Vpr-mediated incorporation of UNG2 into HIV -1 particles ... · Vpr-UNG and HIV-1 replication in macrophages 4 Vpr as a bait (18), the interaction between Vpr and UNG was confirmed

Vpr-UNG and HIV-1 replication in macrophages 1

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the

virus mutation rate and for replication in macrophages

Renxiang Chen2, +, Erwann Le Rouzic3, +, Jessica A. Kearney1, Louis M. Mansky1, 2, *, and

Serge Benichou3, *

Ohio State University Biochemistry Graduate Program, Columbus, Ohio, 432101

; Institute for

Molecular Virology, University of Minnesota, Minneapolis, MN 554552

, and Institut Cochin,

Department of Infectious Diseases, INSERM U567, CNRS UMR8104, Paris, France3

* Corresponding Authors:

Serge Benichou, Institut Cochin, INSERM U567, Bâtiment Gustave Roussy, 27 Rue du

Faubourg Saint-Jacques, 75014 Paris, France. Phone: (33) 1 40 51 65 78; FAX: (33) 1 40 51 65

70; E-mail: [email protected]

Louis M. Mansky, Institute for Molecular Virology, University of Minnesota, 18-242 Moos

Tower, 515 Delaware St. SE, Minneapolis, MN USA. Phone: (612) 626-5525; FAX: (612) 626-

5515; E-mail: [email protected]

+ These authors contributed equally to this work

Keywords: nondividing cells/HIV-1/Vpr/UNG/mutation rate

Running title: Vpr-UNG and HIV-1 replication in macrophages

JBC Papers in Press. Published on April 19, 2004 as Manuscript M403875200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

Human immunodeficiency virus type 1 (HIV-1) is able to infect nondividing cells, such as

macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we

investigated the impact of the recruitment into virus particles of the nuclear form of uracil

DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on

replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to

virion incorporation of a catalytically active enzyme that is directly involved with Vpr in

modulating the virus mutation rate. The lack of UNG in virions during virus replication in

primary monocyte-derived macrophages further exacerbated virus mutant frequencies to a 18-

fold increase compared to the 4-fold increase measured in actively dividing cells. Since the

presence of UNG is also critical for efficient infection of macrophages, these observations

extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis,

that is essential for replication of HIV-1 in nondividing cells.


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Introduction

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96 amino acid non-structural protein

that is associated with virus particles and can accumulate at the nuclear envelope and in the

nucleus of infected cells (1-4). The incorporation of Vpr into particles requires a direct

interaction with the p6 region of the Gag polyprotein precursor (5,6). Several independent

biological activities have been attributed to Vpr during the HIV-1 life cycle. First, expression

of Vpr alters the cell cycle progression by arresting cells in the G2 phase (7-10). Second, Vpr

influences the reverse transcription process of the viral DNA and this can modulate the in vivo

mutation rate of HIV-1 (11,12). Finally, Vpr is required for the infection of nondividing cells,

and this requirement is related, at least in part, to its role in the nuclear translocation of the

preintegration complex (PIC) containing the viral DNA. Vpr possesses an affinity for the

components of the nuclear pore complex (NPC) and it has been proposed that Vpr may

facilitate the nuclear translocation of the PIC across the nuclear envelope (4,13-16). Infection

of nondividing, terminally differentiated macrophages and resting T cells, represents a viral

reservoir in the host which is crucial for subsequent virus spread to lymphoid organs and T-

helper lymphocytes, and finally for AIDS pathogenesis (17).

The HIV-1 Vpr protein has been found to interact with several cellular partners, including

the uracil-DNA glycosylase (UNG) (18), a DNA-repair enzyme involved in the base excision

repair pathway that specifically removes the RNA base uracil from DNA. Uracil can occur in

DNA either by misincorporation of dUTP or by cytosine deamination (19). Two distinct forms

of UNG are generated by alternative splicing, and localize respectively in mitochondria

(UNG1) and in the nucleus (UNG2). Initially identified from a yeast two-hybrid screen using


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Vpr as a bait (18), the interaction between Vpr and UNG was confirmed both in vitro and ex

vivo in Vpr-expressing cells (11,18). While the Trp residue in position 54 located in the

exposed loop connecting the second and the third alpha-helix of HIV-1 Vpr has been shown

critical to maintain the interaction with UNG, the Vpr-binding site was mapped within the

common C-terminal part of UNG2 (11,18). The use of the peptide phage display or yeast two-

hybrid systems revealed that peptides able to bound Vpr had a common WxxF motif (20,21).

Furthermore, UNG proteins contain such a motif in their C-terminal region, which may be

necessary for the interaction with Vpr (20). Vpr has been found to specifically recruit the

nuclear form of UNG into HIV-1 virions (UNG2, commonly named UNG in this report) (11).

Although the viral integrase may also participate in this recruitment (22,23), the Vpr-

dependent packaging of UNG2 into virions strikingly correlated with the ability of Vpr to

influence the mutation rate of HIV-1 (24). This indicated that the interaction between Vpr and

UNG2 may directly influence the reverse transcription accuracy, and thus play a role in the

modulation of the in vivo mutation rate of HIV-1 (11,12).

In this report, we further investigated the specific contribution of UNG2 incorporated into

HIV-1 particles in the early phase of the virus life cycle. To address this question, we

developed an experimental system in which UNG2 was incorporated into virus particles

independently of Vpr by expressing UNG2 as a chimeric protein fused to the C-terminal

extremity of the VprW54R mutant, a Vpr variant that fails to recruit UNG2 into virions and to

influence the virus mutation rate, even though it is incorporated as efficiently as the wild-type

Vpr protein (11). The VprW54R-UNG fusion was efficiently incorporated into HIV-1 virions

and restored a mutation rate equivalent to that observed with wild type Vpr. Since we showed

that VprW54R variant specifically influenced HIV-1 replication in monocyte-derived


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macrophages, these results support the conclusions that the Vpr-dependent recruitment of

UNG2 into virions is directly involved in the modulation of the HIV-1 mutation rate and is

required for efficient virus replication in non dividing cells such as macrophages.


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Experimental procedures

Retroviral vectors and expression plasmids. Most of the retroviral vectors and yeast and

mammalian expression plasmids used in this study have been described previously (5,11)

except plasmids for expression in bacteria of wild type (wt) and mutated forms of UNG2

fused to the glutathione S-transferase (GST) and for expression in mammalian cells of UNG

fused to C-terminus of the wild type or W54R Vpr proteins. The W231xxF234 motif found

within the nuclear UNG2 form (numbering according to (25)) was mutated by PCR-mediated

site-directed mutagenesis using specific primers containing the desired mutations to obtain the

UNGW231A/F234G mutated form. The PCR product was then cloned back into the EcoRI-

XhoI restriction sites of pGadGE (5) and the pGEX-4T1 vectors (Amersham Biosciences) to

obtain plasmids for expression of UNGW231A/F234G fused either to the Gal4 activation

domain (Gal4AD) or to the GST in yeast and in bacteria, respectively. To construct the

plasmids for expression of the Vpr-UNG and VprW54R-UNG fusions, the Vpr and VprW54R

coding sequences were respectively amplified by PCR using a specific set of primers to delete

the stop codon, and the PCR products were cloned back into the BamHI-HindIII restrictions

sites of the pAS1B plasmid (11). The UNG coding sequence was then amplified by PCR to

create a HindIII site at the 5’ end and the products were inserted in-frame into the HindIII-

XhoI sites of either the pAS1B-Vpr or the pAS1B-VprW54R digested plasmids.

Yeast two-hybrid assay. The HF7c yeast reporter strain containing the Gal4-inducible gene,

HIS3, was cotransformed with vectors for expression of the indicated Gal4 DNA binding

domain (Gal4BD) and Gal4AD hybrids, and plated on selective medium lacking tryptophan


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and leucine as reported (5). Double transformants were patched on the same medium and

replica plated on selective medium lacking tryptophan, leucine and histidine for auxotrophy

analysis.

In vitro binding assay. The GST-UNG fusions were expressed in E. Coli BL21 cells

(Invitrogen) after induction with 0.5 mM isopropyl-1-thio-β-galactopyranoside (IPTG) for 3 h

at 30°C. Bacterial pellets were resuspended in phosphate-buffered saline (PBS) containing 2

mM EDTA, 2 mM DTT and an antiprotease cocktail (Sigma). After 1 h incubation at 4 °C with

0.12% lysozyme, bacterial lysis was completed by adding 1% Triton X-100, 10 mM MgCl2, 10

µg/ml RNase A and 20 µg/ml DNAse I. Lysates were centrifuged at 60 000 g for 30 min at

4°C. Supernatants were incubated with glutathione-sepharose beads (Amersham Biosciences)

for 1 h at 4°C. Beads were washed three times with NaCl 1 M containing 0.5% Triton X-100,

and then with PBS. The concentration of the fusion proteins was estimated on a SDS-PAGE

gel stained with Coomassie blue G-250 (BioRad) relatively to a range of bovine serum

albumin standard. Cell lysates from 6.106 HeLa cells expressing HA-Vpr (wt or W54R) were

prepared and incubated with purified GST or GST-UNG proteins as previously described (4).

Bound proteins were then resolved by SDS-PAGE, and then analyzed by western-blotting as

described (4).

Assay for incorporation of Vpr-UNG fusions into HIV-1 particles. Incorporation of the

Vpr-UNG fusions into HIV-1 virions was analyzed as previously described (5), using a virion

packaging assay in which HA-tagged fusions were expressed in trans in virus-producing cells.


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UNG catalytic assay. A catalytic assay was developed to detect the incorporation of UNG

into virus particles. Briefly, we cotransfected (Superfect, Qiagen) a vpr-defective HIV-1 vector

and Vpr and UNG expression plasmids derived from pAS1B (11) into 293T cells. After 48 h,

cell culture supernatants were collected and virions were concentrated by ultracentrifugation

as previously described (5). Virions were resuspended as described (5), and 1 µg of crude

viral protein was used in the UNG assay. The DNA oligonucleotide 5’-

TTTTTTTTTTTTUTTTTTTTTTTTT-3’used for the UNG assay was chosen based on previous

studies (26), and was obtained from Sigma-Genosys (The Woodlands, TX). Uracil-DNA

glycosylase inhibitor (UGI) was obtained from New England Biolabs (Beverly, MA). Assays

of UNG activity were done with the single-stranded DNA oligonucleotide substrate and were

performed in UNG reaction buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol pH

8.0) at 37 °C for 1 h. Apurinic (AP) sites were cleaved by adding one-half volume of 0.5 M

NaOH and one-half volume of 30 mM EDTA and then boiling for 30 min (27). Samples were

then applied to a non-denaturing 20% polyacrylamide gel with electrophoresis at 60 V for 3.5

h, or were applied to a denaturing 19% polyacrylamide gel and running at 400 V for 2 h. Gels

were stained with SYBR Gold (Molecular Probes, Eugene, OR), and nucleic acids were

visualized with an ultraviolet transilluminator.

Analysis of HIV-1 mutant frequencies. The HIV-1 vectors used in these studies have been

previously described (11,12,28). In order to produce vector virus, the HIV vectors were

complemented in trans with pSVgagpol-rre-MPMV, the amphotropic murine leukemia virus

env expression plasmid, pSV-A-MLV-env, and a Vpr expression plasmid derived from

pAS1B (11). HIV-1 vector and expression plasmids were transfected into HeLa cells by using


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Superfect (Qiagen). Infection of HeLa target cells was also done by cocultivation of virus-

producing cells with target cells (29). The protocols for analysis of mutant frequencies have

been previously described with minor modifications (12,28,30). Specifically, infected

peripheral blood mononuclear cells (PBMCs) and monocytes-derived macrophages (MDMs)

were not placed under drug selection and after purification of proviral DNA with the lac

repressor protein, the vector cassette containing the mutation target was PCR amplified prior

to analysis of mutant frequencies in E. coli.

Isolation and infection of PBMCs and MDMs. PBMCs from HIV-1 seronegative donors

were isolated by Ficoll-Hypaque density centrifugation. PHA-stimulated cells were plated in

RPMI/10% FCS containing 10 U/ml IL-2 and streptomycin (100 µg/ml). Peripheral blood

monocytes were isolated from fresh PBMCs by adherence to plastic at 37°C. Following

overnight culture, the adherent cells were removed from plates by gentle scraping and residual

T-lymphocytes were further depleted using anti-CD2 immunomagnetic beads. Cells were

cultured in medium without added growth factors. Mature macrophages were derived by

culturing the purified monocytes 7 to 14 days in RPMI/10% FCS without additional cytokines.

Cells typically became enlarged or spindle shaped with extended processes.

Virus infection of primary cells was done using virus produced from 293T cells

transfected (Superfect, Qiagen) with T-cell tropic or macrophage-tropic HIV-1 molecular

clones expressing either wt or mutant Vpr. CAp24 equivalent amounts of virus produced

from infected cells was used for infection of primary cells and corresponded to a multiplicity

of infection of about 0.05. After overnight incubation at 37°C, the cells were washed twice

and placed in either RPMI/10%FCS (macrophages, resting T-cells) or medium supplemented


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with 10 U/ml IL-2 (stimulated PBMCs/stimulated T-cells). Sampling of cell culture

supernatants was done immediately after washing (Day 0) and on subsequent timepoints.

Amounts of CAp24 produced were determined by ELISA.


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Results

Characterization of the determinants involved in the interaction between Vpr and UNG2.

Previous studies have established a correlation between the property of Vpr to interact with

UNG and to influence the HIV-1 mutation rate (11,24). In particular, a VprW54R variant (see

Fig. 1B), which failed to bind UNG in a yeast two-hybrid assay, also failed to recruit UNG into

HIV-1 virions, and was not able to complement a vpr null mutant HIV-1 in a mutation rate

assay (11). To further characterize the respective molecular determinants of Vpr and UNG

involved in the interaction, we developed an in vitro binding assay using recombinant UNG

expressed in E. coli in fusion with the glutathione-S-transferase (GST-UNG). Since it has been

reported that Vpr binding is related to the presence of a WxxF motif found within the C-

terminal part of UNG2 (a.a. 231-234), a GST-UNGW231A/F234G mutant was included as a

control of specificity in this in vitro binding assay. Purified recombinant GST-UNG and GST-

UNGW231A/F234G fusions were immobilized on GSH-sepharose beads and then incubated

with lysates from cells expressing either HA-tagged wild type (wt) Vpr or VprW54R. Bound

proteins were analyzed by Western blotting with anti-HA (Fig 1A). HA-Vpr specifically

bound to GST-UNG, but not to GST alone. In contrast, VprW54R was not retained on GST-

UNG, and neither wt Vpr nor VprW54R bound to GST-UNGW231A/F234G. These results were

in complete agreement with the yeast two-hybrid data reported in Fig. 1B and 1C. Only wt

UNG, but not UNGW231A/F234G, fused to Gal4AD interacted with Vpr fused to Gal4BD, as

indicated by growth of the HF7c yeast reporter strain on medium without histidine. As

expected, the VprW54R variant failed to interact with UNG in the two-hybrid assay. Together,

these observations provide further data in support that the Trp in position 54 of Vpr is critical


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to maintain the interaction with UNG, and that the W231xxF234 motif of UNG2 is involved in

binding to Vpr.

UNG-associated enzymatic activity is recovered from HIV-1 particles. To determine

whether the UNG incorporated into virions was catalytically active, we developed a simple

assay for UNG activity using a 25-base T homopolymer oligonucleotide substrate containing a

single uracil residue located at position 12 (see Fig. 2A). In the presence of UNG activity, the

uracil residue is excised and leaves the phosphodiester backbone. Heating the sample can

destroy the backbone at that position, resulting in a 12-base product that can be visualized on

a nondenaturing polyacrylamide gel. UNG activity from purified HIV-1 virions was thus

determined by transiently transfecting 293T cells with an HIV-1 vector with either Vpr or

VprW54R in combination with UNG expression plasmids. HIV-1 virions were collected 48 h

later, concentrated, and used in the UNG activity assay (Fig. 2A). Expression of Vpr in virus

producing cells led to UNG enzymatic activity from purified virions. This indicates that the

fully active endogenous UNG was incorporated into HIV-1 virus particles, but expression of

Vpr in combination with a HA-tagged UNG form led to the detection of a higher level of UNG

activity into virions. In contrast, expression in virus producing cells of the VprW54R mutant

alone or in combination with UNG was not associated with the detection of enzymatic activity

from virions. Similarly, very low level of UNG activity was detected from virions produced

from cells co-expressing Vpr and a mutated UNGW231A/F234G form that do not interact with Vpr

(see Fig. 1). As shown in Fig. 2B, no activity was detected when the assays were performed in

the presence of a specific inhibitor of UNG (UGI) in the reaction mixtures (31), demonstrating

that the activity detected into virions is related to a specific recruitment of UNG. These results


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show that Vpr-mediated recruitment of UNG results in the presence of catalytically active

enzyme into HIV-1 virions.

Incorporation of Vpr-UNG fusion proteins into HIV-1 particles. To gain further insight on

the functional role of the recruitment of UNG into HIV-1 virions, we took advantage of the

VprW54R mutant to specifically incorporate UNG without requiring interaction with Vpr by

expressing UNG as a fusion to the carboxy-terminus of VprW54R. We previously reported

that this Vpr mutant failed to recruit UNG2 into virions, even though it is efficiently

incorporated into virions (11). The virion incorporation of the VprW54R-UNG fusion was first

analyzed using a packaging assay in which the fusion was expressed in trans in virus-

producing cells. 293T cells were cotransfected with the HIV-1 vector lacking the vpr gene in

combination with the VprW54R-UNG expression plasmid. Cell- and virion-associated Vpr-

UNG fusions were then assayed by immunoblotting (Fig. 3A). The VprW54R-UNG fusion, as

well as the wild type Vpr-UNG fusion used as a control, were well expressed in virus

producing cells (upper panels), and both fusions were detected from virions purified from the

supernatant of transfected cells (lower panels). Using the same enzymatic assay as described

above (see Fig. 2A), we checked that the VprW54R-UNG and Vpr-UNG fusions incorporated

into virions were catalytically active (Fig. 3B). These results indicate that the Vpr-UNG fusion

proteins are efficiently incorporated into HIV-1 particles and retain UNG enzymatic activity.

The Vpr-UNG fusion proteins can influence the HIV-1 mutant frequency. The VprW54R-

UNG fusion therefore represents a valuable tool for analyzing the direct contribution of UNG

to the modulation of the virus mutation rate. We thus used an HIV-1 mutation rate assay to


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determine if the Vpr-UNG fusions could complement a vpr-defective HIV-1 for mutant

frequencies (11). Briefly, the plasmids for expression of Vpr-UNG fusions were transiently

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 cells, which allowed for a determination of the virus mutant

frequency per round of replication (Fig. 4). In contrast to the fourfold increase in mutant

frequency observed by trans-complementation with the VprW54R mutant, the wild type

fusion but also the VprW54R-UNG fusion gave rise to virus mutant frequencies equivalent to

that observed by complementation with the wild type Vpr protein. These data show that the

VprW54R-UNG fusion can rescue the defective phenotype of VprW54R and modulate HIV-1

mutant frequency as efficiently as Vpr, demonstrating that the recruitment of UNG into

virions is directly responsible for this Vpr function.

Recruitment of UNG into virions is essential for efficient replication of HIV-1 in

macrophages. Since data presented here as well as a previous study demonstrate that the

recruitment of UNG into HIV-1 virions plays an important role at an early step in HIV-1

replication (e.g., viral DNA synthesis) (11), we tested whether this recruitment had a direct

impact on virus replication in primary target cells of HIV-1. The W54R mutation was

introduced into the vpr gene of either a T-cell tropic (NL4-3) or a macrophage tropic (YU-2)

HIV-1 molecular clone. Wild-type and Vpr mutant proviruses were transfected into 293T cells,

cell culture supernatant harvested, adjusted for equal amounts of CA p24 antigen, and used to

infect peripheral blood mononuclear cells (PBMCs) or monocyte-derived macrophages

(MDMs). Virus production was then monitored by measuring the CA p24 antigen every 3


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days (Fig. 5). Whereas HIV-1 expressing Vprwt replicated efficiently in MDMs with a rapid

increase in CA p24 antigen at 9 days postinfection and peaking at 15 days, HIV-1 expressing

VprW54R had a significant replication defect, with only low levels of CA p24 detected 9 days

after infection (Fig. 5A). In contrast, viruses expressing Vprwt or VprW54R both efficiently

replicated in PBMCs (Fig. 5B). In summary, these data show that VprW54R mutation can

influence HIV-1 replication in MDMs, and not in PBMCs, indicating that the Vpr-dependant

incorporation of UNG is important for virus replication in non-dividing cells.

Influence of Vpr on HIV-1 mutant frequency in monocyte-derived macrophages. Because

the replication defect of HIV-1 expressing the VprW54R mutant was specifically apparent in

non-dividing cells, we finally analyzed in MDMs the influence of virion-associated UNG on

virus mutant frequencies. The same HIV-1 mutation rate assay was used, but viruses

containing the lacZα peptide gene as a mutation target were then used to infect MDMs to

determine if the VprW54R mutant could complement a vpr-defective HIV-1. As reported in

Table 1, complementation with Vprwt or with the previously characterized VprR90K (32)

mutant that efficiently interacts with UNG led to an average mutation frequency (0.006 and

0.007 mutation/cycle, respectively), which is equivalent to that observed when HeLa cells were

the targets for infection (see Fig. 4, and (11)). However, VprW54R as well as the lack of Vpr

expression (∆Vpr) led to a 16-18-fold increase in virus mutant frequencies (averages were

0.109 and 0.098, respectively) compared to that observed with Vprwt during infection of

MDMs. This increase is about 4-5 times higher than the increase in virus mutant frequencies

observed when HeLa cells were used as target cells (11). Again, trans-complementation with

either the VprW54R-UNG or Vpr-UNG fusions led to a mutation rate comparable to that


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obtained with Vpr alone, demonstrating that UNG fused to VprW54R restore a normal

mutation phenotype. These observations indicate that virus mutant frequencies are

significantly higher in MDMs when UNG is not packaged into HIV-1 virions and confirm that

the recruitment of UNG is directly responsible for this Vpr function.


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Discussion

The observations described in this report provide important new insights into the

functional role for the interaction of the Vpr auxiliary protein from HIV-1 and the cellular

UNG2 protein, an enzyme implicated in DNA repair. We demonstrate that the Vpr-dependant

incorporation of UNG into HIV-1 particles is directly responsible for the role of Vpr in the in

vivo modulation of the virus mutation rate (11,12). Moreover, our results show that the

incorporation of UNG into virions is critical for efficient replication of HIV-1 in primary non-

dividing cells such as macrophages. This observation parallels the involvement of Vpr in the

nuclear import of viral DNA in non-dividing cells (33), and extends its role to another early

step of the virus life cycle (e.g., viral DNA synthesis) essential for replication of HIV-1 in non-

dividing cells. Several lines of evidences reported here support these conclusions. First, while

the UNG-binding deficient VprW54R variant failed to influence the virus mutation rate, a

VprW54R-UNG fusion was able to influence HIV-1 mutant frequencies in a manner

equivalent to that of wild type Vpr. Second, when the VprW54R variant was introduced into

infectious HIV-1 molecular clones, replication in MDMs was significantly diminished,

whereas virus replication in PBMCs was not altered. Finally, the lack of UNG virion-

incorporation during virus replication in macrophages further exacerbated HIV-1 mutant

frequencies compared to that measured in actively dividing cells.

Using both yeast two-hybrid and biochemical approaches, we confirm that substitution of

the Trp231 and/or Phe234 residues of the WxxF motif of UNG alters its binding to Vpr, while

mutation of the Trp54 residue of HIV-1 Vpr abolishes binding to UNG. Moreover, no UNG

activity was detected into purified virions trans-complemented with either wild type Vpr and


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UNGW231A/F234G or VprW54R and wild type UNG, indicating that the enzymatic activity

detected into virus particles is strictly related to the direct interaction that takes place in virus-

producing cells between Vpr and UNG. Currently, three distinct cellular partners of Vpr

contain a WxxF motif including TFIIB (34), the adenine nucleotide translocator (35) and UNG

(20). The Trp54 residue of HIV-1 is crucial both for binding to UNG and then its recruitment

into viral particles (11,32), but does not participate in the interaction of Vpr with the viral Gag

precursor in virus-producing cells, which allows for the incorporation of Vpr into virions

(5,11). The three-dimensional structure of the complete Vpr polypeptide was recently solved

and confirms that Trp54 is localized between the second and third α-helix. This suggests that

this residue is easily accessible for protein-protein interactions with UNG (36), and that

substitution of W54 does not modify or alter the overall conformation of the HIV-1 Vpr

protein. Indeed, the VprW54R mutant is still able to induce a G2 arrest of the cell cycle (32)

and efficiently localizes at the nuclear envelope through interaction with the hCG1

nucleoporin (data not shown, and (4)).

We therefore took advantage of the VprW54R mutant, which failed to incorporate UNG

into virions (11), to generate a Vpr-UNG fusion protein that allows for an evaluation of the

specific role(s) of UNG recruitment into viral particles on the early steps of HIV-1 infection.

The VprW54R-UNG fusion is efficiently incorporated into virions, and enzymatic assays

performed from purified virions show that the UNG fused to VprW54R was still catalytically

active. These results confirm that Vpr can efficiently target proteins within HIV-1 particles

without affecting the catalytic properties of the cargo (20,37-39). Moreover, the observation

that the Vpr-UNG fusions can restore the mutant frequency phenotype indicates that Vpr and

the virion-associated UNG are directly responsible for the modulation of the virus mutant


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frequency in vivo. The enhanced alteration in virus mutant frequencies observed in primary

macrophages when UNG was not incorporated into virions shows that this phenotype may

have greater biological relevance in nondividing cells than in actively dividing cells. It is of

particular interest to note that virus lacking Vpr or expressing the VprW54R mutant display

analogous mutation rate indicating that no other viral proteins can rescue this Vpr phenotype.

Although it was proposed that the viral integrase (IN) was also able to mediate interaction with

UNG (22,23), our results argue that Vpr is the main viral determinant that allows for the

incorporation of cellular UNG into virus particles. However, preliminary results obtained from

in vitro binding assays suggest that both Vpr and IN associate with UNG to form a trimeric

complex (data not shown), but further analyses are needed to document the dynamic of

interactions between UNG, Vpr, IN as well as RT (23) both in virus-producing cells and then

in target cells.

HIV-1 and other lentiviruses are unusual among retroviruses in their ability to infect

resting or terminally differentiated cells. Vpr from HIV-1 has been related to facilitate nuclear

import of the viral DNA in such non-dividing cells (33). In this report, we have identified that

the virion incorporation of UNG via Vpr also contributes to the ability of HIV-1 to replicate in

primary macrophages assigning another critical role of Vpr during the viral life cycle. This

implies that UNG is a cellular factor that plays an important role in the early steps of the HIV-1

replication cycle (i. e. viral DNA synthesis). In agreement, it has been recently reported that

the misincorporation of uracil into minus strand viral DNA affects the initiation of the plus

strand DNA synthesis in vitro (40). These results suggest that UNG is likely recruited into

HIV-1 particles to subsequently minimize the detrimental accumulation of uracil into the

newly synthesized proviral DNA. While further work is needed to explain the precise


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mechanism for how UNG catalytic activity may specifically influence HIV-1 replication in

macrophages, it is noteworthy that such nondividing cells express low levels of UNG and

contain relatively high levels of dUTP (41). Similarly , most non-primate lentiviruses, such as

feline immunodeficiency virus (FIV), caprine-arthritis-encephalitis virus (CAEV) and equine

infectious anemia (EIAV), have also developed an efficient strategy to reduce accumulation of

uracil into viral DNA. These lentiviruses encode and package a dUTP pyropshophatase

(dUTPase) into virus particles (for review, see (19,41)), an enzyme that hydrolyzed dUTP to

dUMP, and thus maintains a low level of dUTP. Interestingly, replication of FIV, CAEV or

EIAV that lack functional dUTPase activity is severely affected in nondividing host cells (e.g.,

primary macrophages) (42-44). Altogether, these results indicate that uracil misincorporation

in viral DNA strands during reverse transcription is deleterious for the ongoing steps of the

virus life cycle. The presence of a viral dUTPase or a cellular UNG will prevent these

detrimental effects for replication of non-primate and primate lentiviruses in macrophages,

respectively.

It is intriguing to note that two viral auxiliary proteins from HIV-1, Vpr and Vif, act in the

same way to contribute in the fidelity of the synthesis of the viral DNA from the RNA

template, but using two different mechanisms. The Vif protein forms a complex with the

cellular deaminase APOBEC-3G (CEM15) preventing its encapsidation into virions (45-49),

while Vpr binds the DNA repair enzyme, UNG, to recruit it into the particles where it could

start to exert its activity. It is tempting to speculate that action of both viral proteins may

influence the mutation rate during the course of HIV-1 infection, and their balance may play a

key role during disease progression in infected individuals.


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In summary, this report provides strong evidence for a direct role of the cellular UNG2

incorporated into HIV-1 virions in the modulation of virus mutation rate. The requirement of

UNG2 incorporation by Vpr for efficient virus replication in macrophages implies that the

interaction between Vpr and UNG2 could represent an attractive target for antiviral

intervention.


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Acknowledgements

We thank A. Benmerah for continuous support and S. Maire for technical assistance. This

research was supported by Public Health Service grant GM56615 (to L.M.M.) and from the

French Agency for AIDS Research (ANRS) and SIDACTION (to S.B. and E.L.R).


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Figure legends

Figure 1. Characterization of the interaction of Vpr with UNG. (A) In vitro binding

analysis of the Vpr-UNG interaction. HeLa cells were transfected with either 1 (lanes 1 and 3)

or 3 µg (lanes 2 and 4) of plasmids for expression of either HA-tagged Vpr (lanes 1 and 2) or

VprW54R (lanes 3 and 4). Lysates from transfected cells were incubated with equal amounts

of GST, GST-UNG, or GST-UNGW231A/F234G immobilized on GSH-sepharose beads as

indicated at the bottom. Bound proteins were resolved by SDS-PAGE and immunoblotted

with an anti-HA antibody. One-tenth of the imput of the cell lysate from the transfected cells

used for the binding assay was run on the right panel. (B) and (C) Two-hybrid analysis of the

Vpr binding to UNG. HF7c reporter strain expressing either wild type Vpr or VprW54R fused

to Gal4BD, in combination with either wild type UNG or UNGW231A/F234G fused to Gal4AD was

analyzed for histidine auxotrophy. Double transformants were patched on selective medium

with histidine (+His) and then replica plated on medium without histidine (-His). Growth in

the absence of histidine indicates interaction between hybrid proteins. Each patch represents

an independent transformant.

Figure 2. UNG-associated enzymatic activity recovered from HIV-1 particles. (A)

Analysis of UNG activity into virions. Virions produced from cells expressing either wild type

Vpr or VprW54R alone or in combination with wild type UNG or UNGW231A/F234G were

collected from cell supernatants and prepared as described in Materials and Methods. Assays

of UNG activity were performed with a 25 bp single-stranded DNA oligonucleotide substrate

containing an uracil base at position 13 (shown on the right), and AP sites were then cleaved


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by adding 0.5 M NaOH and 30 mM EDTA, and boiling for 30 min. The samples were run on a

20% polyacrylamide denaturing gel. Gels were stained with SYBR Gold, and nucleic acids

were visualized with an ultraviolet transilluminator. Following the alkaline and heat treatment,

the deoxyribose phosphate backbone is hydrolyzed to form two 12 base-pair fragment

products (shown on the right). The control lane contains untreated DNA substrate, while the

∆Vpr lane corresponds to purified virions produced from cells transfected with vpr-defective

HIV-1 vector alone as indicated in Materials and Methods. (B) Analysis of UNG activity in the

presence of UGI inhibitor. Activity was assayed as in (A), but UGI was added to the reaction

mixture where indicated. The samples were then analyzed as described above.

Figure 3. Expression and incorporation into HIV-1 virions of enzymatically active Vpr-

UNG fusions. (A) Virion incorporation of the Vpr-UNG fusion proteins. Virions were

produced, as indicated in the Materials and Methods, from 293T cells cotransfected with an

HIV-1-based vector lacking the vpr gene in combination with plasmids for expression of HA-

tagged Vpr-UNG or VprW54R-UNG fusions. Proteins from cell and virion lysates were

separated by SDS-PAGE and analyzed by Western blotting with anti-HA (Cells and Virions,

upper panels) or anti-CA p24 (Cells and Virions, lower panels). (B) UNG activity from the

Vpr-UNG fusions incorporated into virions. Virions produced from cells expressing either

Vpr-UNG or VprW54R-UNG were collected from cell supernatants and prepared as described

in Materials and Methods. UNG enzymatic activity was assayed as in Figure 2. The control

lane contains untreated DNA substrate.

Figure 4. Influence of Vpr-UNG fusion proteins on HIV-1 mutant frequencies in


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dividing cells. The ability of wild type or mutated Vpr or Vpr-UNG fusions to complement a

vpr-defective HIV-1 was analyzed in a single-cycle replication assay for mutant frequencies.

The plasmids for expression of HA-tagged forms of Vpr, VprW54R, VprW54R-UNG or Vpr-

UNG were transiently 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 as described in the

Materials and Methods. The average mutant frequency of the vpr null mutant HIV-1 in the

absence of Vpr trans-complementation (Control) was 0.15 mutant/cycle. Values are the

means of three independent experiments. Error bars represent 1 standard deviation from the

mean.

Figure 5. The VprW54R mutation specifically influences HIV-1 replication in monocyte-

derived macrophages. The W54R mutation was introduced into the vpr gene of either a T-

cell tropic (NL4-3) or a macrophage tropic (YU-2) HIV-1 molecular clone. Wild type (Vprwt,

circles) and mutated (VprW54R, triangles) viruses produced in cell free supernatant of 293T

cells transfected with proviral DNAs were harvested, adjusted for equal amounts of CA p24

antigen and used to infect MDMs (A) or PBMCs (B). Virus production was then monitored by

measuring the CA p24 antigen every 3 days.


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Table 1. Influence of Vpr variants on HIV-1 mutant frequencies in monocyte-derived

macrophages.

Vpr variant Mutant frequency Fold difference

(mutants/cycle)a (P value)b

∆Vpr 0.098 +/- 0.009 16 (< 0.0001)

Vpr wt 0.006 +/- 0.003

VprR90K 0.007 +/- 0.002 1

VprW54R 0.109 +/- 0.006 18 (< 0.0001)

Vpr-UNG 0.005 +/- 0.003 1 (> 0.9)

VprW54R-UNG 0.008 +/- 0.004 1 (> 0.9)

a Mutant frequencies are averages from three independent experiments +/- standard

deviations.

b P values were determined by chi-square analysis.


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Cell lysateGST-UNGW231A/F234G

WT W54R

Chen et al.

Figure 1

GST-UNGGST

62 -

47.5 -

32.5 -

kDa

Anti-HA Western blotting

18 -

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

VprA.

HA-Vpr

VprVprW54R

UNGUNG

VprVprW54R

UNGGal4BDGal4ADGal4AD

Gal4BD-hybrid Gal4AD-hybrid + His - His

VprVpr

UNGUNGW231A/F234G

Gal4BDVpr

UNGGal4BDUNGW231A/F234G

Gal4AD

Gal4BD-hybrid Gal4AD-hybrid + His - His

B. C.

WT W54R WT W54R WT W54R


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B.

A.

Chen et al.Figure 2

cont

rol

Vpr

Vpr

+ U

NG

Vpr

+ U

NG

W23

1A/F

234G

Vpr

W54

R +

UN

G

contr

olVp

rVp

r + UNG

+ UGI+ UGI

25 bp DNA subtrateTTTTTTTTTTTTUTTTTTTTTTTTT

12 bp productTTTTTTTTTTTT

Vpr + U

NG

-- UGIUGI

∆Vpr

Vpr

W54

Rsubtrate

product


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A.

Vpr-UNG

p24

Vpr-UNG

Pr55

HIV-1 vector (∆vpr)

Vpr-UNG

- -+ +

p41

W54R-UNG

Cells

Virions

Chen et al.Figure 3

cont

rol

Vpr

-UN

G

Vpr-UNG fusions

Vpr

W54

R-U

NG

B.

substrate

product


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Relativemutantfrequency

VprW54R-UNG Vpr-UNG

4.0

3.0

2.0

1.0

Chen et al.Figure 4

VprW54R∆ Vpr Vprwt


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p24

(n

g/m

l)

Days post-infection

VprW54R HIV-1

Vprwt HIV-1

Chen et al.Figure 5

A B

3 6 9 12 15 18 21

MDMs 2000

1000

3 6 9 12 15 18 21

1500

500

PBMCs2000

1000

1500

500


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BenichouRenxiang Chen, Erwann Le Rouzic, Jessica A. Kearney, Louis M. Mansky and Serge

thevirus mutation rate and for replication in macrophagesVpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate

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