UNG2 or VprW54R-UNG2. Virions were collected from cell supernatants; proteins from cell and virion lysates were
analyzed by Western blotting with anti-CAp24 and anti-?-actin as a control.
Guenzel et al.
jvi.asm.orgJournal of Virology
when UNG2 expression was downregulated in both virus-
producing and target cells, indicating the dominant effect of the
downregulation of UNG2 in virus-producing cells.
To confirm the specificity of the decrease in virus infectivity
observed with viruses produced in UNG2-depleted cells, we
checked that this defect was not related to off-target effects of
shUNG2-transduction by complementation with the shRNA-
insensitive murine form of UNG2 (mUNG2) in shUNG2-
transduced human 293T cells. As indicated in Fig. 4D, we deter-
mined by coimmunoprecipitation analysis that mUNG2 was able
(hUNG2). In addition, the shRNA-insensitive mUNG2 version
was equally incorporated into HIV-1 particles when produced in
either human UNG2-depleted 293T cells or shLuc-transduced
control cells (Fig. 4E). When viruses were produced in control
cells overexpressing mUNG2 (Fig. 4F), there was a 2.5-fold in-
crease in virus infectivity, which was equivalent to that observed
by overexpressing human UNG2 in virus-producing 293T cells
(see Fig. 3A). As already reported in Fig. 3C, viruses produced in
produced in control cells (Fig. 4F). In contrast, expression of
a restoration of virus infectivity (Fig. 4F). These results confirm
that virus production in UNG2-depleted cells results in a specific
negative impact on viral infectivity, confirming that incorpora-
FIG 4 Impact of UNG2 depletion on virus infectivity. (A) and (B) Depletion of UNG2 in virus-producing (293T) and target (HeLa-CD4) cells. 293T or
HeLa-CD4 cells were transduced with lentiviruses expressing shRNA against either UNG2 or luciferase used as a control. In panel A, lysates from shRNA-
transduced 293T and HeLa-CD4 cells were analyzed by Western blotting using anti-UNG1/2 and anti-?-actin antibodies. In panel B, shRNA-transduced
in shUNG2-transduced cells. Wild-type GFP reporter viruses were produced in shRNA-transduced 293T cells and then used to infect shRNA-transduced
HeLa-CD4 cells as indicated. Viruses were normalized for CAp24 before infection. The percentages of GFP-positive infected cells were then measured by flow
as target cells. (D) Interaction of Vpr with murine UNG2. 293T cells were transfected with plasmids for expression of GFP-tagged murine (mUNG2) or human
(hUNG2) UNG2 in combination with the HA-tagged Vpr expression plasmid. Control (mock) corresponds to cells that did not express HA-Vpr. At 24 h after
transfection, cell lysates (top and middle panels) were submitted to immunoprecipitation with anti-GFP antibody (bottom panel). Immunoprecipitates were
with anti-Flag (top and middle) and anti-CAp24 (bottom) antibodies. (F) Infectivity of viruses produced in shUNG2-transduced human cells expressing
mUNG2. Wild-type GFP reporter viruses were produced in 293T shRNA-transduced cells expressing or not Flag-tagged mUNG2 and then used to infect
HeLa-CD4 cells. Viruses were normalized for CAp24 before infection. The percentages of GFP-positive infected cells were then measured by flow cytometry 60
h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced cells. Values are the means of at least four independent experiments.
Error bars represent one SD from the mean. Statistical significance was determined by using the Student t test (n.s., P ? 0.05; *, P ? 0.05; **, P ? 0.01).
UNG2 and HIV-1 Infectivity
March 2012 Volume 86 Number 5jvi.asm.org 2539
tion of UNG2 into viral particles is required for maintaining full
itive influence on virus replication. Wild-type replication-
competent HIV-1 was produced in 293T cells and used to infect
HeLa-CD4 cells that were depleted of UNG2 (Fig. 5A). Whereas
viruses replicated efficiently in shLuc-transduced control cells
with a significant CAp24 production detected as soon as 4 days
after infection, a strong replication defect could be observed in
UNG2-depleted cells with low levels of CAp24 detected 6 days
infectivity and replication observed in UNG2-depleted cells was
related to a defect in the reverse transcription process, we quanti-
fied total viral DNA after infection with wt viruses produced in
either UNG2-depleted 293T cells or control shLuc-transduced
cells. As shown in Fig. 5B, a significant reduction of the total viral
compared to the control cells, indicating that UNG2 may affect
of reverse transcripts produced in the early phase of the viral life
Taken together, the data reported in Fig. 4 and 5 show that
previously indicated, we mapped and determined that the
N-terminal region of UNG2 between residues 30 and 90 was re-
quired for modulation of the virus mutation frequency; this re-
gion also contains the determinants for interaction with the p32
31). Here, we investigated the potential role of RPA in the modu-
against RPA2 (shRPA2) were used for specific depletion in virus-
protein was successfully depleted for all three concentrations in
293T cells compared to shLuc-transduced control cells, as evi-
denced both by Western blot and immunofluorescence analyses
(Fig. 6B and C, respectively). Intriguingly, when wt HIV-1 was
of HeLa-CD4 cells, we observed that virus infectivity was signifi-
cantly decreased compared to viruses produced in shLuc-
transduced cells (Fig. 6D), suggesting a potential role for RPA2
during HIV-1 infection.
ing the biological relevance of the recruitment of UNG2 into
HIV-1 particles (9, 17, 18, 22, 37, 40, 48), our results argue that
virus infection of target cells but rather has a positive impact on
virus replication. These positive effects correlate with a positive
influence on the reverse transcription process. While it was re-
cently suggested that UNG2 was specifically required for efficient
infection of primary cells with R5 viruses (17), we observed that
enforced virion recruitment of UNG2, through overexpression of
UNG2 in virus-producing cells, similarly influenced infectivity of
X4 and R5 HIV-1 strains in transformed cell lines and primary
monocyte-derived macrophages, respectively. Conversely, viral
infectivity and spreading were significantly reduced when viruses
UNG2 using specific shRNA, confirming the positive impact of
UNG2 incorporation for the full infectivity of HIV-1 particles. In
addition, we observed that the p32 subunit of RPA, which has
FIG 5 Impact of UNG2 depletion on virus replication and reverse transcription. (A) Virus replication in UNG2-depleted cells. Equivalent amounts of wt
replication-competent viruses were produced in 293T cells and then used for infection of shLuc-transduced (plain line) or shUNG2-transduced (dashed line)
Wild-type replication-competent viruses were produced in shRNA-transduced 293T cells as indicated and then used for infection of HeLa-CD4 cells. Viruses
were normalized for CAp24 before infection. At 7 h after infection, cell samples were collected and subjected to DNA purification, and the total viral DNA was
quantified via quantitative PCR using specific primers for gag. Values are the means of three independent experiments. Error bars represent one SD from the
mean. Statistical significance was determined by using the Student t test (n.s., P ? 0.05; *, P ? 0.05; **, P ? 0.01).
Guenzel et al.
jvi.asm.orgJournal of Virology
also participate in maintaining HIV-1 infectivity.
While we previously reported that the catalytically active form
of UNG2 is incorporated into HIV-1 particles (9), we show here,
through substitution of UNG2 residues required for the catalytic
of the protein to the DNA substrate (29), that the uracil excision
activity of UNG2 incorporated into virions was required neither
for the modulation of the virus mutation rate nor for virus infec-
tivity. These intriguing observations are in agreement with previ-
ous reports showing that HIV-1 particles produced in 293T cells
overexpressing the specific potent catalytic active-site bacterio-
phage PBS1 inhibitor (UGI) of human UNG2 were still fully ef-
(17, 18, 22).
Interestingly, the genome of numerous viruses from the Pox-
viridae and Herpesviridae, such as the cytomegalovirus (CMV)
and the vaccinia virus, contains an open reading frame coding for
a UNG protein with sequence similarities to the mammalian
UNG2 (10, 42). In vaccinia virus, the viral UNG is required for
lacking uracil-removal activity could efficiently replace the wt vi-
Moreover, deletion of UNG in CMV also resulted in a significant
activity of CMV UNG did not appear to be important for replica-
tion, since poor viral replication was unrelated to the uracil con-
role of the Vpr-mediated incorporation of UNG2 into virus par-
ticles for efficient reverse transcription and viral replication may
also be different from uracil removal.
It was also reported that despite the absolute requirement of
the UNG2 protein for an efficient CSR process in B lymphocytes
(42), catalytically inactive mutants of UNG2 were fully proficient
in CSR (3–5). These results indicated that the specific function of
UNG2 in CSR is not related to the uracil removal activity of the
protein and correlate with our observations regarding the specific
role of UNG2 for the modulation of the HIV-1 mutation rate.
Together, these observations support a model in which both the
viral mutation rate and CSR depend on a novel nonenzymatic
function of UNG2. As mentioned above, it was previously re-
ported that Ig class switching was drastically inhibited when Vpr
FIG 6 Impact of RPA2 depletion on virus infectivity. (A) Schematic representation of UNG2 showing the motifs required for interaction with Vpr or with the
p32 subunit (RPA2) of the RPA heterotrimeric complex. (B and C) Depletion of RPA2 in virus-producing cells. 293T cells were transduced with lentiviruses
expressing shRNA against either RPA2 or Luciferase used as a control. In panel B, lysates from shRNA-transduced cells were analyzed by Western blotting with
anti-RPA2 (top) and anti-?-actin (bottom) antibodies. In panel C, shRNA-transduced cells were analyzed by indirect immunofluorescence with anti-RPA2
Virus infectivity. Wild-type GFP reporter viruses were produced in shRNA-transduced 293T cells as indicated and then used to infect HeLa-CD4 cells. Viruses
were normalized for CAp24 before infection. The percentages of GFP-positive infected cells were measured by flow cytometry 60 h later. Infectivity was
normalized to that of viruses produced in shLuc-transduced cells and measured on HeLa-CD4 as target cells. Values are the means of three independent
experiments. Error bars represent one SD from the mean. Statistical significance was determined by using the Student t test (n.s., P ? 0.05; *, P ? 0.05; **, P ?
UNG2 and HIV-1 Infectivity
March 2012 Volume 86 Number 5 jvi.asm.org 2541
was overexpressed in stimulated B cells, indicating that Vpr had a
dominant-negative effect on CSR (3). However, Vpr mutants de-
fective for interaction with UNG2, such as the VprW54R mutant,
failed to influence CSR. Interestingly, it was also reported that
mutations in the conserved UNG2 motif (i.e., WxxF), which is
required for Vpr binding to UNG2 (8, 9), blocked CSR without
affecting its uracil removal activity, indicating that this motif is
suggested that the exogenous Vpr competes with an unknown
function of UNG2 in CSR (3, 5). Therefore, the WxxF motif
within UNG2 is required for binding to the Vpr-like factor in
the UNG2 WxxF motif per se was not necessary for enhancement
of HIV-1 infectivity, but this motif was strictly required for inter-
action with Vpr in virus producing cells in order to recruit UNG2
into virions (9), where it would be subsequently required for effi-
cient reverse transcription and viral replication in target cells. In-
motif of UNG2 (VprW54R-UNG2WxxF/AxxG) was still able to
wt VprW54R-UNG2 fusion.
ipating in the accuracy of the reverse transcription process are
located within a 60-aa region between the N-terminal residues 30
and 90 of the UNG2 protein. It is noteworthy that the same
role of UNG2 in CSR in B lymphocytes (3, 5), suggesting that this
region, which is neither required for uracil-removal activity nor
Vpr binding (29, 41), is involved in a new function of UNG2
shared by CSR and HIV-1 mutation modulation processes. Inter-
estingly, this N-terminal region of UNG2 also contains determi-
nants of the protein required for interaction with the p32 subunit
of the RPA trimeric complex (14, 28, 30, 31). RPA has been re-
ported to be essential for the repair of double-strand breaks by
excision repair (BER) (11, 31). Through tight binding of RPA to
single-stranded DNA (6), it is thought that RPA mediates the co-
ordinated assembly of the DNA repair machinery at sites of DNA
unit RPA-14 is believed to serve a structural purpose in the RPA
heterotrimer. RPA and UNG2 do colocalize in replication foci
UNG2 in rapid postreplicative removal of uracil in single-strand
DNA at the replication fork (14, 28, 30, 31). Given the close func-
tional relationship between UNG2 and RPA for BER, our initial
findings indicated a potential role for RPA during HIV-1 infec-
virus infectivity was significantly reduced, suggesting that RPA2
might be involved in the infection process, probably through di-
rect interaction with UNG2 and independently of its enzymatic
activity. Intriguingly, the same identified N-terminal region
within UNG2 has been reported to bind to proliferating cell nu-
various processes such as DNA replication and repair (19, 33, 34,
45). Since it was demonstrated that UNG2, RPA2, and PCNA
colocalized within replication foci (31), this implies an evident
functional relationship between these proteins. Further analyses
are therefore needed to investigate the potential recruitment of
these additional factors of the DNA repair system for HIV-1 in-
fectivity and replication.
We initially reported data indicating that the interaction be-
ration of the enzyme into viral particles in order to restrict the
errors introduced by the reverse transcriptase during viral DNA
synthesis in the target cells (9, 26), but the specific role of UNG2
incorporation into virions was subsequently questioned by other
studies (18, 40, 48). While our previous studies with the UNG2-
binding deficient VprW54R mutant demonstrated the impor-
tance of UNG2 recruitment for efficient replication in cell types
with low endogenous UNG2 levels (e.g., nondividing cells), our
UNG2 depletion experiments reported in the present study in
actively dividing HeLa-CD4 cells using shRNAs directed against
UNG2 further support the critical requirement of UNG2 recruit-
demonstrating that viral infectivity was significantly reduced
when viruses were produced in UNG2-depleted cells and was re-
stored in the same cells overexpressing the shRNA-insensitive
murine form of UNG2. Moreover, the low infectivity of HIV-1
particles produced in UNG2-depleted cells correlated with a re-
duced amount of viral DNA generated during the reverse tran-
scription process in the target cells. In contrast, enforced virion
producing cells, similarly influenced HIV-1 infectivity in trans-
formed cell lines and primary monocyte-derived macrophages.
Using similar small interfering RNA (siRNA) strategies targeting
as the 293T and HeLa-CD4 MAGI-CCR5 cell lines, or primary
that the recruitment of UNG2 into viral particles has a positive
influence on the reverse transcription process and is required for
cells or in primary macrophages used as target cells. In contrast,
studies by Schröfelbauer et al. (40) and Yang et al. (48) suggested
a model in which incorporation of UNG2 into viral particles
would have a detrimental effect on reverse transcription by intro-
ducing abasic sites into viral DNA in regard to uracil residues
resulting from cytosine deamination by the cytidine deaminase
APOBEC3G. While Schröfelbauer et al. did not directly question
the specific role of UNG2 in the antiviral activity of APOBEC3G
of overexpressed APOBEC3G was partially affected when viruses
were produced in UNG2-depleted 293T cells using an siRNA
contradiction to results reported by us and others by produc-
ing viruses in UNG2-depleted cells which expressed or not
APOBEC3G (17, 37) but also with other reports showing that
APOBEC3G-mediated restriction of HIV-1 in human cells was
independent of UNG (18, 22). Since Yang et al. (48) did not ex-
amine in their siRNA strategy used to analyze the role UNG2
if viruses produced in UNG2-depleted cells overexpressing
APOBEC3G could be rescued through expression of a RNAi-
insensitive form of UNG2, additional investigations are thus re-
quired to further understand this apparent contradiction regard-
of APOBEC restriction factors. Finally, Kaiser and Emerman (18)
reported data indicating that UNG2 had neither a positive nor a
negative impact on HIV-1 infectivity, but viruses were produced
in a UNG2-defective B-lymphoid cell line, and the different cell
Guenzel et al.
jvi.asm.orgJournal of Virology
discrepancy between this report and data reported by us and oth-
ers (17, 37).
In conclusion, the results reported here support the model in
which incorporation of UNG2 into virions has a positive impact
on HIV-1 infectivity and replication and positively modulates the
ingly, direct interaction between UNG2 and RPA2 might partici-
investigations are thus needed to elucidate how incorporation of
process. This could also help to elucidate the novel molecular
function of UNG2 in CSR in B lymphocytes.
We thank Geir Slupphaug (Trondheim, Norway), Tasuku Honjo and
Nasim Begum (Kyoto, Japan), and Didier Trono (Geneva, Switzerland)
for the generous gift of reagents.
This study was supported in part by INSERM, the CNRS, the Univer-
sité Paris-Descartes, the French National Agency for AIDS Research
(ANRS), Sidaction (S.B.), and National Institutes of Health grant
GM56615 (L.M.M.). C.G. is an ANRS fellowship recipient.
1. Ali SI, Shin J-S, Bae S-H, Kim B, Choi B-S. 2010. Replication protein A
32 interacts through a similar binding interface with TIPIN, XPA, and
UNG2. Int. J. Biochem. Cell Biol. 42:1210–1215.
2. Andersen JL, Le Rouzic E, Planelles V. 2008. HIV-1 Vpr: mechanisms of
G2arrest and apoptosis. Exp. Mol. Pathol. 85:2–10.
3. Begum NA, et al. 2007. Requirement of non-canonical activity of uracil
DNA glycosylase for class switch recombination. J. Biol. Chem. 282:731–
4. Begum NA, et al. 2004. Uracil DNA glycosylase activity is dispensable for
immunoglobulin class switch. Science 305:1160–1163.
5. Begum NA, et al. 2009. Further evidence for involvement of a nonca-
Proc. Natl. Acad. Sci. U. S. A. 106:2752–2757.
6. Bochkarev A, Bochkareva E, Frappier L, Edwards AM. 1999. The crystal
structure of the complex of replication protein A subunits RPA32 and
RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J.
7. Bouchet J, et al. 2011. Inhibition of the Nef regulatory protein of HIV-1
by a single-domain antibody. Blood 117:3559–3568.
8. BouHamdan M, et al. 1998. Diversity of HIV-1 Vpr interactions involves
usage of the WXXF motif of host cell proteins. J. Biol. Chem. 273:8009–
9. Chen R, Le Rouzic E, Kearney JA, Mansky LM, Benichou S. 2004.
Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to
modulate the virus mutation rate and for replication in macrophages. J.
Biol. Chem. 279:28419–28425.
10. Chen R, Wang H, Mansky LM. 2002. Roles of uracil-DNA glycosylase
and dUTPase in virus replication. J. Gen. Virol. 83:2339–2345.
11. DeMott MS, Zigman S, Bambara RA. 1998. Replication protein A stim-
ulates long patch DNA base excision repair. J. Biol. Chem. 273:27492–
12. De Silva FS, Moss B. 2008. Effects of vaccinia virus uracil DNA glycosy-
lase catalytic site and deoxyuridine triphosphatase deletion mutations in-
dividually and together on replication in active and quiescent cells and
pathogenesis in mice. Virol. J. 5:145.
13. De Silva FS, Moss B. 2003. Vaccinia virus uracil DNA glycosylase has an
essential role in DNA synthesis that is independent of its glycosylase ac-
cultured cells. J. Virol. 77:159–166.
14. Hagen L, et al. 2008. Cell cycle-specific UNG2 phosphorylations regulate
protein turnover, activity and association with RPA. EMBO J. 27:51–61.
15. Haug T, Skorpen F, Lund H, Krokan HE. 1994. Structure of the gene for
human uracil-DNA glycosylase and analysis of the promoter function.
FEBS Lett. 353:180–184.
16. Iftode C, Daniely Y, Borowiec JA. 1999. Replication protein A (RPA): the
eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol. 34:141–180.
17. Jones KL, et al. 2010. X4 and R5 HIV-1 have distinct post-entry require-
18. Kaiser SM, Emerman M. 2006. Uracil DNA glycosylase is dispensable for
human immunodeficiency virus type 1 replication and does not contrib-
ute to the antiviral effects of the cytidine deaminase Apobec3G. J. Virol.
19. Ko R, Bennett SE. 2005. Physical and functional interaction of human
nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen.
DNA Repair (Amst.) 4:1421–1431.
20. Laguette N, Benichou S, Basmaciogullari S. 2009. Human immunode-
ficiency virus type 1 Nef incorporation into virions does not increase in-
fectivity. J. Virol. 83:1093–1104.
21. Langevin C, et al. 2009. Human immunodeficiency virus type 1 Vpr
modulates cellular expression of UNG2 via a negative transcriptional ef-
fect. J. Virol. 83:10256–10263.
22. Langlois M-A, Neuberger MS. 2008. Human APOBEC3G can restrict
SMUG1. J. Virol. 82:4660–4664.
23. Le Rouzic E, Benichou S. 2005. The Vpr protein from HIV-1: distinct
roles along the viral life cycle. Retrovirology 2:11.
24. Mansky LM. 1996. The mutation rate of human immunodeficiency virus
type 1 is influenced by the vpr gene. Virology 222:391–400.
25. Mansky LM, et al. 2001. Interaction of human immunodeficiency virus
type 1 Vpr with the HHR23A DNA repair protein does not correlate with
multiple biological functions of Vpr. Virology 282:176–185.
26. Mansky LM, Preveral S, Selig L, Benarous R, Benichou S. 2000. The
interaction of vpr with uracil DNA glycosylase modulates the human im-
27. Mer G, Bochkarev A, Chazin WJ, Edwards AM. 2000. Three-
dimensional structure and function of replication protein A. Cold Spring
Harbor Symp. Quant. Biol. 65:193–200.
28. Mer G, et al. 2000. Structural basis for the recognition of DNA repair
proteins UNG2, XPA, and RAD52 by replication factor RPA. Cell 103:
29. Mol CD, et al. 1995. Crystal structure and mutational analysis of human
uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell
30. Nagelhus TA, et al. 1997. A sequence in the N-terminal region of human
uracil-DNA glycosylase with homology to XPA interacts with the
C-terminal part of the 34-kDa subunit of replication protein A. J. Biol.
31. Otterlei M, et al. 1999. Post-replicative base excision repair in replication
foci. EMBO J. 18:3834–3844.
32. Park MS, Ludwig DL, Stigger E, Lee SH. 1996. Physical interaction
tion in mammalian cells. J. Biol. Chem. 271:18996–19000.
33. Prelich G, Kostura M, Marshak DR, Mathews MB, Stillman B. 1987.
The cell-cycle regulated proliferating cell nuclear antigen is required for
SV40 DNA replication in vitro. Nature 326:471–475.
34. Prelich G, et al. 1987. Functional identity of proliferating cell nuclear
antigen and a DNA polymerase-delta auxiliary protein. Nature 326:517–
35. Prichard MN, Duke GM, Mocarski ES. 1996. Human cytomegalovirus
uracil DNA glycosylase is required for the normal temporal regulation of
both DNA synthesis and viral replication. J. Virol. 70:3018–3025.
36. Prichard MN, et al. 2005. Human cytomegalovirus uracil DNA glycosy-
lase associates with ppUL44 and accelerates the accumulation of viral
DNA. Virol. J. 2:55.
37. Priet S, et al. 2005. HIV-1-associated uracil DNA glycosylase activity
controls dUTP misincorporation in viral DNA and is essential to the
HIV-1 life cycle. Mol. Cell 17:479–490.
38. Ranneberg-Nilsen T, et al. 2008. Characterization of human cytomega-
ase processivity factor (UL44). J. Mol. Biol. 381:276–288.
39. Schröfelbauer B, Hakata Y, Landau NR. 2007. HIV-1 Vpr function is
mediated by interaction with the damage-specific DNA-binding protein
DDB1. Proc. Natl. Acad. Sci. U. S. A. 104:4130–4135.
40. Schröfelbauer B, Yu Q, Zeitlin SG, Landau NR. 2005. Human immu-
nodeficiency virus type 1 Vpr induces the degradation of the UNG and
SMUG uracil-DNA glycosylases. J. Virol. 79:10978–10987.
UNG2 and HIV-1 Infectivity
March 2012 Volume 86 Number 5jvi.asm.org 2543
41. Selig L, et al. 1997. Uracil DNA glycosylase specifically interacts with Vpr
of both human immunodeficiency virus type 1 and simian immunodefi-
ciency virus of sooty mangabeys, but binding does not correlate with cell
cycle arrest. J. Virol. 71:4842–4846.
42. Sousa MML, Krokan HE, Slupphaug G. 2007. DNA-uracil and human
pathology. Mol. Aspects Med. 28:276–306.
43. Stanitsa ES, Arps L, Traktman P. 2006. Vaccinia virus uracil DNA
44. Sugiyama T, New JH, Kowalczykowski SC. 1998. DNA annealing by
RAD52 protein is stimulated by specific interaction with the complex of
replication protein A and single-stranded DNA. Proc. Natl. Acad. Sci.
U. S. A. 95:6049–6054.
45. Takasaki Y, Deng JS, Tan EM. 1981. A nuclear antigen associated with
cell proliferation and blast transformation. J. Exp. Med. 154:1899–
46. Wold MS. 1997. Replication protein A: a heterotrimeric, single-stranded
DNA-binding protein required for eukaryotic DNA metabolism. Annu.
Rev. Biochem. 66:61–92.
47. Yan N, O’Day E, Wheeler LA, Engelman A, Lieberman J. 2011. HIV
DNA is heavily uracilated, which protects it from autointegration. Proc.
Natl. Acad. Sci. U. S. A. 108:9244–9249.
48. Yang B, Chen K, Zhang C, Huang S, Zhang H. 2007. Virion-associated
volved in the degradation of APOBEC3G-edited nascent HIV-1 DNA. J.
Biol. Chem. 282:11667–11675.
Guenzel et al.
jvi.asm.org Journal of Virology