HIV-1-Associated Uracil DNA Glycosylase Activity Controls dutp Misincorporation in Viral DNA and Is Essential to the HIV-1 Life Cycle

Transcription

1 Molecular Cell, Vol. 17, , February 18, 2005, Copyright 2005 by Elsevier Inc. DOI /j.molcel HIV-1-Associated Uracil DNA Glycosylase Activity Controls dutp Misincorporation in Viral DNA and Is Essential to the HIV-1 Life Cycle Stéphane Priet, 1,5 Nathalie Gros, 1,6 Jean-Marc Navarro, 1,7 Joëlle Boretto, 2 Bruno Canard, 2 Gilles Quérat, 1,3,4, * and Joséphine Sire 1,3,4, * 1 Pathogénie des Infections à Lentivirus INSERM U372 Université de la Méditerranée 163 Avenue de Luminy, BP Marseille Cedex 9 France 2 Architecture et Fonction des Macromolécules Biologiques Introduction Uracil appears in DNA as a result of dutp misincorporation or of deamination of cytosine (Lindahl and Wood, 1999). Uracil in DNA is counteracted by several defense mechanisms that use uracil DNA glycosylase (UNG) and deoxyuridine triphosphatase (dutpase) enzymes. All free-living organisms express UNG and dutpase. dutpase acts in hydrolyzing dutp in dump and inorganic pyrophosphate, thus preventing dutp misincorporation. Uracil DNA glycosylase acts in removing uracil bases from the sugar backbone of DNA, leaving abasic sites and initiating the uracil base-excision-repair (BER) pathway. The completion of BER requires the sequential participation of enzymes including an AP-endonuclease enzyme that cleaves 5 of the abasic site, a polymerase that cleaves 3 of the abasic site via its lyase activity and then inserts the correct nucleotide, and the XRCC1- ligase 3 complex that seals the remaining nick (Hoeijmakers, 2001). Five distinct enzymes that have uracil CNRS UMR 6098 Université de la Méditerranée ESIL Case Marseille Cedex 9 France 3 Faculté de Médecine Unité des Virus Emergents 27 Bd Jean Moulin Marseille France DNA glycosylase activity are expressed by human cells, namely UNG1, UNG2, TDG, MBD4, and SMUG (Krokan et al., 2002). UNG1 and UNG2 are generated by transcription from two different promoters of the ung gene Summary and the use of alternative splicing (Nilsen et al., 1997). These isoforms differ in their N-terminal sequence, re- Uracilation of DNA represents a constant threat to the sulting in one form that enters the nucleus (UNG2) while survival of many organisms including viruses. Uracil the other form enters the mitochondria (UNG1). may appear in DNA either by cytosine deamination or Genomes of some DNA viruses, such as pox and her- by misincorporation of dutp. The HIV-1-encoded Vif pes viruses, encode both UNG and dutpase enzymes protein controls cytosine deamination by preventing (McGeoch, 1990; Roizman and Sears, 1990). Genomes the incorporation of host-derived APOBEC3G cytidine of -retroviruses, such as Mazon-Pfizer monkey virus deaminase into viral particles. Here, we show that the and murine mammary tumor virus, and genomes of non- host-derived uracil DNA glycosylase UNG2 enzyme, primate lentiviruses, such as ovine Maedi-Visna virus, which is recruited into viral particles by the HIV-1- caprine arthritis-encephalitis virus (CAEV), feline immu- encoded integrase domain, is essential to the viral life nodeficiency virus (FIV), and equine infectious anemia cycle. We demonstrate that virion-associated UNG2 virus (EIAV), encode a dutpase enzyme that acts to counteract the misincorporation of dutp in viral DNA catalytic activity can be replaced by the packaging that otherwise affects viral spread (Lerner et al., 1995; of heterologous dutpase into virion, indicating that Steagall et al., 1995; Turelli et al., 1997). dutpase is UNG2 acts to counteract dutp misincorporation in encoded by most of the members of the lentiviridae the viral genome. Therefore, HIV-1 prevents incorporafamily, but not by primate lentiviruses such as HIV-1. tion of dutp in viral cdna by UNG2-mediated uracil Interestingly, we have reported that the host-derived excision followed by a dntp-dependent, reverse tran- UNG2 enzyme is incorporated into HIV-1 virions via a scriptase-mediated endonucleolytic cleavage and fi- specific interaction with the integrase domain of the nally by strand-displacement polymerization. Our find- Gag-Pol precursor (Priet et al., 2003a; Willetts et al., ings indicate that pharmacologic strategies aimed 1999), although it has been proposed that the virally toward blocking UNG2 packaging should be explored encoded Vpr protein was also able to mediate the incoras potential HIV/AIDS therapeutics. poration of UNG2 (Mansky et al., 2000). We have shown in in vitro assays that packaged UNG2 can process uracil from DNA, indicating that HIV-1 has the ability to control *Correspondence: jsire@marseille.inserm.fr (J.S.); gquerat@marseille. the uracilation of its genome (Priet et al., 2003b). inserm.fr (G.Q.) A series of recent reports described that the uracila- 4 These authors contributed equally to this work. tion of HIV-1 DNA constitutes an innate cellular barrier 5 Present address: Faculté de Médecine de Marseille, Unite des Virus against this pathogen (Harris et al., 2003; Lecossier et Emergents, 27 Bd Jean Moulin, F13385, Marseilles, Cedex 05, al., 2003; Mangeat et al., 2003; Mariani et al., 2003; Yu France. 6 Present address: ENS de Lyon, INSERM U412, 46 Allée d Italie, 69364, Lyon, Cedex 07, France. 7 Present address: Centre d Immunologie de Marseille Luminy, Campus de Luminy, Case 906, 13009, Marseille, France. et al., 2004; Zhang et al., 2003). In the absence of the HIV-1-encoded Vif protein, the host cytidine deaminase APOBEC3G is incorporated into viral particles, converting many cytosines specifically on the minus-strand

2 Molecular Cell 480 cdna to uracils, thus generating a lethal accumulation of G to A hypermutations in the plus strand. In the presence of Vif, APOBEC3G is excluded from viral particles and degraded by the proteasome (Marin et al., 2003; Mehle et al., 2004; Sheehy et al., 2002; Yu et al., 2003). These data highlight the deleterious effects of uracil in the HIV-1 genome and show that HIV-1 has evolved in elaborating viral countermeasures. UNG2 controls the level of uracil in cellular DNA, and we have previously shown that it is packaged into HIV-1 viral particles (Priet et al., 2003a). This led us to ask whether blocking the incorporation of UNG2 into HIV-1 virions might be able to alter viral propagation via the accumulation of uracil in viral transcripts. Results HIV-1 Virus Fails to Replicate in UNG2-Depleted Macrophages To investigate the role of virion-associated UNG2 in the HIV-1 life cycle, we followed the spread of HIV-1 in human primary macrophages depleted of UNG2 by RNA interference-mediated knockdown (Elbashir et al., 2001). The sequence of the UNG2-specific sirna corresponds to a stretch of 19 nucleotides located in exon I A of the human ung gene (Nilsen et al., 1997) so that only UNG2, but not its UNG1 mitochondrial isoform, would be downregulated (Figure 1A). This selective degradation thus provided a good internal control for the specificity of silencing. As a control, we used an sirna (UNG2-random sirna) that had the identical base composition as that of UNG2-specific sirna but with a scrambled sequence. Because the expression of UNG2 in macrophages was too weak to be detected by Western blotting, we used quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to verify that UNG2 mrnas, but not UNG1 mrnas, were depleted in macrophage populations (Figure 1B). The UNG2 depletion remained complete over 28 days post- Figure 1. Depletion of UNG2 From Macrophages by UNG2-Specific transfection (Figure 1C), consistent with the observation sirna Abolishes Viral Replication that sirnas can persist for a long time in nondividing (A) Depiction of the human 5 UNG genomic region with two different cells (Song et al., 2003). promoters, P A and P B, resulting in two distinct splice products, the Macrophages were infected with the wild-type HIV- UNG2 and UNG1 isoforms, respectively. 1 AD8 strain on day 2 after being transfected with sirnas, (B) UNG2 mrna depletion in macrophages. Macrophages were and viral replication was monitored by measuring CAp24 transfected with UNG2-specific (siung2) or UNG2-random (siranantigen in the cell-free supernatant (Figure 1D and Figtive dom) sirnas. UNG2 and UNG1 mrna were measured by quantita- ure S1 available online at RT-PCR on day 1 posttransfection. (C) Long-term depletion of UNG2 mrna in macrophages transfected content/full/17/4/479/dc1/). Viral spread was impaired with UNG2-specific sirna. RNA was purified at the specified times when infected macrophages were depleted of UNG2 in posttransfection, and UNG2 and UNG1 RNA were quantified by realcontrast to untreated or UNG2-random sirna-treated time RT-PCR. macrophages, which efficiently support viral replication. (D) Abortive viral spread in UNG2-depleted macrophages. Macro- Therefore, the expression of UNG2 in macrophages is phages were transfected with UNG2-specific or UNG2-random required for viral replication. sirnas and were infected on day 1 posttransfection with the wild- type HIV-1 AD8 strain. Viral replication was monitored by measurement Depletion of UNG2 in Producer Cells Generates of the CAp24 antigen content in the cell-free supernatant. Results are representative of three independent experiments using sirna- Noninfectious Virus treated macrophages isolated from three distinct healthy donors. The inhibition of viral propagation observed in macrophages depleted of UNG2 might be attributed either to a defect in UNG2-depleted target cells that impedes were transfected with UNG2-random or UNG2-specific early steps of infection or to the production of noninfectious sirnas. By using quantitative real-time RT-PCR, we virus from UNG2-depleted cells. To address the confirmed that UNG2 mrna was significantly reduced, first possibility, MAGI-CCR5 cells containing a lacz reporter and we confirmed by Western blotting that UNG2 ex- under the control of an integrated HIV promoter pression was barely detectable in UNG2-specific sirna-

3 UNG2-Dependent Repair of dutp in HIV-1 DNA 481 treated cells (Figure 2A). UNG2-depleted MAGI-CCR5 cells were then infected with wild-type HIV-1 AD8 viral stock in single round assays (Figure 2B). -galactosidase ( -gal) expression staining showed that HIV-1 virus was able to successfully infect UNG2-depleted cells, indicating that the presence of UNG2 in target cells is not required for the first steps of infection. This finding suggests that the inhibition of viral spread occurred in the second round of infection and could be due to the lack of packaged UNG2. We therefore evaluated the effect of depletion of UNG2 in producer cells on viral replication. UNG2-depleted MAGI-CCR5 cells were transfected with HIV-1 proviral DNA to obtain viral stock deficient for packaged UNG2 (Figure 2C, left). UNG2- deficient virus was then used to infect MAGI-CCR5 target cells for a single round of replication (Figure 2C, right). -gal expression staining showed that UNG2- deficient virus, although displaying normal levels of exogenous reverse transcriptase (RT) activity and CAp24 antigen content (data not shown), failed to infect UNG2- positive target cells. Similar results were obtained when UNG2-deficient virus was used to infect C8166-CCR5 target cells (Figure 2D, left). These data indicate that the presence of UNG2 in target cells was unable to compensate for the lack of packaged UNG2 and that viral infectivity required the presence of UNG2 in the HIV-1 viral particle. We next analyzed the infectivity of HIV-2 ROD, a closely related primate lentivirus produced from UNG2-depleted cells. Because HIV-2 does not have the power to package host UNG2 (Priet et al., 2003a), we expected that its replication would not be affected by the depletion of UNG2. HIV-2 virus produced in the cell-free supernatant of UNG2-depleted MAGI-CCR5 cells was harvested and used to challenge C8166-CCR5 cells (Figure 2D, right). HIV-2 virus produced from UNG2-depleted cells still retained its infectivity, indicating that the transfection of UNG2-specific sirna did not induce obvious defects in cells that might preclude lentiviral replication. Consistently, transfection of a wild-type UNG2-expressing vector engineered to escape the silencing of sirna into UNG2-depleted MAGI-CCR5 producer cells restored HIV-1 infectivity (Figure 2E). Then we investigated whether UNG2 is packaged via an integrase-dependent pathway, as we have demonstrated previously (Priet et al., 2003a, 2003b; Willetts et al., 1999), or whether UNG2 is packaged via its binding to Vpr as described by Mansky et al. (2000). To discriminate between these possibilities, we overexpressed sirna- insensitive UNG2 containing the mutations W231A/ F234G, which impair the association of UNG2 with Vpr, in UNG2-depleted MAGI-CCR5 producer cells (Bou- Hamdan et al., 1998) (Figure 2E). The full restoration of viral infectivity indicates that UNG2 was not packaged via a Vpr-dependent pathway. We next investigated Figure 2. Virus Produced from Cells Depleted of UNG2 by sirna Treatment Is Noninfectious (A) Depletion of UNG2 in MAGI-CCR5 cells. Total RNA or cell lysate was prepared 1 day posttransfection with sirna. Left: UNG2 and UNG1 transcripts were measured by quantitative real-time RT-PCR. Right: the expression of UNG2 and UNG1 proteins in cells was detected on immunoblots probed with anti-ung antibody. Error bars represent standard deviations of three quantifications. (B) UNG2-depleted cells support viral replication. Wild-type HIV-1 AD8 viral stock was used to infect sirna-treated MAGI-CCR5 target cells for a single round of replication. Infectivity was estimated from Tatinduced -gal expression. (C) UNG2-deficient HIV-1 virus is noninfectious. Left: Western blot analysis of virions produced from sirna-treated producer cells by using anti-ung antibody. Right: HIV-1 viral stocks produced from sirna-treated cells were used to infect UNG2-positive MAGI-CCR5 cells. Viral infectivity was estimated as in (B). (D) Depletion of UNG2 in producer cells affects HIV-1, but not HIV-2, replication. HIV-1 (left) or HIV-2 (right) viral stocks produced from sirna-treated cells were used to challenge C8166R5 target cells. Viral infectivity was estimated as in (B). The infectivity of wild-type HIV-2 virus was given the arbitrary score of 100%. (E) Restoration of HIV-1 infectivity. The infectivity of virus produced from UNG2-depleted cells overexpressing either sirna-insensitive wild-type UNG2 or sirna-insensitive Q152L/D154E UNG2, or sirna-insensitive W231A/F234G UNG2 was monitored for -gal ex- pression. Below is a Western blot analysis of extracts from treated producer cells using anti-ung antibody. Error bars represent standard deviations of three quantifications.

4 Molecular Cell 482 whether the ability of UNG2 to remove uracil was required for viral replication. The overexpression of a sirna-insensitive UNG2 containing two inactivating mutations (Q152L/D154E) (Mol et al., 1995) in UNG2- depleted MAGI-CCR5 producer cells failed to restore viral infectivity. The results support the conclusion that the capability of UNG2 to remove uracil is required for viral replication. The Inhibition of UNG2 Activity in Producer Cells Generates Noninfectious Virus To substantiate our results, we explored the infectivity of virions produced from cells in which the UNG2 enzymatic activity was inhibited by the uracil DNA glycosylase inhibitor (Ugi) protein encoded by the uracil-containing genome of bacteriophage PBS-2 (Wang and Mosbaugh, 1989). We bacterially expressed and purified Ugi as a fusion protein with the basic HIV-1 Tat-derived peptide (Nagahara et al., 1998) to obtain a TAT-Ugi recombinant protein that is able to penetrate cells and to inhibit cellular UNG2 when added directly to the cell culture media (Studebaker et al., 2004). 293T cells were transduced with varying concentrations of TAT-Ugi and then transfected with HIV-1 or HIV-2 molecular clones. Three days posttransfection, the inhibition of UNG2 activity was revealed by enzymatic assays (Figure 3A). The residual uracil DNA glycosylase activity observed was probably due to a back-up activity for the excision of uracil from DNA that is insensitive to Ugi as previously reported (Kavli et al., 2002). HIV-1 or HIV-2 virions released in the cell-free supernatant were used to challenge MAGI-CCR5 cells (Figure 3B). The inhibition of UNG2 activity correlated with the loss of HIV-1 infectivity. As expected, HIV-2 infectivity was unaffected. The Loss of Propagation of UNG2-Deficient Virus Figure 3. Ugi-Mediated UNG2 Inhibition in HIV-1 Producer Cells Results from the Greatly Reduced Accumulation Leads to Noninfectious Virus of Newly Synthesized Reverse Transcripts (A) Uracil excision activity in TAT-Ugi-transduced cells. TAT-Ugi recombinant protein was added in the culture medium at the indi- It has been reported that the presence of uracil in viral cated concentration, and cell extracts were prepared 3 days after reverse transcripts synthesized in cells infected with transduction. The uracil excision activity was monitored in serial 10- APOBEC3G-positive Vif virus led to their degradation fold dilutions of either untransduced cell extracts with or without (Mangeat et al., 2003; Mariani et al., 2003). We therefore 1 U of commercial Ugi added in the assays (top) or cell extracts investigated the fate of viral reverse transcripts synthetom) from cells transduced with varying concentrations of TAT-Ugi (bot- sized in cells infected with UNG2-deficient virus. SupT1 using a double-stranded oligonucleotide containing a unique uracil residue. target cells were infected with similar amounts of HIV-1 (B) HIV-1 virus produced from TAT-Ugi-transduced cells is noninfecvirus produced from either UNG2-depleted or UNG2- tious. HIV-1 and HIV-2 viral stocks produced from TAT-Ugi-transpositive cells, and neosynthesized reverse transcripts duced cells were used to infect MAGI-CCR5 cells. Viral infectivity were measured at different times postinfection using was estimated from -gal expression. Error bars represent standard quantitative real-time PCR with specific primers (Figure deviations of three quantifications. 4A). The infection of cells with UNG2-deficient virus generated barely detectable early and late reverse tranthose scripts compared to cells infected with UNG2-positive reporting that transcripts generated by APOscripts virus, indicating either a failure to complete reverse tranin BEC3G-positive Vif virus are uracilated and degraded scription or a degradation of transcripts. These results infected cells (Harris et al., 2003; Lecossier et al., were confirmed by using an sirna-independent apal., 2003; Mangeat et al., 2003; Mariani et al., 2003; Yu et proach to obtain UNG2-deficient virus; i.e., analyzing 2004; Zhang et al., 2003). We conclude that the loss reverse transcripts generated by HIV-1 virus bearing the of accumulation of reverse transcripts generated by mutation L172A in the integrase domain that impairs the UNG2-deficient virus was probably induced by the pres- packaging of UNG2 (Priet et al., 2003b) (Figure S2). Early ence of uracil in proviral DNA. reverse transcripts synthesized within UNG2-deficient viral particles were detected at wild-type levels (Figure Uracilation of Viral DNA Is Due 4B), indicating that there is no defect in the initiation to dutp Misincorporation of the intravirion reverse transcription process in the Uracil opposite to adenine gives rise to nonmutagenic absence of UNG2. The results are in agreement with A:U mismatch, whereas uracil opposite to guanine gives

5 UNG2-Dependent Repair of dutp in HIV-1 DNA 483 Figure 4. Fate and Sequence of Transcripts Generated upon Infection of Cells with UNG2-Deficient Virus Produced by sirna-treated Cells (A) Quantification of newly synthesized reverse transcripts in infected cells. Normalized amounts of UNG2-proficient and UNG2-deficient viral stocks were used to infect SupT1 cells. At the specified times postinfection, total DNA was purified. Newly synthesized cdna was measured by quantitative PCR with specific primers for early and late reverse transcripts. (B) Quantification of newly synthesized reverse transcripts in viral particles. Normalized amounts of wild-type and UNG2-deficient virus obtained from sirna-treated producer cells were lysed, and intravirion quantification of early reverse transcripts was measured by quantitative PCR with specific primers. Error bars represent standard deviations of three quantifications. (C) Sequences of newly synthesized cdna. SupT1 cells were infected with wild-type or UNG2-deficient HIV-1 virus. Cellular DNA was prepared 4 hr postinfection, and the env region was amplified, cloned, and sequenced. The parental sequence is taken as reference. (D) Incorporation of dutp by HIV-1 RT. The 32 P-labeled primer-template substrate was incubated with HIV-1 RT in the presence of either the four dntps or a mixture of datp, dgtp, dctp, and dutp or a mixture of datp, dgtp, dutp, and dttp. DNA products extended by HIV-1 RT were analyzed by using a DNA sequencing gel and visualized by autoradiography. rise to mutagenic G:U mismatch that leads to a G to A misincorporation of dutp opposite to dgmp in the minus- mutation. Usually, G to A mutations are the hallmark of as well as the plus-strand DNA and suggests that uracil present in the minus-strand of HIV-1 DNA. These RT incorporates dutp only opposite to damp. To confirm uracil residues arise from cytosine deamination (Harris this assumption, we incubated a 5 -labeled primeruracil et al., 2003; Lecossier et al., 2003; Mangeat et al., 2003; template DNA substrate with purified HIV-1 RT in the Mariani et al., 2003; Yu et al., 2004; Zhang et al., 2003) but presence of either a mixture of dntps in which dttp may appear upon misincorporation of dutp opposite to was replaced by dutp to test the ability of HIV-1 RT to guanine. Uracil in place of cytosine in the plus-strand incorporate U opposite to A or a mixture of dntps in DNA would lead to C to T mutations. To investigate which dctp was replaced by dutp to test the ability of whether viral transcripts synthesized in the absence of HIV-1 RT to incorporate U opposite to G (Figure 4D). In UNG2 contained mutations, SupT1 cells were infected the presence of a mix in which dttp was replaced by with UNG2-deficient virus, and 4 hr postinfection DNA dutp, complete extension of the primer was observed was recovered, amplified with env specific primers, by using a gel assay, confirming that HIV-1 RT efficiently cloned, and sequenced (Figure 4C). As a control, we incorporated dutp opposite to damp. In contrast, in determined sequences of transcripts from cells infected the presence of a mix in which dctp was replaced by with APOBEC3G-positive Vif, APOBEC3G-negative dutp, polymerization products were shortened due to Vif, W54R Vpr, or L172A integrase virus (Figure S3). the failure to incorporate dutp opposite to dgmp. The W54R Vpr mutation has been reported to block the These results are in agreement with those reported by association of Vpr with UNG2 and to enhance the G to Martinez et al. (1995) and explain why, in the context of A mutation frequency (Mansky et al., 2000). Sequences in vivo infection, UNG2-deficient virus yielded nonmuof a 102 bp region that included the env gene of about tated viral transcripts. ten clones from each virus were determined. With the To confirm that the loss of infectivity of UNG2-defi- exception of sequences derived from APOBEC3G-posi- cient virus was induced by dutp misincorporation during tive Vif virus that showed excessive G to A mutations, the reverse transcription process, we targeted a there was no evidence in the accumulation of G to A or C heterologous dutpase protein into UNG2-deficient particles to T mutations among the classes of virus or a significant in order to hydrolyze dutp, and we measured the difference in the virus mutation frequency. The failure infectivity of the resulting virus. We constructed to detect G to A or C to T mutations indicates that uracil a plasmid expressing the caprine lentivirus (CAEV) did not arise from cytosine deamination or from the dutpase protein fused to the C-terminal part of the

6 Molecular Cell 484 Figure 5. Restoration of Viral Infectivity of UNG2-Deficient Virus by Transfection of dutpase-expressing Vector in UNG2-Depleted Producer Cells (A) Expression of Vpr-dUTPase fusion protein in viral particles. MAGI-CCR5 producer cells were transfected with a plasmid expressing either the Vpr protein alone or a fusion protein consisting of Vpr, the protease cleavage site, and wild-type dutpase or a fusion protein with the G109E mutation in dutpase that impairs catalytic activity. Virion progeny was harvested, and viral lysate was analyzed by Western blot with anti-hiv-1 Vpr or anti-caev dutpase antibody. (B) dutpase expression in UNG2-deficient HIV-1 particles restores infectivity. MAGI-CCR5 cells were depleted of UNG2 by sirna treatment and cotransfected with each Vpr expressing vector and proviral DNA plasmid. Virion progeny was used to infect MAGI-CCR5 cells, and viral infectivity was scored from -gal expression. The molar ratios of transfected Vpr-expressing vector and proviral DNA plasmid were 0.125:1, 0.5:1, and 1:1. Error bars represent standard deviations of three quantifications. (C) UNG2-deficient virus generates uracilated transcripts. Normalized amounts of UNG2-proficient and UNG2-deficient viral stocks either obtained from sirna-treated producer cells (sirna-dependent) or from transfection with a L172A integrase mutant molecular clone (sirnaindependent) were used to infect SupT1 cells. Four hours postinfection, total DNA was purified, and early reverse transcripts were amplified with Taq or Pfu polymerases. HIV-1 Vpr protein to allow its targeting into HIV-1 viral This demonstrates that the loss of infectivity was induced particles (Wu et al., 1995). The fusion protein contains by uracil arising from dutp misincorporation. To the Vpr protein separated from the dutpase protein by confirm that reverse transcripts contained uracil, we the HIV-1 protease cleavage site (Figure 5A). As a control, used the differential property of Taq or Pfu polymerases we constructed a plasmid expressing the dutpase to copy templates bearing uracil residues. Pfu polymer- protein containing the mutation G109E (Turelli et al., ase is unable to readily amplify a uracil-containing tem- 1997), which impairs the catalytic activity. Western blot plate in contrast to Taq polymerase (Suspene et al., analysis confirmed the efficient packaging and cleavage 2004). We amplified, with either Taq or Pfu polymerases, of the Vpr-dUTPase fusion protein by protease (Figure early transcripts synthesized in target cells by wild-type 5A). To explore whether dutpase expression has the virus or sirna-dependent or -independent (the L172A ability to restore viral infectivity of UNG2-deficient viral integrase mutant) UNG2-deficient virus (Figure 5C). particles, UNG2-depleted MAGI-CCR5 producer cells Each of the transcripts was efficiently amplified by Taq were transfected with Vpr-dUTPase expressing vector, polymerase, but only transcripts from wild-type virus and virion progeny was used to challenge untreated were efficiently amplified by Pfu polymerase, demon- MAGI-CCR5 target cells (Figure 5B). The overexpression strating the presence of uracil in viral DNA when virions of wild-type, but not that of the catalytically inactive were depleted from UNG2. G109E dutpase fusion protein, restored viral infectivity in a dose-dependent manner. We also demonstrated HIV-1 RT and UNG2 Recombinant Proteins Can that viral infectivity could be restored by Vpr-dUTPase Process Uracil from Primer-Template Substrate expression in TAT-Ugi-treated producer cells (Figure We next investigated the molecular mechanism by S4). These results show that either the absence of virionassociated which misincorporated dutp in viral DNA was repaired UNG2 or the loss of UNG2 enzymatic activity during the reverse transcription process. Our previous could be compensated for by the presence of virionassociated data showing the direct association of UNG2 with HIV-1 dutpase that acts in hydrolyzing free dutp. RT (Priet et al., 2003b) suggested that UNG2 may act in

7 UNG2-Dependent Repair of dutp in HIV-1 DNA 485 Figure 6. In Vitro Reconstitution of HIV-1-Associated Uracil Repair (A) HIV-1 RT and UNG2 recombinant proteins can repair uracil residues from a uracil-containing DNA primer-dna template substrate. The topmost portion of 6A is the sequence of the DNA/DNA substrate used. The asterisk indicates the 32 P-labeled strand, the box indicates the sequence of the SalI restriction site, and the vertical arrow indicates the SalI cleavage site. The substrate was incubated with recombinant HIV-1 RT and UNG2 in the presence of the four dntps and subjected to SalI digestion. Reaction products were resolved on DNA sequencing gels and visualized by autoradiography. Horizontal arrows indicate the position of the SalI digestion product. and / elimination products are side products of the UNG2 activity (see Figure S2). (B) Depiction of the flow diagram for expected DNA products upon incubation of the tetrahydrofuran (F)-containing DNA substrate with HIV-1 RT followed by SalI digestion. (C) AP-endonuclease activity of HIV-1 RT. The F substrate was incubated with HIV-1 RT and/or APE1 endonuclease in the presence of a mixture containing datp, dgtp, dctp, and ddttp to visualize the de novo primer extension by HIV-1 RT. Where indicated, recovered DNA products were subjected to SalI digestion. Horizontal arrow indicates the position of the SalI product. Note that one-fifth of the reaction assay with APE1 alone was loaded. (D) The RNase H domain of RT is not involved in nicking activity. The F substrate was incubated with either wild-type HIV-1 RT (p66/p51), HIV-1 RT deleted from the RNase H domain (p51/p51), p66/p51 containing a point mutation in the RNase H domain (E478Q), or with the RNase H domain alone in the presence of a mixture containing datp, dgtp, dctp, and ddttp. (E) Repair of abasic site by HIV-1 viral lysate. The F substrate was incubated with highly purified HIV-1 virions (1 g of CAp24 antigen) in the presence of each of the four dntps and subjected to SalI digestion or in the presence of a mixture consisting of datp, dgtp, dctp, and ddttp. Horizontal arrow indicates the position of the SalI product. resistant to hydrolysis by SalI (Wiebauer and Jiricny, 1989). The SalI-cleaved product separated from poly- merized substrate in a gel assay is a measure of repair activity. When the uracil-containing DNA primer/dna template substrate was incubated with both recombinant UNG2 and HIV-1 RT in the presence of the four dntps, it became susceptible to cleavage with SalI, as evidenced by the appearance of the 27-mer DNA product resulting from specific in vitro correction of the G:U mispair to a G:C pair (Figure 6A). The SalI-digest product was not detected when the substrate was incubated with HIV-1 RT alone. Although UNG2-induced abasic sites are chemically unstable and cleave spontaneously on the 3 side via a -elimination reaction, the extension concert with RT to repair misincorporated dutp. To understand the role of the UNG2-RT complex in uracil repair, we used a cell-free system consisting of synthetic uracil-containing primer-template substrates (Figure 6). The substrates are thought to mimic the intermediate DNA products generated during the sequential steps of the reverse transcription process (Telenitsky and Goff, 1997). In our assay, the substrate comprised a uracilcontaining DNA primer annealed with complementary RNA or DNA templates that mimic duplexes recovered during minus- or plus-strand DNA synthesis, respectively. In the duplex, the uracil residue was mismatched with a guanine residue situated within the restriction endonuclease SalI recognition sequence, rendering it

8 Molecular Cell 486 of the resulting 3 -OH end generates a DNA product still inhibitor Ugi, or by using virions bearing the L172A integrase containing the abasic site (Strauss et al., 1997). Our mutation, we have presented evidence that the results showing that UNG2-induced abasic sites were absence of virion-associated UNG2 activity triggers not a substrate for SalI digestion indicate that uracil abortive HIV-1 replication, whatever the target cell types repair did not involve a -elimination reaction. The uracil challenged. Viral spread of UNG2-deficient virus can repair process was also detected when HIV-1 RT and be rescued by the overexpression of the dutpase, an UNG2 were incubated with the uracil-containing DNA enzyme that hydrolyzes dutp to dump and PPi, in primer/rna template heteroduplex (Figure S5), indicating UNG2-depleted producer cells. This demonstrates that that HIV-1 RT and UNG2 have the ability to repair the loss of infectivity was induced by dutp misincorpo- uracils either in DNA/DNA or DNA/RNA duplexes. ration in nascent reverse transcripts. During the reverse To understand how HIV-1 RT can process UNG2- transcription process, dutp misincorporation is a likely induced abasic sites, we used a 35-mer double-stranded event, due to the fact that the approximate size of the oligonucleotide containing a tetrahydrofuranyl (F) resi- dutp pool in macrophages and lymphocytes was estimated due that mimics an abasic site (Masuda et al., 1998) to be 30% of that of dttp (Aquaro et al., 2002; located within the SalI sequence. The F substrate is Cross et al., 1993; Horowitz et al., 1997). dutp misincorporation resistant to spontaneous cleavage arising via the is not mutagenic, but uracil-containing reverse -elimination reaction (Takeshita et al., 1987). We pre- transcripts failed to accumulate in newly infected target dicted that HIV-1 RT would have to cleave 5 to the cells. This phenotype is quite different than that ob- baseless nucleotide to generate a new 3 -hydroxyl (3 - served upon infection by APOBEC3G-positive Vif virus OH) end that would serve to prime further polymerization that results in frequent inactivating G to A mutations, in in a similar manner to that of APE1, the major cellular addition to the decreased abundance of reverse transcripts. AP-endonuclease that cleaves 5 to an abasic site (Strauss et al., 1997). The F substrate was incubated On the basis of our data, we propose a model for with RT or APE1 in the presence of ddttp and the three virion-associated UNG2 function in HIV-1 replication other dntps. We used ddttp as a dttp analog lacking (Figure 7). In cells infected with HIV-1, host-derived a3 -OH end to visualize the de novo DNA extension by UNG2 is packaged into virions during virus assembly RT. The expected intermediate DNA species are de- via a specific association with the integrase domain of picted in Figure 6B. Incubation of substrate with RT the Gag-Pol precursor. dutps are misincorporated into alone induced a 24-mer DNA product that comigrated viral DNA during the reverse transcription process, but with that obtained with RT and APE1, suggesting that the uracil base is recognized and excised by virion- RT exhibited a previously uncharacterized intrinsic AP- associated UNG2. The resulting baseless nucleotide is endonuclease activity (Figure 6C). The abasic site repair then eliminated by the dntp-dependent, RT-mediated process was confirmed by the appearance of the 15- endonucleolytic cleavage that creates a new 3 -OH end mer SalI digest product. However, the RT-associated used to prime further polymerization by strand-displace- AP-endonuclease activity was distinct from the APE1 ment polymerization activity (Hottiger et al., 1994). When activity because of the strict requirement for dntps. UNG2-deficient virus infects a new target cell, dump is Because cleavage by cellular AP-endonucleases does fixed in the minus- as well as the plus-strand DNA, and not require dntps, contamination of RT with bacterial uracil-containing reverse transcript levels are greatly re- AP-endonucleases are unlikely. The nicking activity is duced. Our model suggests a positive role of encapsi- Mg 2 -dependent as shown by its sensitivity to EDTA. dated UNG2 for HIV-1 replication in avoiding uracil mis- Such an AP-endonuclease activity was not observed for incorporation in viral DNA but does not exclude a cellular DNA polymerases, such as DNA polymerase, negative role of cellular UNG2 in the destabilization of although it displayed a deoxyribose phosphate lyase uracilated reverse transcripts once generated in target activity in addition to its conventional polymerase activ- cells. It has been postulated that the presence of uracil ity (Prasad et al., 1998). We also analyzed the nicking in viral DNA could recruit components of the cellular BER activity of either a p51/p51 RT homodimer that lacks pathway, such as uracil DNA glycosylases and apurinic the RNase H domain or a p66/p51 RT heterodimer with endonucleases, which could destroy uracilated viral the E478Q mutation (Julias et al., 2001) that impairs DNA by introducing nicks into the phosphodiester back- RNase H enzymatic activity, or the RNase H domain bone (Cullen, 2003; Gu and Sundquist, 2003). However, alone (Figure 6D). Results demonstrated that the RNase to date, no data directly support this assumption. H domain is not involved in the nicking activity of RT. It is noteworthy that HIV-2 viral spread is not influ- Finally, we confirmed that the RT-associated AP-endo- enced by the depletion of UNG2 or the inhibition of nuclease activity was efficient in virally associated RT UNG2 activity. It is possible that HIV-2 has developed by using highly purified HIV-1 virions (Figure 6E). The an alternative strategy to counteract the accumulation processing of uracil by RT and UNG2 differs from the of uracil in viral DNA by recruiting another member of main mammalian uracil BER pathway (Hoeijmakers, the cellular uracil DNA glycosylase enzyme family or 2001), as AP-lyase and ligase activities are not required by recruiting cellular dutpase. Alternatively, HIV-2 RT because of the strand displacement synthesis property could discriminate dutp from dttp. of RT (Hottiger et al., 1994). dutp is misincorporated during reverse transcription because HIV-1 RT does not discriminate dutp from Discussion dttp (Klarmann et al., 2003) but is efficiently repaired by the action of virion-associated UNG2 and RT. But By using virions produced by cells either depleted of why were uracils arising from APOBEC3G-dependent UNG2 protein by sirna treatment or treated with UNG2 cytosine deamination not processed? The main expla-

9 UNG2-Dependent Repair of dutp in HIV-1 DNA 487 Figure 7. Proposed Model for HIV-1-Associated UNG2 Function In infected cells, the assembling virion encapsidates the host-derived UNG2 enzyme that prevents uracilation of viral genome induced by dutp misincorporation. UNG2 catalyses the excision of uracil at the time when dutp is misincorporated by HIV-1 RT, leading to an abasic site. The apurinic endonuclease activity of RT induces a nick 5 to the abasic site excluding the baseless nucleotide and proceeds further polymerization through its strand displacement synthesis property. The asterisk indicates the baseless nucleotide. Virus released from UNG2- depleted cells is able to infect a new target cell but yields transcripts containing uracil residues that compromise the completion of reverse transcription, thereby impairing viral spread. Uracil in viral transcripts is not mutagenic because RT incorporates dutp only opposite to damp and not to dgmp. nation is that the uracil repair mechanism induced by course of infection. Moreover, Chen et al. (2004) claimed UNG2 and HIV-1 RT is efficient only during the polymerization that G to A mutations observed in Vpr or W54R Vpr process, as it requires viral RNA or DNA strands transcripts were induced by uracil mismatched with as a template. In the case of cytosine deamination, guanine and showed that these viruses were able to which occurs in single-stranded minus DNA (Yu et al., propagate despite the presence of uracil. A number of 2004), uracil repair is inefficient because of the lack of studies have demonstrated that uracil in DNA inhibits viral RNA template that has been hydrolyzed by the viral spread (Harris et al., 2003; Lecossier et al., 2003; RNase H activity of RT. Although not repaired, some Mangeat et al., 2003; Mariani et al., 2003; Yu et al., 2004; uracils provided by cytosine deamination might never- Zhang et al., 2003). One explanation for this discrepancy theless be recognized and excised by virion-associated might be that, indeed, the viral genomic DNA generated UNG2, leading to viral single-stranded cdna with an from Vpr or Vpr-mutated virus has not been subjected abasic site. In this case, abasic site-containing cdna to uracilation. On the basis of our results and of those can be used as a template for plus-strand DNA synthesis from others (Harris et al., 2003; Lecossier et al., 2003; that will lead to G to A hypermutations because HIV-1 Mangeat et al., 2003; Mariani et al., 2003; Yu et al., 2004; RT preferentially incorporates datp opposite to an aba- Zhang et al., 2003), it seems probable that Vpr does not sic site (Cai et al., 1993). play a role in the control of uracils. Intriguingly, our findings differ from those reported by To conclude, HIV-1 has evolved by elaborating two Mansky et al. (2000) and Chen et al. (2004), who found distinct pathways to protect its genome from uracilation: that Vpr modulates the virus mutation rate via its ability one involving the virally encoded Vif protein to exclude to package UNG2 into HIV-1 viral particles. In these APOBEC3G from the budding virion and the other involv- studies, the authors showed that genome from Vpr ing at least three partners, including UNG2 to process virus or a virus expressing the W54R Vpr mutation displayed uracil residues, virally encoded integrase to package increased G to A mutations compared to wild- UNG2 into the budding virion, and virally encoded RT type virus. These data contrast with those reported by to process UNG2-induced abasic sites. This essential Mangeat et al. (2003) and Mariani et al. (2003), who pathway of uracil repair represents an attractive therapeutic showed that no G to A mutations were found in viral target to control HIV-1 infection. transcripts generated in cells infected with Vpr-deficient virus. It is possible that findings from Mansky et al. (2000) Experimental Procedures and Chen et al. (2004) could be related to a bias in sirna Treatment and mrna Quantification the design of the experiments used to measure virus MAGI-CCR5 cells and macrophages were depleted of UNG2 by mutation rate. G to A mutations were detected in the transfection with Lipofectamine 2000 (Invitrogen) and doublelacz gene of an HIV-1-derived shuttle vector, but not in stranded UNG2-specific sirnas (100 nm) on days 1 and 2 before reverse transcripts synthesized during the natural transfection with HIV proviral clones or infection with HIV virus.

10 Molecular Cell 488 Quantitative real-time PCR was performed with an ABI Prism 7000 virus (50 ng of CAp24 antigen) were used for intravirion early reverse apparatus (Applied Biosystems). mrnas were extracted from untreated transcripts quantification. or sirna-treated cells using the RNeasy kit (Qiagen) and quantified by real-time RT-PCR. The sequence of UNG2-specific Sequencing of Newly Synthesized Viral cdna sirnas (Eurogentec) was as follows: sense, 5 -AUCGGCCAGAAGA Sequencing of newly synthesized reverse transcripts was performed CGCUCUdTdT-3, which corresponds to the first exon (nucleotides as previously described (Lecossier et al., 2003). Briefly, SupT1 cells ) of the UNG genomic region (GenBank accession number were infected as reported for real-time PCR assays, and cellular X89398) (Nilsen et al., 1997) that is spliced out of UNG1 mrna. The DNA was extracted 4 hr postinfection. Heat-inactivated virus was sequence of the UNG2-random sirna was as follows: sense, 5 used in a real-time PCR assay as a control to ensure that plasmid GCUGCAAGCCGAAUAGCUAdTdT-3. The sequence of the UNG2 DNA remaining from transfection represented less than 1% of reand UNG1 primers were as follows: UNG2 sense, 5 -CCTCCTCAGC verse transcripts. A 158 bp fragment of the env gene (nt ) TCCAGGATGA-3 ; UNG2 antisense, 5 -TCGCTTCCTGGCGGG-3 ; was amplified with high fidelity polymerase using the forward primer UNG2 probe, 5 -(FAM)-CGGCCAGAAGACGCTCTACTCCTTTTTC- ENV and reverse primer ENV-rev (Lecossier et al., 2003) through (TAMRA)-3 ; UNG1 sense, 5 -CGCTCCAGTTTAGAACCTAATTCC- 40 cycles (95 C, 15 min; 95 C, 15 s; 60 C, 1 min). Amplicons were 3 ; UNG1 antisense, 5 -AGGCAGAAGACGCCCATTT-3 ; UNG1 agarose gel purified, cloned into pcr4-topo plasmid (Invitrogen), probe, 5 -(FAM)-AATTCCCGGACCGGGCCCAG-(TAMRA)-3. Actin and sequenced using the flanking M13 forward primer. mrna quantification was used as an internal control (Butler et al., 2001). Purification of Recombinant Proteins and Antibodies Purification of C-terminal, His 6 -tagged HIV-1 RT p66/p51 hetero- Virus Production, Infections, and Titrations dimer was performed as previously reported (Boretto et al., 2001). Virus stocks were prepared by transfection of MAGI-CCR5 cells Purification of N-terminal, His 6 -tagged human UNG2 was performed with HIV molecular clones using FuGene 6 transfection reagent as described elsewhere (Slupphaug et al., 1995) from the His 6 - (Roche). All infections were performed using spinoculation (O Dohtagged UNG2-expressing plasmid provided by G. Slupphaug. Ugi erty et al., 2000). Peripheral blood mononuclear cells isolated from open reading frame (Wang and Mosbaugh, 1989), a gift from J. healthy donors were seeded in 25 cm 2 flasks, and 5 days later Cohen, was cloned into ptrchis2c (Invitrogen) that was modified nonadherent cells were removed. Monocyte-derived macrophages by in-frame insertion of the TAT-HA tag (Nagahara et al., 1998). The were then infected with either 1 g (Figure 1D), or 0.1and 0.01 g UGI-TAT-His fusion protein was expressed in E. coli BL21 DE3 and (Figure S1) of CAp24 antigen. Spreading of infection in macrophages purified to near homogeneity on nickel-chelating, ProBond resin was monitored twice a week by measurement of CAp24 antigen (Invitrogen) under denaturing conditions using 8 M urea as described (HIV-1 Ag EIA, Bio-Rad) in the cell-free supernatant. Levels of infecti- (Nagahara et al., 1998). Urea was flash exchanged against 1 M NaCl vity were determined by challenging MAGI-CCR5 cells or on a HiTrap Q sepharose column (Pharmacia Biotech) and eluted C8166R5 cells with 90 or 25 ng, respectively, of CAp24 as recommended by the manufacturer. Eluted protein was desalted antigen for each HIV stock. Highly purified virions were prepared in phosphate-buffered saline and flash-frozen. Anti-UNG2 antibody as previously described (Priet et al., 2003b). was obtained by immunization of rabbits with UNG2 expressing DNA vector and boosted by purified His 6 -tagged UNG2. Specific Plasmid Constructs immunoglobulins were purified by affinity chromatography through HIV-1 AD8 and HIV-2 ROD proviral clones were provided by K. Peden UNG2-coupled beads. Rabbit polyclonal anti-vpr antibody was oband P. Sonigo, respectively. HIV-1 AD8 Vif was obtained by changing tained through the AIDS Research and Reference Reagent Program. the start codon of the vif gene from ATG to ACG by a PCR strategy. Polyclonal anti-caev dutpase antibody was obtained by DNA im- This mutation does not affect the overlapping pol open reading munization of rabbits and boosted with CAEV dutpase peptide and frame. HIV-1 AD8 containing the W54R mutation was constructed by purified His-tagged protein. a PCR strategy. cdnas encoding the wild-type and the catalytically impaired CAEV dutpase (the G109E mutant) were cloned into the Cell Transduction with Recombinant TAT-Ugi and Uracil DNA plr2p-vpr expression plasmid (Wu et al., 1995), a gift from J. Kap- Glycosylase Activity in Cell Extract pes, to obtain the Vpr-dUTPase fusion proteins. Silent mutations 293T cells ( ) were transduced by addition of increasing were introduced by PCR into the UNG2-encoding cdna to allow amounts (0.5, 1, and 2 M) of recombinant TAT-Ugi into the culture the sirna-insensitive expression of the wild-type UNG2 protein. medium for 2 days before being transfected with the HIV molecular This mutated cdna was cloned into the pcdna 3.1expression plasclone. Cell lysate from TAT-Ugi transduced cells was prepared, and mid (Invitrogen). Mutations were introduced by PCR into the sirnauracil DNA glycosylase activity was measured as described (Di Noia insensitive UNG2 expression plasmid to allow the expression of a and Neuberger, 2002). catalytically impaired UNG2 (the Q152L/D154E mutant) (Mol et al., 1995) or the expression of a UNG2 protein that failed to bind Vpr (the W231A/F234G mutant) (BouHamdan et al., 1998). The plasmid dutp Polymerization by HIV-1 RT pcmv4 containing the cdna of APOBEC3G was a gift from M. Malim The 36/64-mer DNA primer/dna template substrate comprised a and was used to express the HA-tagged APOBEC3G protein. 5 -end-labeled 36-mer primer oligonucleotide annealed with a 3-fold molar excess of the unlabeled complementary 64-mer template oligonucleotide Real-Time PCR Quantification of Newly Synthesized Viral cdna (5 -ATGCGAATATTGAAGCTTAGACGTCATCGCGTGCA Reverse transcripts synthesized in infected cells or within viral partiincubated GATCTGTCGACGGAGGAGGGTGGAGTGGA-3 ). The substrate was cles were quantified as described elsewhere (Butler et al., 2001) for 30 min at 37 C with 10 nm of RT in the polymerization with slight modifications. Briefly, SupT1 cells ( cells) were buffer containing 20 mm HEPES (ph 7.8), 60 mm KCl, 10 mm MgCl 2, infected with equivalent amounts (250 ng of CAp24 antigen per mm dithiothreitol, 0.05% Triton X-100, and 100 g/ml bovine serum cells) of DNase-treated viral stocks produced from UNG2-deficient albumin (Ozyme) in the presence of each of the four dntps (100 or UNG2-positive MAGI-CCR5 cells. At indicated times, cells M) or of a mixture containing dutp in place of either dttp or dctp. were removed, DNA was extracted (DNeasy kit, Qiagen), and early (R-U5) and late (R-gag) reverse transcripts were quantified. Heat- Determination of Uracil Residues in Reverse Transcripts inactivated virus was used as a control. For quantification of early Uracil residues within reverse transcripts synthesized in infected reverse transcripts within viral particles, viral particles were concen- cells were determined as previously described (Suspene et al., trated by ultracentrifugation; resuspended in a buffer containing ). Briefly, total DNA extracted from infected cells and used to mm Tris-HCl (ph 8.3), 50 mm KCl, 2 mm MgCl 2, and 750 U of DNase quantify reverse transcripts (see above) were also amplified with I (Invitrogen); and incubated for 30 min at 37 C. Viral particles were Taq (Promega) or Pfu (Stratagene) polymerases by using primers then diluted 10-fold in TNE buffer and ultracentrifuged. Pelleted viral specific for early reverse transcripts. PCRs were performed in 100 particles were lysed in TNE buffer containing 0.2% Triton X-100, l total volume with 0.5 U of Taq or 5 U of Pfu polymerases through and CAp24 antigen content was measured. Equivalent amounts of 20 cycles (94 C, 2 min; 94 C, 10 s; 60 C, 10 s; 60 C, 1 min). PCR