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• W2024302505 abstract "Cellular cytidine deaminases APOBEC3 family is a group of potent inhibitors for many exogenous and endogenous retroviruses. It has been demonstrated that they induce G to A hypermutations in the nascent retroviral DNA, resulting from the cytosine (C) to uracil (U) conversions in minus-stranded viral DNA. In this report, we have demonstrated that the result of C to U conversion in minus-stranded DNA of human immunodeficiency virus type 1 (HIV-1) could trigger a degradation of nascent viral DNA mediated by uracil DNA glycosylases-2 (UNG2) and apurinic/apyrimidinic endonuclease (APE). Since antiviral activity of APOBEC3G is partially affected by UNG2 inhibitor Ugi or UNG2-specific short-interfering RNA in virus-producing cells but not target cells, the virion-associated UNG2 most likely mediates this process. Interestingly, as APE-specific short-interfering RNA can also partially inhibit the anti-HIV-1 activity of APOBEC3G in virus-producing cells but not in target cells and APE molecules can be detected within HIV-1 virions, it seems that the required APE is also virion-associated. Furthermore, the in vitro cleavage experiment using uracil-containing single-stranded DNA as a template has demonstrated that the uracil-excising catalytic activity of virion-associated UNG2 can remove dU from the uracil-containing viral DNA and leave an abasic site, which could be further cleaved by virion-associated APE. Based upon our observations, we propose that the degradation of APOBEC3G-edited viral DNA mediated by virion-associated UNG2 and APE during or after reverse transcription could be partially responsible for the potent anti-HIV-1 effect by APOBEC3G in the absence of vif. Cellular cytidine deaminases APOBEC3 family is a group of potent inhibitors for many exogenous and endogenous retroviruses. It has been demonstrated that they induce G to A hypermutations in the nascent retroviral DNA, resulting from the cytosine (C) to uracil (U) conversions in minus-stranded viral DNA. In this report, we have demonstrated that the result of C to U conversion in minus-stranded DNA of human immunodeficiency virus type 1 (HIV-1) could trigger a degradation of nascent viral DNA mediated by uracil DNA glycosylases-2 (UNG2) and apurinic/apyrimidinic endonuclease (APE). Since antiviral activity of APOBEC3G is partially affected by UNG2 inhibitor Ugi or UNG2-specific short-interfering RNA in virus-producing cells but not target cells, the virion-associated UNG2 most likely mediates this process. Interestingly, as APE-specific short-interfering RNA can also partially inhibit the anti-HIV-1 activity of APOBEC3G in virus-producing cells but not in target cells and APE molecules can be detected within HIV-1 virions, it seems that the required APE is also virion-associated. Furthermore, the in vitro cleavage experiment using uracil-containing single-stranded DNA as a template has demonstrated that the uracil-excising catalytic activity of virion-associated UNG2 can remove dU from the uracil-containing viral DNA and leave an abasic site, which could be further cleaved by virion-associated APE. Based upon our observations, we propose that the degradation of APOBEC3G-edited viral DNA mediated by virion-associated UNG2 and APE during or after reverse transcription could be partially responsible for the potent anti-HIV-1 effect by APOBEC3G in the absence of vif. APOBEC3 is a family of cytidine deaminases that has been identified as the host factor to restrict various retroviruses, endogenous retroviruses, and long interspersed nucleotide element (LINE) elements (1Sheehy A.M. Gaddis N.C. Choi J.D. Malim M.H. Nature. 2002; 418: 646-650Crossref PubMed Scopus (1922) Google Scholar, 2Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (921) Google Scholar, 3Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1252) Google Scholar, 4Jarmuz A. Chester A. Bayliss J. Gisbourne J. Dunham I. Scott J. Navaratnam N. Genomics. 2002; 79: 285-296Crossref PubMed Scopus (593) Google Scholar, 5Bishop K.N. Holmes R.K. Sheehy A.M. Davidson N.O. Cho S.J. Malim M.H. Curr. Biol. 2004; 14: 1392-1396Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 6Doehle B.P. Schafer A. Cullen B.R. Virology. 2005; 80: 12102-12108Crossref Scopus (27) Google Scholar, 7Noguchi C. Ishino H. Tsuge M. Fujimoto Y. Imamura M. Takahashi S. Chayama K. Hepatology. 2005; 41: 626-633Crossref PubMed Scopus (130) Google Scholar, 8Yu Q. Chen D. Konig R. Mariani R. Unutmaz D. Landau N.R. J. Biol. Chem. 2004; 279: 53379-53386Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 9Zheng Y.H. Irwin D. Kurosu T. Tokunaga K. Sata T. Peterlin B.M. J. Virol. 2004; 78: 6073-6076Crossref PubMed Scopus (393) Google Scholar, 10Esnault C. Heidmann O. Delebecque F. Dewannieux M. Ribet D. Hance A.J. Heidmann T. Schwartz O. Nature. 2005; 433: 430-433Crossref PubMed Scopus (281) Google Scholar, 11Bogerd H.P. Wiegand H.L. Doehle B.P. Lueders K.K. Cullen B.R. Nucleic Acids Res. 2006; 34: 89-95Crossref PubMed Scopus (223) Google Scholar). It is closely related to APOBEC1, a cytidine deaminase that causes a specific cytosine to uracil change in the apolipoprotein B mRNA, and to an activation-induced deaminase (AID) enzyme that causes hypermutation of immunoglobulin genes (12Navaratnam N. Sarwar R. Int. J. Hematol. 2006; 83: 195-200Crossref PubMed Scopus (75) Google Scholar). The similarities of the catalytic domains in these proteins strongly suggest that APOBEC3 edits the nucleic acids of various retrotransposons. Among these cytidine deaminases, APOBEC3G is the most extensively studied enzyme. It can be efficiently incorporated into human immunodeficiency virus type 1 (HIV-1) 4The abbreviations used are: HIV-1, human immunodeficiency virus type 1; UNG2, uracil DNA glycosylases-2; AP, apurinic/apyrimidinic; APE, AP endonuclease; siRNA, short-interfering RNA; RT, reverse transcription; GFP, green fluorescent protein; CAT, chloramphenicol acetyltransferase. particles and causes extensive cytosine to uracil conversion in the viral minus-stranded DNA during reverse transcription (2Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (921) Google Scholar, 3Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1252) Google Scholar, 13Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (510) Google Scholar). The significant C to U conversion in minus-stranded DNA is apparently correlated with the decreased viral infectivity. Two consequences could occur after the C to U conversion. First, uracil in minus-stranded DNA could be excised by recruited uracil DNA glycosylase-2 (UNG2), a host DNA repair enzyme. The resulting abasic site could be further cleaved by apurinic/apyrimidinic endonucleases (APE). The vif-defective viruses generated from the restrictive cells are unable to effectively process reverse transcription, or the completed reverse transcripts are readily subjected to degradation in the target cells, which is consistent with this hypothesis (3Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1252) Google Scholar, 14von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar, 15Sova P. Volsky D.J. J. Virol. 1993; 67: 6322-6326Crossref PubMed Google Scholar, 16Dornadula G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar, 17Simon J.H. Malim M.H. J. Virol. 1996; 70: 5297-5305Crossref PubMed Google Scholar). Second, C to U conversion in minus-stranded DNA could lead to G to A hypermutation in the plus-stranded DNA. These double-stranded DNA harboring G to A hypermutation would encode viral proteins containing aberrant premature stop codons or mutated proteins and would lead to accumulated genomic damage in viral replication (13Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (510) Google Scholar). In this report, we have designed a series of experiments to test the first hypothesis. We have shown that in the presence of inhibitors of cellular UNG2 or APE, respectively, the antiviral activity of APOBEC3G can be decreased. Our findings suggest that DNA repair enzymes encapsidated within the virions directly participate in the degradation of uracilated-viral DNA induced by APOBEC3G and that this degradation process is correlated to the antiviral activity of APOBEC3G. Plasmid Construction—An infectious HIV-1 clone with a deletion in the vif region (234-bp deletion), pNL4-3Δvif, and APOBEC3G expressing vectors pcDNA3-APOBEC3G and pSLX-GFP were constructed previously (2Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (921) Google Scholar). The DNA of Ugi was PCR-amplified from pZWtac1 with a 5′-primer containing a MluI site and a 3′-primer containing a XhoI site and a c-Myc tag encoding EQKLISEEDL (18Wang Z.G. Smith D.G. Mosbaugh D.W. Gene (Amst.). 1991; 99: 31-37Crossref PubMed Scopus (30) Google Scholar). The PCR product (∼260 bp) was digested with MluI and XhoI. The resulting DNA fragment was inserted into the pSLX vector to generate pSLX-Ugi-Myc. The APE DNA was amplified by RT-PCR from mRNA extracted from 293T cells with primers containing EcoRI or XhoI sites. The 3′-primer also contains a c-Myc tag. The RT-PCR product was then digested with EcoRI and XhoI followed by insertion into the pSLV vector to generate pSLX-APE-Myc. RNA Interference—The chemically synthesized siRNAs used in the experiments were purchased from Dharmacom. The UNG2-specific siRNA and APE-specific siRNA were siGENOME SMARTpool. The luciferase-specific siRNA or GFP-specific siRNA served as negative controls. Conversely, the siLentGene U6 cassette RNA interference system (Promega) was used to generate U6 promoter-controlled APE-specific siRNAs. The APE siRNA pool was designed by the Dharmacon siDESIGN program. Based upon the protocol, various oligonucleotides were designed as the downstream primers: oligonucleotide 1A (for antisense RNA), 5′-CAA AAA CTG TAA AAA GCC TAT GCC GTA AGA AAC CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 1S (for sense RNA), 5′-CAA AAA CTG TAA AAA GGT TTC TTA CGG CAT AGG CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 2A, 5′-CAA AAA CTG TAA AAA GTT ACC AGC ACA AAC GAG CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 2S, 5′-CAA AAA CTG TAA AAA GCT CGT TTG TGC TGG TAA CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 3A, 5′-CAA AAA CTG TAA AAA GCA TTA GGT ACA TAT GCT CGG TGT TTC GTC CTT TCC ACA AGA-3′, oligonucleotide 3S, 5′-CAA AAA CTG TAA AAA GAG CAT ATG TAC CTA ATG CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 4A, 5′-CAA AAA CTG TAA AAA GGA ACT TGC GAA AGG CTT CGG TGT TTC GTC CTT TCC ACA AGA-3′; oligonucleotide 4S, 5′-CAA AAA CTG TAA AAA GAA GCC TTT CGC AAG TTC CGG TGT TTC GTC CTT TCC ACA AGA-3′. The sequences of APE are labeled bold. These various downstream primers were used for PCR amplification, along with the upstream primer and the template supplied by manufacturer. The PCR products were then inserted into the vectors to generate plasmids expressing various sense and antisense siRNAs, controlled by the U6 promoter. After transfection into 293T cells, these sense and antisense transcripts would complement each other and form various double-stranded siRNAs. Viral Infection—Viral stocks were produced in 293T cells by transfection with 2 μg of pNL4-3Δvif and 2 μg of pNL4-3Δvif plus 1 μg of pcDNA3-APOBEC3G, respectively, or in combination with the vectors expressing various genes such as Ugi or GFP, as well as various siRNAs, using Lipofectamine 2000 (Invitrogen) or FuGENE 6 (Roche Applied Science). Viruses in the supernatants were harvested at 48 h after transfection. After treatment with RQ1 DNase to remove input plasmids, the viruses were passed through a 0.45-μm filter and further quantitated with HIV-1 p24 via enzyme-linked immunosorbent assay. The viral infection was then performed with HLCD4-CAT as the target cells, which contain stably integrated, silent copies of the HIV-1 LTR promoter linked to the CAT gene. At 48 h after infection, the infected cells were harvested and subjected to CAT analysis, as described previously (2Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (921) Google Scholar). Cytidine Deaminase Assay—HIV-1Δvif viruses were harvested from the supernatant of 293T cells at 48 h after transfection, filtered by a 0.45 μm filter, and purified by rate-zonal sedimentation followed by equilibrium density centrifugation, as described previously (19Zhang H. Zhang Y. Spicer T.P. Abbott L.Z. Abbott M. Poiesz B.J. AIDS Res. Hum. Retroviruses. 1993; 9: 1287-1296Crossref PubMed Scopus (88) Google Scholar). The isolated viruses were further washed by cold phosphate-buffered saline and ultracentrifuged for 1 h at 25,000 × g. The viruses were then quantified by p24 enzyme-linked immunosorbent assay detection and mixed with a lysing buffer containing 50 mm Tris (pH 8.0), 40 mm KCl, 50 mm NaCl, 5 mm EDTA, 10 mm dithiothreitol, and 0.1% (v/v) Triton X-100. Conversely, various oligodeoxynucleotides were 5′-32P-labeled through incubation with T4 kinase and [γ-32P]ATP. The sequences for various oligonucleotides were modified from previous designs (13Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (510) Google Scholar, 20Petersen-Mahrt S.K. Neuberger M.S. J. Biol. Chem. 2003; 278: 19583-19586Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 21Conticello S.G. Harris R.S. Neuberger M.S. Curr. Biol. 2003; 13: 2009-2013Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar)(see Fig. 4A). The purified deoxyoligonucleotides (105 cpm) were incubated with 1 μg (p24 antigen equivalent) of viral lysate in buffer R (40 mm Tris (pH 8.0), 40 mm KCl, 50 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol, and 10% glycerol). After incubation for 4 h at 37 °C, the reactions were then terminated by extraction with phenol/chloroform/isoamyl alcohol (25:24:1). The extracted deoxyoligonucleotides were then electrophoresized on 15% Tris-borate-EDTA-urea PAGE gel and followed by direct autoradiography. A 32P-labeled oligodeoxynucleotide oligo(U) that contains a dU instead of a dC at position 18 (see Fig. 4A) was treated with 5 units of UNG2 and subsequently 2 n NaOH in parallel to serve as a size marker for the cleaved products. Virion-associated UNG2 and APE Catalytic Activity Assay—The lysate of HIV-1 virions (1 μg of p24 antigen equivalent) prepared as described above was mixed with 105 cpm 5′-32P-labeled oligodeoxynucleotide oligo(U) with and without recombinant UNG2 or APE (Novus Bio) at various concentrations in a buffer containing 20 mm Tris (pH 8.0), 10 mm MgCl2, 20 mm NaCl, 2 mm dithiothreitol, and 0.5 mm ATP. After incubation for 2 h, the cleaved products were electrophoresized on 15% Tris-borate-EDTA-urea PAGE and followed by direct autoradiography. Immunoblotting Analysis—The lysate of 293T or HLCD4-CAT (3–5 μg totally protein) or the lysate (100–250 ng of p24 antigen equivalent) of purified HIV-1Δvif viruses were subjected to detecting UNG2, APE, or Ugi-Myc. After electrophoresis in SDS-PAGE gel, rabbit polyclonal anti-UNG2 antibody (a gift of Dr. G. Slupphaug), mouse monoclonal anti-APE antibody (Novus Biologicals), and mouse anti-c-Myc monoclonal antibody (Sigma-Aldrich) were used, respectively, for detecting different proteins. Quantitative PCR Analysis of HIV-1 Reverse Transcripts—The HIV-1Δvif and HIV-1Δvif/APOBEC3G viruses were produced in 293T cells with and without co-transfected with Ugi-expressing vectors or APE-specific siRNA (SMARTpool) (100 nm). C8166 cells were infected with normalized viruses. After 24 h, cellular DNA was extracted and amplified by PCR with various primer pairs followed by Southern blotting analysis, as described in previous studies (22Zhang H. Dornadula G. Pomerantz R.J. J. Virol. 1996; 70: 2809-2824Crossref PubMed Google Scholar). The gag region was detected by the primer/probe set SK38-SK39/SK19, and the RU5-PBS-5NC region was detected by the primer/probe set M667-M661/SK31. Expression of UNG2-inhbitor Ugi or UNG2-specific siRNA in Virus-producing Cells Can Partially Block Antiviral Activity of APOBEC3G—It has been demonstrated that virion-associated APOBEC3G can convert dC to dU in newly synthesized minus-stranded viral DNA. The dU in newly synthesized viral DNA could be removed by virion-associated UNG2 and leave an abasic site in the DNA chain. This abasic site could be further cleaved by APE. If this hypothesis is correct, inhibition of viri-on-associated UNG2 could lead to less abasic site(s) in newly synthesized viral DNA chains and less degradation of newly synthesized viral DNA, and subsequently, less viral infectivity. Therefore we employed Ugi, a bacteriophage-encoded short polypeptide that can tightly bind to the DNA-binding site of UNG2, to inhibit the catalyzing activity of UNG2 in either virus-producing cells or viral target cells (23Bennett S.E. Mosbaugh D.W. J. Biol. Chem. 1992; 267: 22512-22521Abstract Full Text PDF PubMed Google Scholar, 24Handa P. Acharya N. Varshney U. Nucleic Acids Res. 2002; 30: 3086-3095Crossref PubMed Scopus (24) Google Scholar, 25Acharya N. Roy S. Varshney U. J. Mol. Biol. 2002; 321: 579-590Crossref PubMed Scopus (15) Google Scholar, 26Acharya N. Kumar P. Varshney U. Microbiology (Reading). 2003; 149: 1647-1658Crossref PubMed Scopus (17) Google Scholar, 27Chen R. Le Rouzic E. Kearney J.A. Mansky L.M. Benichou S. J. Biol. Chem. 2004; 279: 28419-28425Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 28Priet S. Gros N. Navarro J.M. Boretto J. Canard B. Querat G. Sire J. Mol. Cell. 2005; 17: 479-490Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We have transfected Ugi-expressing vector in virus-producing 293T cells or viral target cells (HLCD4-CAT), respectively. The Ugi protein can be detected in 293T cells (Fig. 1A, middle left panel). Its expression does not affect the expression of UNG2 in cells and the incorporation of UNG2 into virions (Fig. 1A, upper panel). Ugi can also be found in HIV-1Δvif virions, possibly because Ugi forms a strong complex with UNG2 in the virus-producing cells and subsequently is packaged into the virions (Fig. 1A, middle right panel). As shown in Fig. 1B, the viruses generated from HIV-1Δvif in the presence of APOBEC3G do not have any infectivity (lane 2). However, when vectors expressing Ugi were transfected to 293T virus-producing cells, the infectivity of HIV-1Δvif in the presence of APOBEC3G can be partially rescued (lane 8). The vector only (cytomegalovirus immediate early promoter) or GFP-expressing vector cannot rescue the virus infectivity (lanes 4 and 6). Conversely, transfection of the vector expressing Ugi-Myc into HLCD4-CAT, the virus target cells, cannot significantly rescue the viral infectivity, indicating that UNG2, which removes dU in the minus-stranded viral DNA, comes from virus-producing cells (Fig. 1C). To verify these observations, we have simultaneously performed similar experiments six times, and the results have been shown in Fig. 1D. The level of viral infectivity rescued by Ugi expression at different ratios to APOBEC3G (1:1, 0.5:1, and 0.25:1) in 293T cells is significantly higher than that without Ugi expression (Fig. 1D, lanes 5, 6, and 7 versus lane 2)(p < 0.001, t test). However, the level of viral infectivity rescued by Ugi expression in HLCD4-CAT cells is not significantly higher than that without Ugi expression (Fig. 1D, lane 8 versus lane 2). These quantitative data further demonstrate that Ugi at various concentrations is able to partially rescue the infectivity of HIV-1Δvif in the presence of APOBEC3G. To confirm that APOBEC3G protein in our experimental system is not overexpressed, we have compared the APOBEC3G expression that resulted from transfection with various amount of APOBEC3G-expressing plasmid with the concentration of APOBEC3G in “non-permissive” primary CD4 T-cells and “permissive” Supt-1 T-cells. Fig. 1E demonstrates that the APOBEC3G expression in our experiment (1 μg of APOBEC3G-expressing plasmid) is lower than the APOBEC3G expression in primary CD4 T-cells, the native target cells for HIV-1 replication. If a very low amount of APOBEC3G-expressing plasmid (0.01 μg) is transfected into 293T cells to generate APOBEC3G protein (Fig. 1E), we have also found that the expression level of APOBEC3G is as low as that in Supt1 T-cells. This amount of APOBEC3G cannot significantly inhibit the infectivity of HIV-1Δvif viruses (Fig. 1F). To further verify that the inhibition of UNG2 in virus-producing cells can rescue the infectivity of HIV-1Δvif in the presence of APOBEC3G, we transfected UNG2-specific siRNA (SMARTpool) into the virus-producing cells. As indicated in Fig. 2A, UNG2-specific siRNA can effectively decrease the expression of UNG2 in 293T cells. When transfected into 293T virus-producing cells, it can also significantly rescue the infectivity of HIV-1Δvif in the presence of APOBEC3G (Fig. 2B, lane 6 versus lane 3). However, transfection into HLCD4-CAT, the viral target cells, cannot effectively rescue the viral infectivity (Fig. 2B, lane 9). Viral Infectivity Suppressed by APOBEC3G Can Be Partially Rescued by APE-specific siRNA—The excision of uracils from DNA by UNG2 results in abasic sites in the minus-stranded viral DNA. The abasic sites could be subsequently cleaved by APE. However, there is no direct evidence showing that the cellular APE participates in the degradation of uracilated-DNA in any viruses. To test the possible involvement of APE in APOBEC3G-induced cDNA degradation, the APE-specific siRNAs (SMARTpool) was chemically synthesized and transfected to 293T cells, and its potent APE-suppressing activity was confirmed by Western blotting (Fig. 3A). To determine the cells in which APE plays a role, APE-specific siRNA was transfected into 293T virus-producing cells or HLCD4-CAT target cells before viral infection. The viral infectivity was analyzed by CAT assay, as described above. APE-specific siRNA could partially rescue the viral infectivity blocked by APOBEC3G when co-transfected into virus-producing cells (Fig. 3B, lane 6), whereas having much less effect in HLCD4-CAT target cells (Fig. 3C, lane 6). These results indicate that APE is involved in the degradation of APOBEC3G-edited viral DNA. Again, this result was further confirmed by multiple experiments, using either the chemically synthesized SMARTpool of APE-specific siRNA or the U6 promoter-driven APE-specific siRNA pool (Fig. 3D). Our data suggest that APE in virus-producing cells, like UNG2, should be encapsidated in viruses and directly participates in the degradation of newly synthesized viral DNA. With an immunoblotting assay, APE was indeed detected in purified HIV-1Δvif viruses (Fig. 3E). Uracil-containing DNA Can Be Degraded by Virion-associated UNG2 and APE Enzymes—It has been proven that APOBEC3G deaminates the single-stranded oligonucleotide in vitro (13Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (510) Google Scholar). To examine the cytidine deaminase activity of APOBEC3G in vitro, we have used a similar system. Oligonucleotides containing cytidine from one to five were incubated with the lysate of Δvif/APOBEC3G viruses (Fig. 4A). We found that the cleaved fragments were clearly detected when 5′-32P-labeled single oligonucleotides containing more than two cytidines were incubated with the lysate from HIV-1ΔVif viruses containing APOBEC3G (Fig. 4B). This result indicates that all the required elements for dC to dU deamination, removal of dU from the oligonucleotide and cleavage of the abasic site, are all enclosed in the virions. To further determine whether the viri-on-encapsidated UNG2 and APE are catalytic, we developed an experiment to monitor the virion-associated UNG2 and APE enzymatic activity by the similar method previously described (27Chen R. Le Rouzic E. Kearney J.A. Mansky L.M. Benichou S. J. Biol. Chem. 2004; 279: 28419-28425Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In this experiment, 5′-32P-labeled uracil-containing oligodeoxynucleotide (oligo(U)) was treated by the viral lysate. If both the virion-associated UNG2 and the virion-associated APE are catalytically active, we assumed that a short length broken DNA fragment should be separated by denaturing PAGE gel without the addition of UNG2 and APE. As shown in Fig. 4C, when 5′-32P-labeled uracil-containing DNA was incubated only with the lysate from Δvif/APOBEC3G viruses or Δvif viruses, the degraded products were clearly detected (lanes 3 and 8), suggesting that the virion-associated UNG2 and APE have the enzymatic ability to degrade the uracil-containing DNA. As additional recombinant UNG2 can enhance the degradation (Fig. 4C, lanes 4, 5, 9, and 10), the virion-associated UNG2 is likely not sufficient to catalyze the removal of dU. However, the addition of APE did not significantly improve the digestion rate (Fig. 4C, lanes 6, 7, 11, and 12), suggesting that virion-associated APE is sufficient to cleave the abasic site. Furthermore, to test whether the uracil excision catalytic activity of virion-associated UNG2 can be inhibited by virion-associated Ugi, viral lysate from Ugi-co-transfected Δvif/APOBEC3G viruses were prepared, and a similar experiment as described in the legend for Fig. 4C was performed. As shown in Fig. 4D, the cleaved products were much less when uracil-containing oligodeoxynucleotide was incubated with the lysate of Δvif/APOBEC3G/Ugi viruses (Fig. 4D, lane 3) than that with the lysate from Δvif/APOBEC3G viruses (Fig. 4D, lane 2). Inhibition of UNG2 and APE Significantly Decrease APOBEC3G-induced Degradation of Newly Synthesized Reverse Transcripts—It has been shown that the newly synthesized viral DNA by reverse transcription was very low in Δvif viruses from non-permissive cells (3Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1252) Google Scholar, 14von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar, 16Dornadula G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar, 17Simon J.H. Malim M.H. J. Virol. 1996; 70: 5297-5305Crossref PubMed Google Scholar, 29Mariani R. Chen D. Schrofelbauer B. Navarro F. Konig R. Bollman B. Munk C. Nymark-McMahon H. Landau N.R. Cell. 2003; 114: 21-31Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). To examine the effect of UNG2 and APE inhibitors upon the viral DNA synthesis of HIV-1Δvif/APOBEC3G viruses in the target cells, a semiquantitative PCR was used (22Zhang H. Dornadula G. Pomerantz R.J. J. Virol. 1996; 70: 2809-2824Crossref PubMed Google Scholar, 30Chen K. Huang J. Zhang C. Huang S. Nunnari G. Wang F.X. Tong X. Gao L. Nikisher K. Zhang H. J. Virol. 2006; 80: 7645-7657Crossref PubMed Scopus (129) Google Scholar). HIV-1Δvif/APOBEC3G viruses were generated from 293T cells with and without various co-transfections. These viruses were then allowed to infect C8166, a CD4 T-cell line. At 24 h after infection, cellular DNA was extracted and amplified by PCR. The late part of minus-stranded DNA (gag region) and plus-stranded DNA (RU5-PBS-5NC region) of Δvif/APOBEC3G viruses were monitored. Fig. 5 indicates that the DNA accumulation of ΔVif/APOBEC3G viruses significantly decreased (lanes 2 and 4). However, Ugi- or APE-specific siRNA can significantly rescue the accumulation of viral DNA (Fig. 5, lanes 6 and 10), indicating that UNG2 and APE are involved in the degradation of APOBEC3G-edited viral DNA. To prove the possible involvements of UNG2 and APE in the antiviral function of APOBEC3G, we have blocked the enzymatic activity of UNG2 and APE, respectively, by the UNG2 inhibitor Ugi, UNG2-specific siRNA, or APE-specific siRNAs. Our data have shown that after catalytically enzymatic activity or expression of UNG2 or APE is decreased in virus-producing cells rather than in virus-targeting cells, the antiviral activity of APOBEC3G is partially inhibited. These findings demonstrate that most of the uracil bases in minus-stranded DNA generated by APOBEC3G will be first removed by excision of virion-associated UNG2 and then degraded by virion-associated APE before integration. This hypothesis is supported by the early observations that less full-length viral DNA is synthesized during the reverse transcriptions of Δvif/APOBEC3G viruses (3Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1252) Google Scholar, 14von Schwedler U. Song J. Aiken C. Trono D. J. Virol. 1993; 67: 4945-4955Crossref PubMed Google Scholar, 15Sova P. Volsky D.J. J. Virol. 1993; 67: 6322-6326Crossref PubMed Google Scholar, 16Dornadula G. Yang S. Pomerantz R.J. Zhang H. J. Virol. 2000; 74: 2594-2602Crossref PubMed Scopus (39) Google Scholar, 17Simon J.H. Malim M.H. J. Virol. 1996; 70: 5297-5305Crossref PubMed Google Scholar). Our data suggest that the subsequent degradation pathway perhaps plays a role in the antiviral activity of APOBEC3G. The G to A hypermutations observed by our group and others in HIV-1 plus-stranded DNA" @default.
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