- Email: [email protected]
Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1
BBRC Biochemical and Biophysical Research Communications 294 (2002) 509–517 www.academicpress.com
Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1 Hidehiro Takahashi,a,* Hirofumi Sawa,b Hideki Hasegawa,a Yuko Shoya,a Tetsutaro Sata,a William W. Hall,c Kazuo Nagashima,b and Takeshi Kurataa a
Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan b Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, and CREST, JST, Japan c Department of Medical Microbiology, University College Dublin, Belfield, Dublin 4, Ireland Received 7 May 2002
Abstract Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I–RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: ATP; Topoisomerase I; HIV-1; Reverse transcription
Human immunodeficiency virus type 1 (HIV-1) replication is regulated not only at the viral entry stage, in which specific interactions of HIV gp120 with the primary receptor including CD4 and a number of chemokine receptors are critical [1–3] but also at the post-entry steps (reverse transcription, nuclear entry, integration, and transcription), where other cellular factors are involved [4–6]. HIV-1 particles contain two copies of a positivesense, single-stranded RNA genome and the genomic RNA exists in the form of a dimer. Two copies of genomic RNA in particles, which are substantially nicked [7–10], would require some regulatory factors for efficient reverse transcription. After thermal melting the HIV-1 RNA dimer yields only a small amount of the intact monomer and the remaining dimer degrades into *
Corresponding author. Fax: +81-3-5285-1189. E-mail address: [email protected] (H. Takahashi).
small RNA fragments which are recognized as smears on either gel electrophoresis [8,11–13] or Northern blot analysis [9,10,14]. These observations suggest that the retroviral genomic RNA is rapidly and substantially cleaved following viral assembly and budding and that some viral and/or cellular factors are required to facilitate and complete proviral DNA synthesis. HIV-1 particles are known to carry a number of cellular components which might be involved in suppression of small fragments of viral RNAs [15]. It has been reported that cellular topoisomerase I is present in retroviral particles and appears to play an important role in the replication of HIV-1 RNA [16,17]. We found that a hairpin-loop formation of RNA might be essential to form a complex of topoisomerase I and RNA; however, neither linearized nor double-stranded RNA could bind with topoisomerase I (unpublished data). Furthermore, we have previously demonstrated that topoisomerase I enhanced HIV-1 cDNA synthesis with
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 0 3 - X
510
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
in vitro reverse transcriptase (RT) assays [18]. Thus, it seems that topoisomerase I deeply affects replication of HIV-1 RNA. Therefore, it is hypothesized that topoisomerase I carried in HIV-1 virions plays an important role in HIV-1 cDNA synthesis in vivo. To investigate this, experiments such as indicator cell assays and endogenous reverse transcription (ERT) assays were performed. We show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I– RNA complex and enhance cDNA sythesis in vitro. In the present study, it is clearly demonstrated that cDNA synthesis of HIV-1 RNA is augmented by topoisomerase I and ATP.
Materials and methods The efficiency of infection of HIV-1 carrying wt or mutant topoisomerase I into MAGIC5 cells. HIV-1 proviral DNA (pIndie-C1, 1 lg) [19] and a mammalian expression vector (pcDNA4c-wt, pcDNA4c-Y723F, pcDNA4c-R488A, or pcDNA4c-K532A) carrying wt topoisomerase I, Y723F, R488A, or K532A cDNA (1 lg) were co-transfected into 293T cells using the MBS Mammalian Transfection Kit (Stratagene, CA). Thirty-six hours after transfection virus stocks were harvested by centrifugation of the supernatants, the p24gag concentration was measured using a HIV-1 p24 antigen ELISA Kit (Zepto Metrix, NY). As an indicator cell, about 104 HeLa-CD4-CCR5-LTR-b-gal (MAGIC5) cells [19,20] were plated on 96-well tissue culture plates and inoculated with serially diluted virus stocks. About 48 h after infection, cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-b-D -galactoside and the stained cells were counted as described earlier [21]. Expression of wt and mutant topoisomerase I were examined by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting using an anti-Xpress epitope antibody (Invitrogen, CA). As topoisomerase I mutants, Tyr-723, which is essential for endoribonuclease function [22–24], was substituted with Phe (Y723F), and Arg-488 or Lys-532, which is essential for ligase activity [23–25], was substituted with Ala (R488A or K532A). Endogenous reverse transcriptase (ERT) assay. Ten lg of uncleaved plasmid, pNL43 including intact open reading frames of all the HIV-1 accessory genes [26], was transfected into 106 of M8166 cells using a diethylaminoethyl (DEAE)-dextran transfection method. HIV-1 in the supernatant was further amplified by incubation with uninfected M8166 cells for three days, after which the supernatant was ultracentrifuged at 35,000 rpm for 3 h using a 50.2 TI rotor (Beckman, CA). Pellets were suspended in Tris-buffered saline [TBS; 25 mM Tris–HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl] and the protein concentration was adjusted to 1 mg/mL. Reaction mixtures [1 lg (Fig. 3), 5 lg (Fig. 2C) or 10 lg (Figs. 2A and B) of virion protein, 100 mM Tris–HCl (pH 8.0), 3 mM MgCl2 , and 0.2 mM each of deoxynucleoside triphosphate (dNTP), 0.01% NP40, nucleotide (Figs. 2 and 3) and purified wt or mutant topoisomerase I (Figs. 2C and 3) in 40 lL solution] were incubated at 37 °C for 3.5 h and the reaction was stopped by addition of 300 lL of the stop buffer [50 mM Tris–HCl (pH 8.0), 0.5% SDS, 10 mM EDTA and 100 lg=mL proteinase K]. The samples was then incubated at 56 °C for 1 h and treated with phenol/chloroform. The supernatant
Fig. 1. Influence of wt or mutant topoisomerase I on HIV-1 infectivity. The number of foci of MAGIC5 cells (per 100 pg of p24) infected with HIV-1 carrying recombinant wt or mutant topoisomerase I with Xpress epitope tag is represented as a bar graph (upper column). Expression levels of wt and mutant topoisomerase I were confirmed by SDS–PAGE followed by immunoblotting using an anti-Xpress epitope antibody (lower). Each bar represents the average of two independent experiments.
containing DNA products was precipitated in ethanol, centrifuged, resuspended with 0.3 N NaOH, and subjected to 0.8% agarose gel electrophoresis in an alkaline running buffer (30 mM NaOH and 2 mM EDTA). Following electrophoresis, DNA products were blotted onto nylon membranes (Pall, NY) and subjected to Southern blot hybridization. Extrachromosomal DNA from HIV-1-infected M8166 cells was extracted by the high salt precipitation method [27] and used as a positive control in Southern blot hybridization. Southern blot hybridization. Nylon membranes blotted with DNA were hybridized with 32 P-labeled 1.3 kb of HIV-1 gag fragment of pNL43 digested with BglII (679–2096 nt) in Quick Hybrisol (Stratagene). After hybridization, membranes were washed twice with 2 SSC (1 SSC; 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at 65 °C for 10 min, followed by two washings in 0:1 SSC containing 0.1% SDS at 65 °C for 30 min. Hybridized signals were visualized and quantitated with the FLA2000 (Fuji film, Japan). Expression and purification of recombinant proteins and enzyme. Human topoisomerase I cDNA was amplified by PCR, using the primers TF (50 -CGT CCC TCC GAATTC ATG AGT GGG GAC CA30 ) and TR (50 -GCC TCT TGA GCGGCC GCT AAA ACT CAT AGT CA-30 ) (underlined sequences correspond to EcoRI and NotI recognition sites, respectively). Three recombinant human topoisomerase I mutants were synthesized; Arg-488 or Lys-532 in wt topoisomerase I were substituted for Ala (designated R488A and K532A, respectively) and Tyr-723 was substituted for Phe (Y723F). Topoisomerase I mutant constructs were made by the overlap extension method [28]. Briefly described, primers 488F (50 -AAG CTT GCT CTG GCA GCA GGC AAT GAA-30 ), 488R (50 -TCC ATT GCC TGC TGC CAG AGC AAG
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
511
Fig. 3. Influence of wt or mutant topoisomerase I on HIV-1 cDNA synthesis. One lg of virions was incubated with 1 mM ATP and 2.5, 5, or 10 ng of purified K532A, R488A, Y723F, or wt topoisomerase I. No enzyme was added in the control reaction ()). Full-length HIV-1 cDNA is indicated by a black arrow.
Fig. 2. ERT assays in the presence of various amounts of nucleotides. Reaction mixtures contained 10 lg (A, B) or 5 lg (C) of virion and various amounts of nucleotides. (A) 0.1 or 1 mM of ADP, AMP-PNP, or ATP; (B) 1 mM of UTP, CTP or GTP, 0.1 or 1 mM of GTP-cS, or ATP; (C) 0.01, 0.1 or 1 mM of AMP-PNP in the absence of wt topoisomerase I, or 0.01 mM AMP-PNP in the presence of various amounts of wt topoisomerase I (1.25, 2.5, 5 or 10 ng) as shown in the panel. No nucleotide or enzyme was present in the control reaction ()). The synthesized cDNA was hybridized and detected with a 32 P-labeled HIV-1 gag probe. Black arrow indicates the position of full-length HIV-1 cDNA.
CTT-30 ), 532F (50 -TTT GAC TTC CTC GGG GCG GAC TCC ATC AGA TAC-30 ), 532R (50 -GTA TCT GAT GGA GTC CGC CCC GAG GAA GTC AAA-30 ), 723F (50 -TCC AAA CTC AAT TTT CTG GAC CCT AGG-30 ), and 723R (50 -CCT AGG GTC CAG AAA ATT GAG TTT GGA-30 ) (underlining denotes mutations) were used for amplification of mutant fragments; TF/488R and 488F/TR were used for R488A, TF/532R and 532F/TR for K532A, and TF/723R and 723F/TR for Y723F, respectively. These amplified fragments were combined in a subsequent fusion reaction. Wt and mutant human topoisomerase I cDNAs were subcloned into baculovirus transfer vectors with six Histag-sequences at the N-terminus (pFASTBAC HTa, BRL, MD) or a mammalian expression vector with an Xpress epitope tag at the N-terminus of the cloning site (pcDNA4/HisMax C, Invitrogen). Recombinant baculovirus was prepared according to manufacturer’s instructions. The nuclei of infected insect cells were resuspended with phosphate buffer [20 mM phosphate (pH 7.5), 1 M NaCl, 10 mM imidazole, and 1 tablet of protease inhibitor cocktail without EDTA
(Roche Diagnostics, IN)]. Following precipitation of polynucleotides with 6% polyethylene glycol 6000, supernatants was applied to HiTrap chelating columns (Amersham Pharmacia, UK). Five hundred mM of imidazole-eluted fractions were applied to 1.0 ml of either hydrophobic columns, Hitrap Phenyl FF (Amersham), or cation exchange columns, UNO S-1 (Bio-Rad, CA). Wt and mutant topoisomerase I were eluted from the columns by either low concentration ammonium sulfate or high concentration sodium chloride and separated by SDS–PAGE. After staining with SYPRO (Molecular Probes, OR), polyacrylamide gel was visualized with the FLA 2000. Wt HIV-1 RT (p66) was expressed in Escherichia coli (E. coli) using a His6 -tagged expression vector [pQE-9, (Qiagen)] encoding HIV-1 p66 (corresponding to 2250–4229 nt of pNL43). Proteins were purified using a HiTrap chelating column (Amersham Pharmacia) and cation-exchange chromatography (UNO S1, BIORAD). Preparation of RNA substrate. Precursor RNAs were prepared by in vitro transcription from PCR-amplified DNA with T7 RNA polymerase. To synthesize short RNA substrates, DNA fragments containing the T7 promoter and either of the p7 transcripts (76 nucleotides, nt), which are the processed proteins originating from HIV-1 p15gag or p15gag (394 nt), were amplified by PCR using pNL43 [26] as a template DNA with the following primers: p7F (50 -TAATAC GACTCACTA TAG GGA ACA AAT CCA GCT ACC ATA ATG ATA-30 ) and P7R (50 -ATT GAA ACA CTT AAC AGT CTT-30 ) (corresponding to 1900–1971 nt of pNL43); and P15F (50 -TAATAC GACTCACTATAG GGA ACA AAT CCA GCT ACC ATA ATG ATA-30 ) and P15R (50 -TTG TGA CGA GGG GTC GTC GCT GCC30 ) (underlining denotes T7 promoter, 1900–2289 nt). For doublestranded p15 substrate the primers were P15 antisense F (50 -TAATAC GACTCACTATAG GGA TTG TGA CGA GGG GTC GTC GCT GCC-30 ) and P15 antisense R (50 -ACA AAT CCA GCT ACC ATA ATG ATA-30 ) (underlining denotes T7 promoter). The precursor RNAs were 50 -end-labeled with adenosine ½c-32 Ptriphosphate and polynucleotide kinase after dephosphorylation with calf intestine
512
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
phosphatase (CIP). The precursor RNAs were purified by electrophoresis on 12% polyacrylamide/7 M urea denaturing gels using a Mini Whole Gel Eluter (Bio-Rad). Concentrations of RNAs stained by SYBER Green II (Molecular Probes) were analyzed with the FLA 2000. Analysis of binding of topoisomerase I to RNA substrates and cDNA synthesis. Ten ng of 50 -end-labeled RNA was heated in a Tris buffer [50 mM Tris–HCl (pH 8.0), 0.2 mM EDTA] at 95 °C for 4 min and quenched on ice for 5 min. Samples (20 lL) were incubated with 0.5% Brij35 (Sigma–Aldrich, WI) and 100 lg=mL nuclease free bovine serum albumin (BSA, Roche Diagnostics, IN) at 37 °C for 60 min to form structures. For investigation of the binding and dissociation of topoisomerase I to RNA substrates, 10 ng of p7 RNAs was cleaved in the above conditions and cleaved RNAs were incubated with topoisomerase I proteins at 37 °C for 10 min. Topoisomerase I–RNA mixtures were further incubated with either ATP, adenosine diphosphate (ADP), adenosine50 -[c-thio]triphosphate (ATP-cS) or adenosine 50 -[b; c-imido] triphosphate (AMP-PNP) at 37 °C for 2 min. Following addition of glycerol (final concentration, 10%) and dye (xylene cyanol and bromophenol blue), samples were electrophoresed onto a native 6% polyacrylamide gel containing 1 TBE (90 mM Tris–borate, 2 mM EDTA). Topoisomerase–RNA complexes were visualized and quantified with the FLA 2000. For analysis of cDNA synthesis, oligonucleotide primer (50 -TTG TGA CGA GGG GTC GCT GCC AAA GAG TGA-30 ) (corresponding to 2260–2289 nt of pNL43) was end-labeled with adenosine ½c-32 Ptriphosphate (NEN Research Product, UK) and T4 Polynucleotide Kinase (New England Biolabs, MA). Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was heated in the Tris buffer [50 mM Tris–HCl (pH 8.0), 0.2 mM EDTA] at 95 °C for 4 min and quenched on ice for 4 min. Samples (20 lL) were incubated with 0.5% Brij35, 100 lg=mL BSA, 5 mM MgCl2 , and topoisomerase I at 37 °C for 60 min. One nanogram of purified reverse transcriptase (RT) and either ATP or ADP was added to the annealed template-primer and allowed to bind for 4 min at 37 °C. Synthesis was initiated with the addition of solution containing dNTPs (80 lM, final concentration). The reactions were terminated by addition of an equal volume of a stop solution (90% formamide, 50 mM EDTA, 1.0% SDS, xylene cyanol, and bromophenol blue), heated at 95 °C for 2 min, and fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea.
Results Topoisomerase I mutants, R488A, and K532A inhibited the infectivity of HIV-1 Previously, we demonstrated that topoisomerase I enhanced cDNA synthesis in vitro [18]. To investigate the role of topoisomerase I in cDNA synthesis and replication of HIV-1, the infectivity of HIV-1 was initially examined in the presence of wild-type (wt) topoisomerase I and its mutants lacking endoribonuclease or ligase activities using a HeLa-CD4-CCR5-LTR-b-gal (MAGIC5) indicator cell system [20,29]. Compared with wt enzyme, topoisomerase I mutants, both R488A and K532A, significantly reduced the infectivity of HIV-1 by 75%, while Y723F had no effect, suggesting that HIV-1 infectivity was dependent on an activity of topoisomerase I which was lost by the mutations at 488-R and 532-K (Fig. 1). In addition, the infectivity of VSV-G
pseudotyped HIV-1 particles [30] in which nef was replaced with a luciferase indicator gene was also inhibited in the presence of R488A or K532A in producer cells (data not shown). The results suggest that the mutant topoisomerase I, R488A and K532A, reduced viral infectivity at a post-entry level. ATP and AMP-PNP activate HIV-1 cDNA synthesis We described that topoisomerase I enhances synthesis of a part of HIV-1 cDNA which is 1.48 kb in length with in vitro reverse transcription (RT) assays [18], while in this study we employed an ERT assay using detergentdisrupted HIV-1 virions to examine the physiological role of topoisomerase I in the full-length (9.4 kb) HIV-1 cDNA synthesis. It has been reported that HIV-1 virions carry topoisomerase I, which has DNA relaxation activity after disruption of virus particles [16,17], and that monoclonal antibodies against topoisomerase I inhibit cDNA synthesis with ERT assays [31]. Furthermore, ATP or non-hydrolyzable analogs have been considered to combine with topoisomerase I and enhance the topoisomerase I activity for DNA relaxation by the dissociation of the enzyme from substrates [32]. We thus investigated whether ATP could influence cDNA synthesis by activation of endogenous topoisomerase I with ERT assay. As shown in Fig. 2A, 0.1 or 1 mM ATP and adenosine 50 -[b; c-imido]triphosphate (AMP-PNP) activated HIV-1 full-length (9.7 kb) cDNA synthesis in a concentration-dependent manner, while 0.01, 0.1, and 1 mM ADP had subtle effects on cDNA production. Next, the effects of 0.1 or 1 mM guanosine 50 -(a-thio) triphosphate (GTP-cS) and 1 mM of other NTPs (UTP, CTP, and GTP) on cDNA synthesis were also examined by ERT assay and it was clearly demonstrated that neither GTP-cS nor other NTPs had no influence on full-length cDNA synthesis by comparison with ATP (Fig. 2B), suggesting that ATP which might activate endogenous virion-associated topoisomerase I exclusively enhanced cDNA synthesis in ERT assay. Next, the efficiency of cDNA synthesis in the presence of exogenous topoisomerase I (15, 62.5, 125, and 250 ng/ ml) and an ATP-analog, AMP-PNP (0.01, 0.1, and 1 mM), was investigated by ERT assay (Fig. 2C). In the absence of exogenous topoisomerase I, the synthesis of full-length cDNAs was increased in an AMP-PNPconcentration-dependent manner. Concentrations of topoisomerase I and 0.01 mM of AMP-PNP synergistically enhanced the efficiency of cDNA synthesis. In ERT assay, two major products reflecting fulllength and 6-kb transcripts were recognized (Fig. 2). The 6-kb subgenomic cDNA has been shown to hybridize with the 50 end of a negative-strand-specific probe, but not with the 30 end of an env-specific probe [33], suggesting that the 6-kb cDNA lacks the 30 region by incorrect jumping of R region.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
Wt and Y723F topoisomerase I enhances cDNA synthesis, while R488A and K532A attenuate it in the presence of ATP The infectivity of HIV-1 was reduced in the presence of the ligase activity mutants of topoisomerase I, R488A and K532A (Fig. 1); therefore, the effect of wt and endoribonuclease activity mutant topoisomerase I, Y723F (62.5, 125, or 250 ng/ml) on cDNA synthesis in the presence of 1 mM of ATP was examined by ERT assay. Wt or Y723F topoisomerase I resulted in an increased production of full-length cDNA in a concentrationdependent manner (Fig. 3). In contrast, both R488A and K532A mutants attenuated cDNA synthesis compared with the control, suggesting that these mutants work as a dominant negative form against the cDNA synthesis. These results showed that an activity of human topoisomerase I which was lost in R488A and
513
K532A were necessary for the activation and completion of HIV-1 cDNA synthesis in ERT assay. In vitro cDNA synthesis assay reveals that ATP dissociates topoisomerase I from RNA and excess amounts of topoisomerase I inhibit cDNA synthesis MAGIC5 cell assay (Fig. 1) and VSV-G pseudotyped HIV-1 assay (data not shown) revealed that an activity of topoisomerase I was required at the post-entry step; also, ERT assays (Figs. 2 and 3) showed that topoisomerase I and ATP increased cDNA synthesis. To analyze the relationship between topoisomerase I and ATP for in vitro cDNA synthesis, we initially examined the influence of ATP or its non-hydrolyzable analogs on the binding of topoisomerase I to RNA by native gel electrophoresis. ATP (3 mM) completely dissociated wt topoisomerase I from RNAs, while 3 mM ATP-cS and
Fig. 4. In vitro cDNA synthesis assay indicating the dissociation of topoisomerase I from RNA and inhibition of cDNA synthesis in the presence of excess amounts of topoisomerase I. (A) Dissociation of 2:7 lg=ml of wt topoisomerase I from p7 RNA transcripts in the presence of (0.1, 0.3, 1, or 3 mM) ATP-cS, (0.03, 0.1, 0.3, 1, or 3 mM) AMP-PNP or ATP was analyzed by native gel electrophoresis. The positions of topoisomerase I–RNA complex (Topo I–RNA complex) and precursor (Prec) and cleaved transcripts are indicated. (B), Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was incubated with 0.1, 0.3, 0.9, or 2:7 lg=ml of K532A or Y723F and 50 ng/ml of reverse transcriptase and fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea. The reaction was performed in the absence of ATP, AMP-PNP, and ATPcS. The position of remaining primers which were not used for cDNA is indicated as Primer. (C) The relative ratios of signal intensity of full-length to remaining primer were calculated and are demonstrated as a line graph, with the ratio in the presence of 0:1 lg=ml of topoisomerase I mutant being 100%. Open circles and closed circles represent the relative ratios of K532A and Y723F, respectively.
514
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
AMP-PNP had little influence on the binding of topoisomerase I (Fig. 4A). This result suggested that the dissociation of topoisomerase I from RNA was probably dependent on the charge close to the c site of ATP. Next, the effects of topoisomerase I on in vitro cDNA synthesis in the absence of ATP were examined. A reaction of in vitro cDNA synthesis was performed in the presence of 50 -labeled oligonucleotides as a primer, synthesized RNA as a template for reverse transcriptase, purified reverse transcriptase, dNTP, and large amounts (0.1, 0.3, 0.9, or 2:7 lg=ml) of wt or mutant topoisomerase I and analyzed by denaturing gel electrophoresis. As shown in Figs. 4B and C, the high concentration (2:7 lg=ml) of wt (data not shown), Y723F and K532A topoisomerase I reduced cDNA synthesis by 40%. It is
hypothesized that the binding of excess amounts of topoisomerase I with template RNA results in inhibition of cDNA synthesis. ATP results in enhancement of HIV-1 cDNA synthesis in the presence of excess amounts of topoisomerase I As excess amounts of topoisomerase I suppressed in vitro cDNA synthesis, the effect of ATP, expected to dissociate topoisomerase I from RNA substrate (Fig. 4A) and non-hydrolyzable analogs, on cDNA synthesis was investigated using in vitro cDNA synthesis assay. In the presence of a high concentration (2:7 lg=ml) of wt or K532A topoisomerase I, 2 mM of ATP enhanced cDNA synthesis much more effectively than did non-
Fig. 5. Analysis of cDNA synthesis in vitro. Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was incubated with wt (A), K532A (A, C), or Y723F topoisomerase I (B). Purified RT [50 ng/ml (A, B) or 6, 12, 25, and 50 ng/ml (C)] and either 2 mM of ATP (A–C), AMP-PNP (A), ATP-cS (A) or ADP (B, C) were added to the annealed template-primer and allowed to bind. Synthetic cDNA was fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea. The relative ratios of signal intensity of full-length to remaining primer were compared with regard to the ratio at time point 0 (A, B) or without RT ()) (C) as 1 and the fold increases are demonstrated in a line graph.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
hydrolyzable analogs in a time-dependent manner (Fig. 5A). Wt and K532A topoisomerase I similarly affected cDNA synthesis; therefore, it seemed that synthesis of full-length cDNA was dependent on the ATP-induced dissociation of topoisomerase I from RNA template. The effect of 2 mM ATP and ADP on cDNA synthesis was next investigated in the presence of 2:7 lg=ml Y723F topoisomerase I by in vitro cDNA synthesis assay (Fig. 5B). As shown in Fig. 5B, ATP activated cDNA synthesis in a time-dependent manner, while ADP had only a slight enhancing effect on the efficiency of cDNA synthesis, suggesting that the charge close to the c site of ATP was important for cDNA synthesis. In addition, we have shown the in vitro cDNA synthesis assay without topoisomerase I in the Fig. 5B. Fulllength cDNA was synthesized even in the absence of topoisomerase I, suggesting that topoisomerase I was not essential for cDNA synthesis; however, topoisomerase I in cooperation with ATP remarkably increased cDNA synthesis. Topoisomerase I mutants, R488A and K532A lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Finally, the efficiency of cDNA synthesis in the various concentrations (6, 12, 25, and 50 ng/ml) of reverse transcriptase (RT) in the presence of K532A topoisomerase I was investigated (Fig. 5C). It was demonstrated that in the presence of K532A (2:7 lg=ml), ATP was more effective in activation of cDNA synthesis than ADP and the efficiency of cDNA synthesis of both groups was dose-dependent to RT. Thus, HIV-1 cDNA synthesis in the presence of topoisomerase I was regulated by ATP and RT.
Discussion Previously, we demonstrated that topoisomerase I could enhance the reverse transcription of short HIV-1 templates in vitro in the presence of HIV-1 nucleocapsid proteins [18]. In the present study, we have extended these observations and first have demonstrated that the topoisomerase I mutants R488A and K532A reduced the infectivity of HIV-1 (Fig. 1). As the decrease of infectivity by topoisomerase I mutants was also observed with VSVG pseudotyped HIV-1 particles (data not shown), the step where topoisomerase I worked for HIV-1 replication was expected to be at the post-entry level. We confirmed that topoisomerase I affected HIV-1 cDNA synthesis and that ATP cooperated with topoisomerase I and enhanced cDNA synthesis using ERT assay (Figs. 2 and 3). Therefore, this is proof that topoisomerase I carried in HIV-1 virions plays an important role in HIV-1 cDNA
515
synthesis in vivo. In addition, although large amounts of topoisomerase I suppressed in vitro cDNA synthesis (Figs. 4B and C), ATP which causes dissociation of topoisomerase I from RNA substrates (Fig. 4A) enhanced in vitro cDNA synthesis even in the presence of excess amounts of topoisomerase I (Fig. 5). These results partly explain the relation of topoisomerase I and ATP in cDNA synthesis. Topoisomerase of the vaccinia virus has been shown to exhibit both endoribonuclease and ligase activities. Replacement of Tyr-274 with Phe and Arg-130 or Lys-167 with Alanine of the vaccinia virus topoisomerase results in the loss of ribonuclease activity and both ribonuclease and ligase activity, respectively [22,25]. Tyr-274, Arg-130, and Lys-167 residues that are well conserved in every member of the type IB topoisomerase family correspond to Tyr-723, Arg-488, and Lys-532 of human topoisomerase I, respectively [24], suggesting that Y723F human topoisomerase I mutant lacks ribonuclease activity and both R488A and K532A mutants are devoid of ribonuclease and ligase activity. As wt topoisomerase I and Y723F enhanced cDNA synthesis but R488A and K532A suppressed replication and cDNA synthesis of HIV-1, it is reasonable to assume that the ligase activity of topoisomerase I is essential for replication and enhancement of cDNA synthesis of HIV-1, while the ribonuclease activity of the enzyme is not necessary. The results of ERT assay were different from those obtained by in vitro cDNA synthesis assay in two points: initially, K532A topoisomerase I mutant could not enhance cDNA synthesis in the presence of ATP (Fig. 3); and second, AMP-PNP as well as ATP could enhance cDNA synthesis in the presence of endogenous topoisomerase I (Figs. 2A and C). These discrepancies seemed to be due to the differences of RNA template conditions between ERT assay and in vitro cDNA synthesis assay. For ERT assay, HIV-1 full-length genomic RNA (9.4 kb) was applied, while for in vitro cDNA synthesis assay, the synthesized RNA, HIV-1 p7 transcripts (76 nt) and p15gag (394 nt) were used as the template. There are some differences in requirements between in vitro cDNA synthesis and ERT assay. It has been reported that retroviral genomic RNAs in virions are less stable than viral mRNA in the cell [34–36] and HIV-1 RNA genomes in the virions are fragmented into many small RNA polynucleotides [10]. In vitro cDNA synthesis assay does not need the ribonuclease and ligase activity of topoisomerase I but requires dissociation of topoisomerase I from RNA substrate (Fig. 5). Therefore, K532A which lacks ribonuclease and ligase activity could have dissociated from RNA substrate and enhanced in vitro cDNA synthesis as well as wt topoisomerase I. On the contrary, for ERT assay the ligase activity of topoisomerase I was quite important for enhancing HIV-1 replication. Thus,
516
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
K532A and R488A topoisomerase I mutants were inactive in ERT assay as shown in Fig. 3, while K532A was active as well as wt and Y723F in in vitro cDNA synthesis (Fig. 5). As described above, there were some differences in requirements between ERT assay and in vitro cDNA synthesis and the different phenomena were observed even in the cases in which the same nucleotide including AMP-PNP was applied to different assays. For in vitro cDNA synthesis, dissociation of topoisomerase I from RNA was required and ATP was more effective than ATP-cS or AMP-PNP probably due to the negative charge close to the c phosphate of ATP. However, as dissociation of topoisomerase I from RNA was not so important for ERT assay, it is most likely that ATP as well as AMP-PNP activated the religation of topoisomerase I and increased cDNA in ERT assay. It has been reported that Rous sarcoma virus RNA [37] and HIV-1 RNA (data not shown) were tightly bound with topoisomerase I; therefore, it is hypothesized that during viral budding, partially dimerized genomic RNA bound with topoisomerase I is packaged into virions in which nucleocapsid protein (NCp7) and other proteins enhance the formation of a complex and both ATP and topoisomerase I are involved in cDNA synthesis by reverse transcriptase after viral entry into target cells. The cellular ATP would regulate cDNA synthesis by dissociation of topoisomerase I with RNA. It has been demonstrated that the presence of only incomplete proviral DNA species in HIV-1-infected quiescent T cells appeared to complete the process of reverse transcription after cellular activation [38,39], and that even after activation, cells may not release progeny virus particles [40]. Hence, it appears that processes closely linked to certain cell cycle phases are also involved in the regulation of reverse transcription in vivo. In this regard, the cytoplasmic concentration of ATP is expected to affect reverse transcription directly. However, it is unclear if or how the concentration and turnover of ATP in the cytoplasm or nucleus are regulated during the cell cycle. Marked quantitative and qualitative changes in thymocyte energy metabolism are known to occur between resting and proliferating states [41,42], and mitogen stimulation induces a twofold increase in the ATP turnover rate in rat thymocytes. More information on the concentration and distribution of ATP during different phases of the cell cycle should help our understanding of HIV-1 reverse transcription and perhaps virus replication. The binding of topoisomerase I to genomic RNA and enhancement of cDNA synthesis would be involved in the rate of retrovirus recombination. In HIV-1 replication, recombination is considered to be an essential step in virus replication and this is estimated as two or three crossovers per genome per replication cycle [43]. Thus, it would be anticipated that topoisomerase I activity could
be important in recombination events which occur primarily during minus-strand DNA synthesis. Further studies will determine the exact role and importance of topoisomerase I in HIV-1 replication.
Acknowledgments We thank Dr. Hiroshi Yoshikura for suggestions and critical reading on the manuscript. This work was supported in part by grants from the Ministry of Education, Science, Technology, Sports and Culture and by grants from the Ministry of Health, Labour and Welfare, Japan, and the Japanese Foundation for AIDS Prevention.
References [1] M.T. Dittmar, A. McKnight, G. Simmons, P.R. Clapham, R.A. Weiss, P. Simmonds, Nature 385 (1997) 495–496. [2] Y. Feng, C.C. Broder, P.E. Kennedy, E.A. Berger, Science 272 (1996) 872–877. [3] P. Wei, M.E. Garber, S.M. Fang, W.H. Fischer, K.A. Jones, Cell 92 (1998) 451–462. [4] A. McKnight, P.R. Clapham, R.A. Weiss, Virology 201 (1994) 8–18. [5] R. Shibata, H. Sakai, M. Kawamura, K. Tokunaga, A. Adachi, J. Gen. Virol. 76 (Pt 11) (1995) 2723–2730. [6] J.H. Simon, G.A. Schockmel, P. Illei, W. James, J. Gen. Virol. 75 (Pt 10) (1994) 2615–2623. [7] J.L. Clever, T.G. Parslow, J. Virol. 71 (1997) 3407–3414. [8] B. Berkhout, J.L. van Wamel, J. Virol. 70 (1996) 6723–6732. [9] T. Hayashi, T. Shioda, Y. Iwakura, H. Shibuta, Virology 188 (1992) 590–599. [10] A. Mizuno, E. Ido, T. Goto, T. Kuwata, M. Nakai, M. Hayami, AIDS Res. Hum. Retroviruses 12 (1996) 793–800. [11] M. Haddrick, A.L. Lear, A.J. Cann, S. Heaphy, J. Mol. Biol. 259 (1996) 58–68. [12] J. Goncalves, Y. Korin, J. Zack, D. Gabuzda, J. Virol. 70 (1996) 8701–8709. [13] A.L. Lear, M. Haddrick, S. Heaphy, Virology 212 (1995) 47–57. [14] R.J. Gorelick, S.M. Nigida Jr., J.W. Bess Jr., L.O. Arthur, L.E. Henderson, A. Rein, J. Virol. 64 (1990) 3207–3211. [15] L.O. Arthur, J.W. Bess Jr., R.C. Sowder, R.E. Benveniste, D.L. Mann, J.C. Chermann, L.E. Henderson, Science 258 (1992) 1935–1938. [16] D. Jardine, G. Tachedjian, S. Locarnini, C. Birch, AIDS Res. Hum. Retroviruses 9 (1993) 1245–1250. [17] E. Priel, S.D. Showalter, M. Roberts, S. Oroszlan, S. Segal, M. Aboud, D.G. Blair, EMBO J. 9 (1990) 4167–4172. [18] H. Takahashi, M. Matsuda, A. Kojima, T. Sata, T. Andoh, T. Kurata, K. Nagashima, W.W. Hall, Proc. Natl. Acad. Sci. USA 92 (1995) 5694–5698. [19] N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T.N. Dhole, Y. Takebe, M. Matsuda, M. Tatsumi, AIDS Res. Hum. Retroviruses 15 (1999) 1321–1324. [20] A. Hachiya, S. Aizawa-Matsuoka, M. Tanaka, Y. Takahashi, S. Ida, H. Gatanaga, Y. Hirabayashi, A. Kojima, M. Tatsumi, S. Oka, Antimicrob. Agents Chemother. 45 (2001) 495–501. [21] J. Kimpton, M. Emerman, J. Virol. 66 (1992) 2232–2239. [22] J. Sekiguchi, S. Shuman, Mol. Cell 1 (1997) 89–97. [23] A.D. Jensen, J.Q. Svejstrup, Eur. J. Biochem. 236 (1996) 389–394. [24] J. Wittschieben, S. Shuman, Nucl. Acids Res. 25 (1997) 3001–3008. [25] S. Shuman, Mol. Cell 1 (1998) 741–748.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517 [26] A. Adachi, H.E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, M.A. Martin, J. Virol. 59 (1986) 284–291. [27] B. Hirt, J. Mol. Biol. 26 (1967) 365–369. [28] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease, Gene 77 (1989) 51–59. [29] M. Tobiume, M. Takahoko, M. Tatsumi, M. Matsuda, J. Virol. Methods 97 (2001) 151–158. [30] K. Tokunaga, M.L. Greenberg, M.A. Morse, R.I. Cumming, H.K. Lyerly, B.R. Cullen, J. Virol. 75 (2001) 6776–6785. [31] H. Takahashi, T. Iwata, Y. Kitagawa, Y. Shoya, R.H. Takahashi, K. Nagashima, T. Kurata, Hybridoma 19 (2000) 331–334. [32] J. Sekiguchi, S. Shuman, J. Biol. Chem. 269 (1994) 29760–29764. [33] K. Borroto-Esoda, L.R. Boone, Antiviral Res. 23 (1994) 235–249. [34] M. Butsch, K. Boris-Lawrie, J. Virol. 76 (2002) 3089–3094.
517
[35] J.G. Levin, M.J. Rosenak, Proc. Natl. Acad. Sci. USA 73 (1976) 1154–1158. [36] N. Dorman, A. Lever, J. Virol. 74 (2000) 11413–11417. [37] J.H. Weis, A.J. Faras, Virology 114 (1981) 563–566. [38] W.A. O’Brien, A. Namazi, H. Kalhor, S.H. Mao, J.A. Zack, I.S. Chen, J. Virol. 68 (1994) 1258–1263. [39] H. Schuitemaker, N.A. Kootstra, R.A. Fouchier, B. Hooibrink, F. Miedema, EMBO J. 13 (1994) 5929–5936. [40] S. Tang, B. Patterson, J.A. Levy, J. Virol. 69 (1995) 5659–5665. [41] F. Buttgereit, M.D. Brand, Biochem. J. 312 (Pt 1) (1995) 163–167. [42] T. Nilsson, V. Schultz, P.O. Berggren, B.E. Corkey, K. Tornheim, Biochem. J. 314 (Pt 1) (1996) 91–94. [43] A.E. Jetzt, H. Yu, G.J. Klarmann, Y. Ron, B.D. Preston, J.P. Dougherty, J. Virol. 74 (2000) 1234–1240.
Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1 Hidehiro Takahashi,a,* Hirofumi Sawa,b Hideki Hasegawa,a Yuko Shoya,a Tetsutaro Sata,a William W. Hall,c Kazuo Nagashima,b and Takeshi Kurataa a
Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan b Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, and CREST, JST, Japan c Department of Medical Microbiology, University College Dublin, Belfield, Dublin 4, Ireland Received 7 May 2002
Abstract Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I–RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: ATP; Topoisomerase I; HIV-1; Reverse transcription
Human immunodeficiency virus type 1 (HIV-1) replication is regulated not only at the viral entry stage, in which specific interactions of HIV gp120 with the primary receptor including CD4 and a number of chemokine receptors are critical [1–3] but also at the post-entry steps (reverse transcription, nuclear entry, integration, and transcription), where other cellular factors are involved [4–6]. HIV-1 particles contain two copies of a positivesense, single-stranded RNA genome and the genomic RNA exists in the form of a dimer. Two copies of genomic RNA in particles, which are substantially nicked [7–10], would require some regulatory factors for efficient reverse transcription. After thermal melting the HIV-1 RNA dimer yields only a small amount of the intact monomer and the remaining dimer degrades into *
Corresponding author. Fax: +81-3-5285-1189. E-mail address: [email protected] (H. Takahashi).
small RNA fragments which are recognized as smears on either gel electrophoresis [8,11–13] or Northern blot analysis [9,10,14]. These observations suggest that the retroviral genomic RNA is rapidly and substantially cleaved following viral assembly and budding and that some viral and/or cellular factors are required to facilitate and complete proviral DNA synthesis. HIV-1 particles are known to carry a number of cellular components which might be involved in suppression of small fragments of viral RNAs [15]. It has been reported that cellular topoisomerase I is present in retroviral particles and appears to play an important role in the replication of HIV-1 RNA [16,17]. We found that a hairpin-loop formation of RNA might be essential to form a complex of topoisomerase I and RNA; however, neither linearized nor double-stranded RNA could bind with topoisomerase I (unpublished data). Furthermore, we have previously demonstrated that topoisomerase I enhanced HIV-1 cDNA synthesis with
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 0 3 - X
510
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
in vitro reverse transcriptase (RT) assays [18]. Thus, it seems that topoisomerase I deeply affects replication of HIV-1 RNA. Therefore, it is hypothesized that topoisomerase I carried in HIV-1 virions plays an important role in HIV-1 cDNA synthesis in vivo. To investigate this, experiments such as indicator cell assays and endogenous reverse transcription (ERT) assays were performed. We show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I– RNA complex and enhance cDNA sythesis in vitro. In the present study, it is clearly demonstrated that cDNA synthesis of HIV-1 RNA is augmented by topoisomerase I and ATP.
Materials and methods The efficiency of infection of HIV-1 carrying wt or mutant topoisomerase I into MAGIC5 cells. HIV-1 proviral DNA (pIndie-C1, 1 lg) [19] and a mammalian expression vector (pcDNA4c-wt, pcDNA4c-Y723F, pcDNA4c-R488A, or pcDNA4c-K532A) carrying wt topoisomerase I, Y723F, R488A, or K532A cDNA (1 lg) were co-transfected into 293T cells using the MBS Mammalian Transfection Kit (Stratagene, CA). Thirty-six hours after transfection virus stocks were harvested by centrifugation of the supernatants, the p24gag concentration was measured using a HIV-1 p24 antigen ELISA Kit (Zepto Metrix, NY). As an indicator cell, about 104 HeLa-CD4-CCR5-LTR-b-gal (MAGIC5) cells [19,20] were plated on 96-well tissue culture plates and inoculated with serially diluted virus stocks. About 48 h after infection, cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-b-D -galactoside and the stained cells were counted as described earlier [21]. Expression of wt and mutant topoisomerase I were examined by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting using an anti-Xpress epitope antibody (Invitrogen, CA). As topoisomerase I mutants, Tyr-723, which is essential for endoribonuclease function [22–24], was substituted with Phe (Y723F), and Arg-488 or Lys-532, which is essential for ligase activity [23–25], was substituted with Ala (R488A or K532A). Endogenous reverse transcriptase (ERT) assay. Ten lg of uncleaved plasmid, pNL43 including intact open reading frames of all the HIV-1 accessory genes [26], was transfected into 106 of M8166 cells using a diethylaminoethyl (DEAE)-dextran transfection method. HIV-1 in the supernatant was further amplified by incubation with uninfected M8166 cells for three days, after which the supernatant was ultracentrifuged at 35,000 rpm for 3 h using a 50.2 TI rotor (Beckman, CA). Pellets were suspended in Tris-buffered saline [TBS; 25 mM Tris–HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl] and the protein concentration was adjusted to 1 mg/mL. Reaction mixtures [1 lg (Fig. 3), 5 lg (Fig. 2C) or 10 lg (Figs. 2A and B) of virion protein, 100 mM Tris–HCl (pH 8.0), 3 mM MgCl2 , and 0.2 mM each of deoxynucleoside triphosphate (dNTP), 0.01% NP40, nucleotide (Figs. 2 and 3) and purified wt or mutant topoisomerase I (Figs. 2C and 3) in 40 lL solution] were incubated at 37 °C for 3.5 h and the reaction was stopped by addition of 300 lL of the stop buffer [50 mM Tris–HCl (pH 8.0), 0.5% SDS, 10 mM EDTA and 100 lg=mL proteinase K]. The samples was then incubated at 56 °C for 1 h and treated with phenol/chloroform. The supernatant
Fig. 1. Influence of wt or mutant topoisomerase I on HIV-1 infectivity. The number of foci of MAGIC5 cells (per 100 pg of p24) infected with HIV-1 carrying recombinant wt or mutant topoisomerase I with Xpress epitope tag is represented as a bar graph (upper column). Expression levels of wt and mutant topoisomerase I were confirmed by SDS–PAGE followed by immunoblotting using an anti-Xpress epitope antibody (lower). Each bar represents the average of two independent experiments.
containing DNA products was precipitated in ethanol, centrifuged, resuspended with 0.3 N NaOH, and subjected to 0.8% agarose gel electrophoresis in an alkaline running buffer (30 mM NaOH and 2 mM EDTA). Following electrophoresis, DNA products were blotted onto nylon membranes (Pall, NY) and subjected to Southern blot hybridization. Extrachromosomal DNA from HIV-1-infected M8166 cells was extracted by the high salt precipitation method [27] and used as a positive control in Southern blot hybridization. Southern blot hybridization. Nylon membranes blotted with DNA were hybridized with 32 P-labeled 1.3 kb of HIV-1 gag fragment of pNL43 digested with BglII (679–2096 nt) in Quick Hybrisol (Stratagene). After hybridization, membranes were washed twice with 2 SSC (1 SSC; 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at 65 °C for 10 min, followed by two washings in 0:1 SSC containing 0.1% SDS at 65 °C for 30 min. Hybridized signals were visualized and quantitated with the FLA2000 (Fuji film, Japan). Expression and purification of recombinant proteins and enzyme. Human topoisomerase I cDNA was amplified by PCR, using the primers TF (50 -CGT CCC TCC GAATTC ATG AGT GGG GAC CA30 ) and TR (50 -GCC TCT TGA GCGGCC GCT AAA ACT CAT AGT CA-30 ) (underlined sequences correspond to EcoRI and NotI recognition sites, respectively). Three recombinant human topoisomerase I mutants were synthesized; Arg-488 or Lys-532 in wt topoisomerase I were substituted for Ala (designated R488A and K532A, respectively) and Tyr-723 was substituted for Phe (Y723F). Topoisomerase I mutant constructs were made by the overlap extension method [28]. Briefly described, primers 488F (50 -AAG CTT GCT CTG GCA GCA GGC AAT GAA-30 ), 488R (50 -TCC ATT GCC TGC TGC CAG AGC AAG
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
511
Fig. 3. Influence of wt or mutant topoisomerase I on HIV-1 cDNA synthesis. One lg of virions was incubated with 1 mM ATP and 2.5, 5, or 10 ng of purified K532A, R488A, Y723F, or wt topoisomerase I. No enzyme was added in the control reaction ()). Full-length HIV-1 cDNA is indicated by a black arrow.
Fig. 2. ERT assays in the presence of various amounts of nucleotides. Reaction mixtures contained 10 lg (A, B) or 5 lg (C) of virion and various amounts of nucleotides. (A) 0.1 or 1 mM of ADP, AMP-PNP, or ATP; (B) 1 mM of UTP, CTP or GTP, 0.1 or 1 mM of GTP-cS, or ATP; (C) 0.01, 0.1 or 1 mM of AMP-PNP in the absence of wt topoisomerase I, or 0.01 mM AMP-PNP in the presence of various amounts of wt topoisomerase I (1.25, 2.5, 5 or 10 ng) as shown in the panel. No nucleotide or enzyme was present in the control reaction ()). The synthesized cDNA was hybridized and detected with a 32 P-labeled HIV-1 gag probe. Black arrow indicates the position of full-length HIV-1 cDNA.
CTT-30 ), 532F (50 -TTT GAC TTC CTC GGG GCG GAC TCC ATC AGA TAC-30 ), 532R (50 -GTA TCT GAT GGA GTC CGC CCC GAG GAA GTC AAA-30 ), 723F (50 -TCC AAA CTC AAT TTT CTG GAC CCT AGG-30 ), and 723R (50 -CCT AGG GTC CAG AAA ATT GAG TTT GGA-30 ) (underlining denotes mutations) were used for amplification of mutant fragments; TF/488R and 488F/TR were used for R488A, TF/532R and 532F/TR for K532A, and TF/723R and 723F/TR for Y723F, respectively. These amplified fragments were combined in a subsequent fusion reaction. Wt and mutant human topoisomerase I cDNAs were subcloned into baculovirus transfer vectors with six Histag-sequences at the N-terminus (pFASTBAC HTa, BRL, MD) or a mammalian expression vector with an Xpress epitope tag at the N-terminus of the cloning site (pcDNA4/HisMax C, Invitrogen). Recombinant baculovirus was prepared according to manufacturer’s instructions. The nuclei of infected insect cells were resuspended with phosphate buffer [20 mM phosphate (pH 7.5), 1 M NaCl, 10 mM imidazole, and 1 tablet of protease inhibitor cocktail without EDTA
(Roche Diagnostics, IN)]. Following precipitation of polynucleotides with 6% polyethylene glycol 6000, supernatants was applied to HiTrap chelating columns (Amersham Pharmacia, UK). Five hundred mM of imidazole-eluted fractions were applied to 1.0 ml of either hydrophobic columns, Hitrap Phenyl FF (Amersham), or cation exchange columns, UNO S-1 (Bio-Rad, CA). Wt and mutant topoisomerase I were eluted from the columns by either low concentration ammonium sulfate or high concentration sodium chloride and separated by SDS–PAGE. After staining with SYPRO (Molecular Probes, OR), polyacrylamide gel was visualized with the FLA 2000. Wt HIV-1 RT (p66) was expressed in Escherichia coli (E. coli) using a His6 -tagged expression vector [pQE-9, (Qiagen)] encoding HIV-1 p66 (corresponding to 2250–4229 nt of pNL43). Proteins were purified using a HiTrap chelating column (Amersham Pharmacia) and cation-exchange chromatography (UNO S1, BIORAD). Preparation of RNA substrate. Precursor RNAs were prepared by in vitro transcription from PCR-amplified DNA with T7 RNA polymerase. To synthesize short RNA substrates, DNA fragments containing the T7 promoter and either of the p7 transcripts (76 nucleotides, nt), which are the processed proteins originating from HIV-1 p15gag or p15gag (394 nt), were amplified by PCR using pNL43 [26] as a template DNA with the following primers: p7F (50 -TAATAC GACTCACTA TAG GGA ACA AAT CCA GCT ACC ATA ATG ATA-30 ) and P7R (50 -ATT GAA ACA CTT AAC AGT CTT-30 ) (corresponding to 1900–1971 nt of pNL43); and P15F (50 -TAATAC GACTCACTATAG GGA ACA AAT CCA GCT ACC ATA ATG ATA-30 ) and P15R (50 -TTG TGA CGA GGG GTC GTC GCT GCC30 ) (underlining denotes T7 promoter, 1900–2289 nt). For doublestranded p15 substrate the primers were P15 antisense F (50 -TAATAC GACTCACTATAG GGA TTG TGA CGA GGG GTC GTC GCT GCC-30 ) and P15 antisense R (50 -ACA AAT CCA GCT ACC ATA ATG ATA-30 ) (underlining denotes T7 promoter). The precursor RNAs were 50 -end-labeled with adenosine ½c-32 Ptriphosphate and polynucleotide kinase after dephosphorylation with calf intestine
512
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
phosphatase (CIP). The precursor RNAs were purified by electrophoresis on 12% polyacrylamide/7 M urea denaturing gels using a Mini Whole Gel Eluter (Bio-Rad). Concentrations of RNAs stained by SYBER Green II (Molecular Probes) were analyzed with the FLA 2000. Analysis of binding of topoisomerase I to RNA substrates and cDNA synthesis. Ten ng of 50 -end-labeled RNA was heated in a Tris buffer [50 mM Tris–HCl (pH 8.0), 0.2 mM EDTA] at 95 °C for 4 min and quenched on ice for 5 min. Samples (20 lL) were incubated with 0.5% Brij35 (Sigma–Aldrich, WI) and 100 lg=mL nuclease free bovine serum albumin (BSA, Roche Diagnostics, IN) at 37 °C for 60 min to form structures. For investigation of the binding and dissociation of topoisomerase I to RNA substrates, 10 ng of p7 RNAs was cleaved in the above conditions and cleaved RNAs were incubated with topoisomerase I proteins at 37 °C for 10 min. Topoisomerase I–RNA mixtures were further incubated with either ATP, adenosine diphosphate (ADP), adenosine50 -[c-thio]triphosphate (ATP-cS) or adenosine 50 -[b; c-imido] triphosphate (AMP-PNP) at 37 °C for 2 min. Following addition of glycerol (final concentration, 10%) and dye (xylene cyanol and bromophenol blue), samples were electrophoresed onto a native 6% polyacrylamide gel containing 1 TBE (90 mM Tris–borate, 2 mM EDTA). Topoisomerase–RNA complexes were visualized and quantified with the FLA 2000. For analysis of cDNA synthesis, oligonucleotide primer (50 -TTG TGA CGA GGG GTC GCT GCC AAA GAG TGA-30 ) (corresponding to 2260–2289 nt of pNL43) was end-labeled with adenosine ½c-32 Ptriphosphate (NEN Research Product, UK) and T4 Polynucleotide Kinase (New England Biolabs, MA). Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was heated in the Tris buffer [50 mM Tris–HCl (pH 8.0), 0.2 mM EDTA] at 95 °C for 4 min and quenched on ice for 4 min. Samples (20 lL) were incubated with 0.5% Brij35, 100 lg=mL BSA, 5 mM MgCl2 , and topoisomerase I at 37 °C for 60 min. One nanogram of purified reverse transcriptase (RT) and either ATP or ADP was added to the annealed template-primer and allowed to bind for 4 min at 37 °C. Synthesis was initiated with the addition of solution containing dNTPs (80 lM, final concentration). The reactions were terminated by addition of an equal volume of a stop solution (90% formamide, 50 mM EDTA, 1.0% SDS, xylene cyanol, and bromophenol blue), heated at 95 °C for 2 min, and fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea.
Results Topoisomerase I mutants, R488A, and K532A inhibited the infectivity of HIV-1 Previously, we demonstrated that topoisomerase I enhanced cDNA synthesis in vitro [18]. To investigate the role of topoisomerase I in cDNA synthesis and replication of HIV-1, the infectivity of HIV-1 was initially examined in the presence of wild-type (wt) topoisomerase I and its mutants lacking endoribonuclease or ligase activities using a HeLa-CD4-CCR5-LTR-b-gal (MAGIC5) indicator cell system [20,29]. Compared with wt enzyme, topoisomerase I mutants, both R488A and K532A, significantly reduced the infectivity of HIV-1 by 75%, while Y723F had no effect, suggesting that HIV-1 infectivity was dependent on an activity of topoisomerase I which was lost by the mutations at 488-R and 532-K (Fig. 1). In addition, the infectivity of VSV-G
pseudotyped HIV-1 particles [30] in which nef was replaced with a luciferase indicator gene was also inhibited in the presence of R488A or K532A in producer cells (data not shown). The results suggest that the mutant topoisomerase I, R488A and K532A, reduced viral infectivity at a post-entry level. ATP and AMP-PNP activate HIV-1 cDNA synthesis We described that topoisomerase I enhances synthesis of a part of HIV-1 cDNA which is 1.48 kb in length with in vitro reverse transcription (RT) assays [18], while in this study we employed an ERT assay using detergentdisrupted HIV-1 virions to examine the physiological role of topoisomerase I in the full-length (9.4 kb) HIV-1 cDNA synthesis. It has been reported that HIV-1 virions carry topoisomerase I, which has DNA relaxation activity after disruption of virus particles [16,17], and that monoclonal antibodies against topoisomerase I inhibit cDNA synthesis with ERT assays [31]. Furthermore, ATP or non-hydrolyzable analogs have been considered to combine with topoisomerase I and enhance the topoisomerase I activity for DNA relaxation by the dissociation of the enzyme from substrates [32]. We thus investigated whether ATP could influence cDNA synthesis by activation of endogenous topoisomerase I with ERT assay. As shown in Fig. 2A, 0.1 or 1 mM ATP and adenosine 50 -[b; c-imido]triphosphate (AMP-PNP) activated HIV-1 full-length (9.7 kb) cDNA synthesis in a concentration-dependent manner, while 0.01, 0.1, and 1 mM ADP had subtle effects on cDNA production. Next, the effects of 0.1 or 1 mM guanosine 50 -(a-thio) triphosphate (GTP-cS) and 1 mM of other NTPs (UTP, CTP, and GTP) on cDNA synthesis were also examined by ERT assay and it was clearly demonstrated that neither GTP-cS nor other NTPs had no influence on full-length cDNA synthesis by comparison with ATP (Fig. 2B), suggesting that ATP which might activate endogenous virion-associated topoisomerase I exclusively enhanced cDNA synthesis in ERT assay. Next, the efficiency of cDNA synthesis in the presence of exogenous topoisomerase I (15, 62.5, 125, and 250 ng/ ml) and an ATP-analog, AMP-PNP (0.01, 0.1, and 1 mM), was investigated by ERT assay (Fig. 2C). In the absence of exogenous topoisomerase I, the synthesis of full-length cDNAs was increased in an AMP-PNPconcentration-dependent manner. Concentrations of topoisomerase I and 0.01 mM of AMP-PNP synergistically enhanced the efficiency of cDNA synthesis. In ERT assay, two major products reflecting fulllength and 6-kb transcripts were recognized (Fig. 2). The 6-kb subgenomic cDNA has been shown to hybridize with the 50 end of a negative-strand-specific probe, but not with the 30 end of an env-specific probe [33], suggesting that the 6-kb cDNA lacks the 30 region by incorrect jumping of R region.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
Wt and Y723F topoisomerase I enhances cDNA synthesis, while R488A and K532A attenuate it in the presence of ATP The infectivity of HIV-1 was reduced in the presence of the ligase activity mutants of topoisomerase I, R488A and K532A (Fig. 1); therefore, the effect of wt and endoribonuclease activity mutant topoisomerase I, Y723F (62.5, 125, or 250 ng/ml) on cDNA synthesis in the presence of 1 mM of ATP was examined by ERT assay. Wt or Y723F topoisomerase I resulted in an increased production of full-length cDNA in a concentrationdependent manner (Fig. 3). In contrast, both R488A and K532A mutants attenuated cDNA synthesis compared with the control, suggesting that these mutants work as a dominant negative form against the cDNA synthesis. These results showed that an activity of human topoisomerase I which was lost in R488A and
513
K532A were necessary for the activation and completion of HIV-1 cDNA synthesis in ERT assay. In vitro cDNA synthesis assay reveals that ATP dissociates topoisomerase I from RNA and excess amounts of topoisomerase I inhibit cDNA synthesis MAGIC5 cell assay (Fig. 1) and VSV-G pseudotyped HIV-1 assay (data not shown) revealed that an activity of topoisomerase I was required at the post-entry step; also, ERT assays (Figs. 2 and 3) showed that topoisomerase I and ATP increased cDNA synthesis. To analyze the relationship between topoisomerase I and ATP for in vitro cDNA synthesis, we initially examined the influence of ATP or its non-hydrolyzable analogs on the binding of topoisomerase I to RNA by native gel electrophoresis. ATP (3 mM) completely dissociated wt topoisomerase I from RNAs, while 3 mM ATP-cS and
Fig. 4. In vitro cDNA synthesis assay indicating the dissociation of topoisomerase I from RNA and inhibition of cDNA synthesis in the presence of excess amounts of topoisomerase I. (A) Dissociation of 2:7 lg=ml of wt topoisomerase I from p7 RNA transcripts in the presence of (0.1, 0.3, 1, or 3 mM) ATP-cS, (0.03, 0.1, 0.3, 1, or 3 mM) AMP-PNP or ATP was analyzed by native gel electrophoresis. The positions of topoisomerase I–RNA complex (Topo I–RNA complex) and precursor (Prec) and cleaved transcripts are indicated. (B), Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was incubated with 0.1, 0.3, 0.9, or 2:7 lg=ml of K532A or Y723F and 50 ng/ml of reverse transcriptase and fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea. The reaction was performed in the absence of ATP, AMP-PNP, and ATPcS. The position of remaining primers which were not used for cDNA is indicated as Primer. (C) The relative ratios of signal intensity of full-length to remaining primer were calculated and are demonstrated as a line graph, with the ratio in the presence of 0:1 lg=ml of topoisomerase I mutant being 100%. Open circles and closed circles represent the relative ratios of K532A and Y723F, respectively.
514
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
AMP-PNP had little influence on the binding of topoisomerase I (Fig. 4A). This result suggested that the dissociation of topoisomerase I from RNA was probably dependent on the charge close to the c site of ATP. Next, the effects of topoisomerase I on in vitro cDNA synthesis in the absence of ATP were examined. A reaction of in vitro cDNA synthesis was performed in the presence of 50 -labeled oligonucleotides as a primer, synthesized RNA as a template for reverse transcriptase, purified reverse transcriptase, dNTP, and large amounts (0.1, 0.3, 0.9, or 2:7 lg=ml) of wt or mutant topoisomerase I and analyzed by denaturing gel electrophoresis. As shown in Figs. 4B and C, the high concentration (2:7 lg=ml) of wt (data not shown), Y723F and K532A topoisomerase I reduced cDNA synthesis by 40%. It is
hypothesized that the binding of excess amounts of topoisomerase I with template RNA results in inhibition of cDNA synthesis. ATP results in enhancement of HIV-1 cDNA synthesis in the presence of excess amounts of topoisomerase I As excess amounts of topoisomerase I suppressed in vitro cDNA synthesis, the effect of ATP, expected to dissociate topoisomerase I from RNA substrate (Fig. 4A) and non-hydrolyzable analogs, on cDNA synthesis was investigated using in vitro cDNA synthesis assay. In the presence of a high concentration (2:7 lg=ml) of wt or K532A topoisomerase I, 2 mM of ATP enhanced cDNA synthesis much more effectively than did non-
Fig. 5. Analysis of cDNA synthesis in vitro. Ten ng of unlabeled p15 RNA and 50 -end-labeled oligonucleotide primer was incubated with wt (A), K532A (A, C), or Y723F topoisomerase I (B). Purified RT [50 ng/ml (A, B) or 6, 12, 25, and 50 ng/ml (C)] and either 2 mM of ATP (A–C), AMP-PNP (A), ATP-cS (A) or ADP (B, C) were added to the annealed template-primer and allowed to bind. Synthetic cDNA was fractionated by electrophoresis on a denaturing 15% acrylamide gel containing 7 M urea. The relative ratios of signal intensity of full-length to remaining primer were compared with regard to the ratio at time point 0 (A, B) or without RT ()) (C) as 1 and the fold increases are demonstrated in a line graph.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
hydrolyzable analogs in a time-dependent manner (Fig. 5A). Wt and K532A topoisomerase I similarly affected cDNA synthesis; therefore, it seemed that synthesis of full-length cDNA was dependent on the ATP-induced dissociation of topoisomerase I from RNA template. The effect of 2 mM ATP and ADP on cDNA synthesis was next investigated in the presence of 2:7 lg=ml Y723F topoisomerase I by in vitro cDNA synthesis assay (Fig. 5B). As shown in Fig. 5B, ATP activated cDNA synthesis in a time-dependent manner, while ADP had only a slight enhancing effect on the efficiency of cDNA synthesis, suggesting that the charge close to the c site of ATP was important for cDNA synthesis. In addition, we have shown the in vitro cDNA synthesis assay without topoisomerase I in the Fig. 5B. Fulllength cDNA was synthesized even in the absence of topoisomerase I, suggesting that topoisomerase I was not essential for cDNA synthesis; however, topoisomerase I in cooperation with ATP remarkably increased cDNA synthesis. Topoisomerase I mutants, R488A and K532A lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Finally, the efficiency of cDNA synthesis in the various concentrations (6, 12, 25, and 50 ng/ml) of reverse transcriptase (RT) in the presence of K532A topoisomerase I was investigated (Fig. 5C). It was demonstrated that in the presence of K532A (2:7 lg=ml), ATP was more effective in activation of cDNA synthesis than ADP and the efficiency of cDNA synthesis of both groups was dose-dependent to RT. Thus, HIV-1 cDNA synthesis in the presence of topoisomerase I was regulated by ATP and RT.
Discussion Previously, we demonstrated that topoisomerase I could enhance the reverse transcription of short HIV-1 templates in vitro in the presence of HIV-1 nucleocapsid proteins [18]. In the present study, we have extended these observations and first have demonstrated that the topoisomerase I mutants R488A and K532A reduced the infectivity of HIV-1 (Fig. 1). As the decrease of infectivity by topoisomerase I mutants was also observed with VSVG pseudotyped HIV-1 particles (data not shown), the step where topoisomerase I worked for HIV-1 replication was expected to be at the post-entry level. We confirmed that topoisomerase I affected HIV-1 cDNA synthesis and that ATP cooperated with topoisomerase I and enhanced cDNA synthesis using ERT assay (Figs. 2 and 3). Therefore, this is proof that topoisomerase I carried in HIV-1 virions plays an important role in HIV-1 cDNA
515
synthesis in vivo. In addition, although large amounts of topoisomerase I suppressed in vitro cDNA synthesis (Figs. 4B and C), ATP which causes dissociation of topoisomerase I from RNA substrates (Fig. 4A) enhanced in vitro cDNA synthesis even in the presence of excess amounts of topoisomerase I (Fig. 5). These results partly explain the relation of topoisomerase I and ATP in cDNA synthesis. Topoisomerase of the vaccinia virus has been shown to exhibit both endoribonuclease and ligase activities. Replacement of Tyr-274 with Phe and Arg-130 or Lys-167 with Alanine of the vaccinia virus topoisomerase results in the loss of ribonuclease activity and both ribonuclease and ligase activity, respectively [22,25]. Tyr-274, Arg-130, and Lys-167 residues that are well conserved in every member of the type IB topoisomerase family correspond to Tyr-723, Arg-488, and Lys-532 of human topoisomerase I, respectively [24], suggesting that Y723F human topoisomerase I mutant lacks ribonuclease activity and both R488A and K532A mutants are devoid of ribonuclease and ligase activity. As wt topoisomerase I and Y723F enhanced cDNA synthesis but R488A and K532A suppressed replication and cDNA synthesis of HIV-1, it is reasonable to assume that the ligase activity of topoisomerase I is essential for replication and enhancement of cDNA synthesis of HIV-1, while the ribonuclease activity of the enzyme is not necessary. The results of ERT assay were different from those obtained by in vitro cDNA synthesis assay in two points: initially, K532A topoisomerase I mutant could not enhance cDNA synthesis in the presence of ATP (Fig. 3); and second, AMP-PNP as well as ATP could enhance cDNA synthesis in the presence of endogenous topoisomerase I (Figs. 2A and C). These discrepancies seemed to be due to the differences of RNA template conditions between ERT assay and in vitro cDNA synthesis assay. For ERT assay, HIV-1 full-length genomic RNA (9.4 kb) was applied, while for in vitro cDNA synthesis assay, the synthesized RNA, HIV-1 p7 transcripts (76 nt) and p15gag (394 nt) were used as the template. There are some differences in requirements between in vitro cDNA synthesis and ERT assay. It has been reported that retroviral genomic RNAs in virions are less stable than viral mRNA in the cell [34–36] and HIV-1 RNA genomes in the virions are fragmented into many small RNA polynucleotides [10]. In vitro cDNA synthesis assay does not need the ribonuclease and ligase activity of topoisomerase I but requires dissociation of topoisomerase I from RNA substrate (Fig. 5). Therefore, K532A which lacks ribonuclease and ligase activity could have dissociated from RNA substrate and enhanced in vitro cDNA synthesis as well as wt topoisomerase I. On the contrary, for ERT assay the ligase activity of topoisomerase I was quite important for enhancing HIV-1 replication. Thus,
516
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517
K532A and R488A topoisomerase I mutants were inactive in ERT assay as shown in Fig. 3, while K532A was active as well as wt and Y723F in in vitro cDNA synthesis (Fig. 5). As described above, there were some differences in requirements between ERT assay and in vitro cDNA synthesis and the different phenomena were observed even in the cases in which the same nucleotide including AMP-PNP was applied to different assays. For in vitro cDNA synthesis, dissociation of topoisomerase I from RNA was required and ATP was more effective than ATP-cS or AMP-PNP probably due to the negative charge close to the c phosphate of ATP. However, as dissociation of topoisomerase I from RNA was not so important for ERT assay, it is most likely that ATP as well as AMP-PNP activated the religation of topoisomerase I and increased cDNA in ERT assay. It has been reported that Rous sarcoma virus RNA [37] and HIV-1 RNA (data not shown) were tightly bound with topoisomerase I; therefore, it is hypothesized that during viral budding, partially dimerized genomic RNA bound with topoisomerase I is packaged into virions in which nucleocapsid protein (NCp7) and other proteins enhance the formation of a complex and both ATP and topoisomerase I are involved in cDNA synthesis by reverse transcriptase after viral entry into target cells. The cellular ATP would regulate cDNA synthesis by dissociation of topoisomerase I with RNA. It has been demonstrated that the presence of only incomplete proviral DNA species in HIV-1-infected quiescent T cells appeared to complete the process of reverse transcription after cellular activation [38,39], and that even after activation, cells may not release progeny virus particles [40]. Hence, it appears that processes closely linked to certain cell cycle phases are also involved in the regulation of reverse transcription in vivo. In this regard, the cytoplasmic concentration of ATP is expected to affect reverse transcription directly. However, it is unclear if or how the concentration and turnover of ATP in the cytoplasm or nucleus are regulated during the cell cycle. Marked quantitative and qualitative changes in thymocyte energy metabolism are known to occur between resting and proliferating states [41,42], and mitogen stimulation induces a twofold increase in the ATP turnover rate in rat thymocytes. More information on the concentration and distribution of ATP during different phases of the cell cycle should help our understanding of HIV-1 reverse transcription and perhaps virus replication. The binding of topoisomerase I to genomic RNA and enhancement of cDNA synthesis would be involved in the rate of retrovirus recombination. In HIV-1 replication, recombination is considered to be an essential step in virus replication and this is estimated as two or three crossovers per genome per replication cycle [43]. Thus, it would be anticipated that topoisomerase I activity could
be important in recombination events which occur primarily during minus-strand DNA synthesis. Further studies will determine the exact role and importance of topoisomerase I in HIV-1 replication.
Acknowledgments We thank Dr. Hiroshi Yoshikura for suggestions and critical reading on the manuscript. This work was supported in part by grants from the Ministry of Education, Science, Technology, Sports and Culture and by grants from the Ministry of Health, Labour and Welfare, Japan, and the Japanese Foundation for AIDS Prevention.
References [1] M.T. Dittmar, A. McKnight, G. Simmons, P.R. Clapham, R.A. Weiss, P. Simmonds, Nature 385 (1997) 495–496. [2] Y. Feng, C.C. Broder, P.E. Kennedy, E.A. Berger, Science 272 (1996) 872–877. [3] P. Wei, M.E. Garber, S.M. Fang, W.H. Fischer, K.A. Jones, Cell 92 (1998) 451–462. [4] A. McKnight, P.R. Clapham, R.A. Weiss, Virology 201 (1994) 8–18. [5] R. Shibata, H. Sakai, M. Kawamura, K. Tokunaga, A. Adachi, J. Gen. Virol. 76 (Pt 11) (1995) 2723–2730. [6] J.H. Simon, G.A. Schockmel, P. Illei, W. James, J. Gen. Virol. 75 (Pt 10) (1994) 2615–2623. [7] J.L. Clever, T.G. Parslow, J. Virol. 71 (1997) 3407–3414. [8] B. Berkhout, J.L. van Wamel, J. Virol. 70 (1996) 6723–6732. [9] T. Hayashi, T. Shioda, Y. Iwakura, H. Shibuta, Virology 188 (1992) 590–599. [10] A. Mizuno, E. Ido, T. Goto, T. Kuwata, M. Nakai, M. Hayami, AIDS Res. Hum. Retroviruses 12 (1996) 793–800. [11] M. Haddrick, A.L. Lear, A.J. Cann, S. Heaphy, J. Mol. Biol. 259 (1996) 58–68. [12] J. Goncalves, Y. Korin, J. Zack, D. Gabuzda, J. Virol. 70 (1996) 8701–8709. [13] A.L. Lear, M. Haddrick, S. Heaphy, Virology 212 (1995) 47–57. [14] R.J. Gorelick, S.M. Nigida Jr., J.W. Bess Jr., L.O. Arthur, L.E. Henderson, A. Rein, J. Virol. 64 (1990) 3207–3211. [15] L.O. Arthur, J.W. Bess Jr., R.C. Sowder, R.E. Benveniste, D.L. Mann, J.C. Chermann, L.E. Henderson, Science 258 (1992) 1935–1938. [16] D. Jardine, G. Tachedjian, S. Locarnini, C. Birch, AIDS Res. Hum. Retroviruses 9 (1993) 1245–1250. [17] E. Priel, S.D. Showalter, M. Roberts, S. Oroszlan, S. Segal, M. Aboud, D.G. Blair, EMBO J. 9 (1990) 4167–4172. [18] H. Takahashi, M. Matsuda, A. Kojima, T. Sata, T. Andoh, T. Kurata, K. Nagashima, W.W. Hall, Proc. Natl. Acad. Sci. USA 92 (1995) 5694–5698. [19] N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T.N. Dhole, Y. Takebe, M. Matsuda, M. Tatsumi, AIDS Res. Hum. Retroviruses 15 (1999) 1321–1324. [20] A. Hachiya, S. Aizawa-Matsuoka, M. Tanaka, Y. Takahashi, S. Ida, H. Gatanaga, Y. Hirabayashi, A. Kojima, M. Tatsumi, S. Oka, Antimicrob. Agents Chemother. 45 (2001) 495–501. [21] J. Kimpton, M. Emerman, J. Virol. 66 (1992) 2232–2239. [22] J. Sekiguchi, S. Shuman, Mol. Cell 1 (1997) 89–97. [23] A.D. Jensen, J.Q. Svejstrup, Eur. J. Biochem. 236 (1996) 389–394. [24] J. Wittschieben, S. Shuman, Nucl. Acids Res. 25 (1997) 3001–3008. [25] S. Shuman, Mol. Cell 1 (1998) 741–748.
H. Takahashi et al. / Biochemical and Biophysical Research Communications 294 (2002) 509–517 [26] A. Adachi, H.E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, M.A. Martin, J. Virol. 59 (1986) 284–291. [27] B. Hirt, J. Mol. Biol. 26 (1967) 365–369. [28] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease, Gene 77 (1989) 51–59. [29] M. Tobiume, M. Takahoko, M. Tatsumi, M. Matsuda, J. Virol. Methods 97 (2001) 151–158. [30] K. Tokunaga, M.L. Greenberg, M.A. Morse, R.I. Cumming, H.K. Lyerly, B.R. Cullen, J. Virol. 75 (2001) 6776–6785. [31] H. Takahashi, T. Iwata, Y. Kitagawa, Y. Shoya, R.H. Takahashi, K. Nagashima, T. Kurata, Hybridoma 19 (2000) 331–334. [32] J. Sekiguchi, S. Shuman, J. Biol. Chem. 269 (1994) 29760–29764. [33] K. Borroto-Esoda, L.R. Boone, Antiviral Res. 23 (1994) 235–249. [34] M. Butsch, K. Boris-Lawrie, J. Virol. 76 (2002) 3089–3094.
517
[35] J.G. Levin, M.J. Rosenak, Proc. Natl. Acad. Sci. USA 73 (1976) 1154–1158. [36] N. Dorman, A. Lever, J. Virol. 74 (2000) 11413–11417. [37] J.H. Weis, A.J. Faras, Virology 114 (1981) 563–566. [38] W.A. O’Brien, A. Namazi, H. Kalhor, S.H. Mao, J.A. Zack, I.S. Chen, J. Virol. 68 (1994) 1258–1263. [39] H. Schuitemaker, N.A. Kootstra, R.A. Fouchier, B. Hooibrink, F. Miedema, EMBO J. 13 (1994) 5929–5936. [40] S. Tang, B. Patterson, J.A. Levy, J. Virol. 69 (1995) 5659–5665. [41] F. Buttgereit, M.D. Brand, Biochem. J. 312 (Pt 1) (1995) 163–167. [42] T. Nilsson, V. Schultz, P.O. Berggren, B.E. Corkey, K. Tornheim, Biochem. J. 314 (Pt 1) (1996) 91–94. [43] A.E. Jetzt, H. Yu, G.J. Klarmann, Y. Ron, B.D. Preston, J.P. Dougherty, J. Virol. 74 (2000) 1234–1240.