Generation and Characterization of Infectious Chimeric Clones between Human Immunodeficiency Virus Type 1 and Simian

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1 JOURNL OF VROLOGY, Dec. 1990, p X/90/ $02.00/0 Copyright 1990, merican Society for Microbiology Vol. 6, No. 12 Generation and Characterization of nfectious Chimeric Clones between Human mmunodeficiency Virus Type 1 and Simian mmunodeficiency Virus from an frican Green Monkey RR SHBT,1 HROYUK SK,' TKHRO KYOMSU,1 KNOR SHMOTO,1 MSNOR HYM,2 ND KO DCH1* Laboratory of Gene nalysis, Department of Viral Oncology,1 and Research Center for mmunodeficiency Virus,2 nstitute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 18 May 1990/ccepted 21 ugust 1990 series of chimeric clones of human immunodeficiency virus type 1 and simian immunodeficiency virus isolated from an frican green monkey was constructed in vitro. n transient transfection experiments, all clones produced virion-associated reverse transcriptase, gag proteins, and env proteins. Eight out of 10 chimeric viruses clearly grew in the human CD+ cell line C8166. Susceptibility of other CD+ cell lines, MT-, 3.01, and Molt clone 8, to infection with these viruses was also demonstrated. Human immunodeficiency virus type 1 (HV-1) and type 2 (HV-2) have been isolated from patients with acquired immunodeficiency syndrome and people at risk for the disease (3, 8, 31, 39). Recently, simian immunodeficiency viruses (SVs), closely related to HVs, also have been found in several species of nonhuman primates (, 10, 12, 21, 32, 36, 38, 51). Nucleotide sequence analyses of molecular clones have revealed that these lentiviruses share a similar genome organization represented by 5' long terminal repeat (LTR)-gag-pol-vif-(vpx)-(vpr)-tat-rev-(vpu)-env-nef-3' LTR (6, 19, 20, 2, 26, 27, 35, 0, 2, 52, 5). HV-1 has a unique gene, vpu, but does not contain vpx. vpx is conserved among HV-2 and various SVs other than an SV from a mandrill. ll HVs and SVs carry vpr, with the exception of an SV from an frican green monkey (SVGM). Despite the differences in the central regions of genomes, the biological properties of HV and SV isolates are very similar. They can infect human or simian CD+ cells in vitro and induce cytopathic effects (CPEs) in the infected cells. Some strains of SVs, after a long-term incubation period, cause a chronic debilitating disease in monkeys that is similar to human acquired immunodeficiency syndrome (5, 11, 30). We recently showed, by transfection and infection experiments, that the phenotypes of HV-2 and SVGM mutants generated in vitro were similar to those of HV-1 mutants, except for some with mutations in the central regions of the genomes (3, ). Furthermore, tat and rev trans activators could be exchanged among various viruses in transient expression systems (2, 9, 15, 2, 28, 3, 1, 3, 53). These observations indicate that primate lentiviruses are functionally related, as predicted from the similarity of genome organization. Compatibility of genes derived from distinct subgroups of lentiviruses within the viral replicative cycle has not yet been reported. There has been no information on functional complementation between HV and SV genes in a productive infection. To gain a better understanding of comparative virology and to find virus clones with potential use in acquired immunodeficiency syndrome research, we created a number of chimeric clones of HV-1 and SVGM and * Corresponding author. monitored them for their gene expression by transfection and infection experiments. MTERLS ND METHODS Cell culture and transfection. CD+ human leukemia cell lines 3.01 (18), Molt clone 8 (M-8) (29), and MT- (25), and a subclone of C8166 (7) were maintained in RPM 160 medium supplemented with 10% heat-inactivated fetal calf serum. human colon carcinoma cell line, SW80 (1), was maintained in Dulbecco modified Eagle medium containing 10% heat-inactivated fetal calf serum. For transfection, uncleaved plasmid DN was introduced into SW80 cells by the calcium phosphate coprecipitation method (22, 55). nfection. Culture supernatants of virus-producing cells were filtered (0.5-,um pore size), and appropriate volumes were added to CD+ leukemia cells as previously described (18). RT assays. Virion-associated reverse transcriptase (RT) activity was measured as previously described (56). Western immunoblotting. Lysates of transfected SW80 cells or infected leukemia cells were prepared as described previously (56), and proteins were resolved on 12% or 10% sodium dodecyl sulfate-polyacrylamide gels, followed by electrophoretic transfer onto nitrocellulose membranes. The membranes were incubated at room temperature with sera from individuals infected with HV-1 or SVGM overnight and with 1251-labeled protein for 3 h, washed, and visualized by autoradiography (56). DN constructs. The infectious molecular clones pnl32 (HV-1) and ps212 (SVGM) were described previously (1, ) (Fig. 1). ll recombinant clones of the two were constructed by standard recombinant DN technology with the restriction sites present in pnl32 and ps212 (Fig. 1). DN fragments with incompatible end structures were ligated by blunt-end ligations or with linkers (8-bp Cla and Xho linkers; Takara Shuzo Co. Ltd., Kyoto, Japan) (Fig. 1B). The resultant recombinant clones (prcns) were numbered serially. dditionally, tat mutants of prcns-1 (designated prcns-1-bm) and prcns-2 (prcns-2-st) were constructed. The former contained a deletion that was the same as that of the HV-1 tat mutant pnlbm (3) (16-bp deletion between Ban and Mva sites within the first coding exon of tat; Los lamos data bank, Los lamos, N.M.), and 5861

2 5862 SHBT ET L. pnl32 (HV-1) ps212 (SVagm) --gag 5 'LTR -rev 'LTR vif po... -tat m_ nef vpr vpu env Nar EooR Ssp Hpa Xho 5'LTR VPc env 3'LTR Ul -----rev l e vif J. VROL. Nar Kpn Stu Hind f B a 3 N Nak v )Qf f v h r- _- ~~ ~ _=U St V Ec --&Jr _ StEC XM EvyKp 5 -_- EC"K fyxh 6 -_ SsaSt fyh 7 _ Ss1St HiyHp 8 Sa,Kp 9 ^ mu e 10 _ FG. 1. Structures of the chimeric clones between HV-1 and SVGM. () Genome organization of HV-1 (pnl32) and SVGM (ps212). LTRs and open reading frames of the clones are based on nucleotide sequence data from Los lamos data bank for pnl32 and from a published article (20) for ps212. The arrowhead indicates the location of the env in-frame stop codon in ps212 (20). Restriction sites used to make recombinants are also indicated. The positions of restriction sites in viral genomes are as follows. For pnl32: Narl, primer binding region; EcoR, vpr; Ssp, vpu; Hpa, gp1 region of env; Xho, nef. For ps212: Narl, primer binding region; Kpn, vif; Stu, tat; Hind, gpl region of env (downstream from the in-frame stop codon); fll, nef. (B) Genome organization of chimeric clones prcns-1 to -10 (1 to 10, respectively). LTRs and open reading frames are represented by black boxes (derived from pnl32) or white boxes (from SVGM) as in panel. Speckled boxes represent truncated open reading frames due to recombination. rrowheads with letters indicate the restriction sites used for the construction of recombinants (Na, Nar; Ec, EcoR; Ss, Ssp; Hp, Hpa; Xh, Xho; St, Stu; Kp, Kpn; Hi, Hind; f, fll). ncompatible sites were joined as follows: prcns-1, blunt end ligation of Xho and fl; prcns-2, Xho linker insertion into the fll site; prcns-3, blunt end ligation of Stu (changed to a Cla site by a linker) and EcoR; prcns-, same as prcns-3; prcns-5, blunt end ligation of EcoR and Kpn; prcns-6, blunt end ligation of EcoR and Kpn and an Xho linker insertion into the fll site; prcns-7, Cla linker insertions into Ssp and Stu sites and an Xho linker insertion into the fl site; prcns-8, Cla linker insertions into Ssp and Stul sites and blunt end ligation of Hindlll and Hpa; prcns-9, blunt end ligation of Ssp (changed to a Cla site by a linker) and Kpn and an Xho linker insertion into the fll site; prcns-10, Cla linker insertion into the Stu site (indicated by an arrow) of prcns-9. f,xh the latter carried an insertion that was the same as that of the SVGM tat mutant ps-st (). RESULTS Transient gene expression of the chimeric clones. Previous works by us and others clearly indicate that, among various HV and SV genes, three structural genes (gag, pol, and env) and two regulatory genes (tat and rev) are essential for virus replication, whereas the other five genes are dispensable (13, 1, 16, 17, 23, 33, 37, 3-50). Taking these observations into account, we generated a series of chimeric clones with possible infectivity at the restriction sites present in virus genomes (Fig. 1). To determine whether various combinations of viral genes could affect the replication potential of recombinants, 10 constructs were made (Fig. 1B). Basically, our recombinants were classified into four major groups. Recombinants prcns-1 and -2 were

3 VOL. 6, 1990 NFECTOUS CHMERC CLONES BETWEEN HV-1 ND SVGM 5863 B NS1 NS2 Cr D NLBM S-St NS1BM NS2-St NL S Cr * * * - *0* * * FG. 2. Transient RT production in SW80 cells transfected with the chimeric clones. Samples of 15,ug () or 0,ug (B) of viral DN were transfected into SW80 cells, and RT production in the culture fluids 8 h later was determined. () RT production directed by pnl32 (32), ps212 (212), prcns-1 (NS1), and prcns-2 (NS2) along with that by their tat mutants, pnlbm (NL&BM), ps-st (S-St), prcns-lbm (NS1&BM), and prcns-2-st (NS2-St). (B) RT production by pnl32 (NL), ps212 (S), and prcns-1 to -10 (1 to 10, respectively). Cr, RT activity of the culture fluids from mock-transfected cells. HV-1 with SVGM LTRs and SVGM with HV-1 LTRs, respectively (no nef). The genomes of prcns-3 and - (HV-1 LTR at the 5' site) consisted of the 5' half of SVGM (gag, pol, vif, and vpx) and the 3' portion of HV-1 (tat, rev, vpu, env, and nef). n addition to the 5' portion of HV-1 (gag, pol, and vif), prcns-5 to -10 contained the following genes: vpx, tat, rev, and env of SVGM (prcns-5 and -6); vpr and tat (first coding exon) of HV-1 and rev and env of SVGM (prcns-7 and -8); vpr and tat (first coding exon) of HV-1 and vpx, tat, rev, and env of SVGM (prcns-9 and -10). There were also some structural differences with respect to the presence of intact SVGM nef (prcns-5 and -6), HV-1 nef (prcns-7 and -8), and SVGM tat (prcns-9 and -10). We tested the abilities of various chimeras to express viral genes by transfection experiments. SW80 cells were trans fected with the clones and monitored for marker gene expression. The tat activity in the proviral contexts of chimeric clones was evaluated by comparing RT production in the transfected cells (Fig. 2). Consistent with our previous report (3), mutations in tat of prcns-1 and -2, which contained a heterologous combination of LTR and tat, drastically reduced RT production, indicating that tat was active in the chimeric clones. Chimeric structures also had little effect on RT production by the other recombinants (Fig. 2B). The results of Western blot analysis of viral proteins specified by recombinants were also as expected (Fig. 3). The HV-1 products p2 (gag), p55 (gag precursor), and gpl20 (env) were recognized by the human antiserum (Fig. 3, lane b), whereas the monkey antiserum reacted with SVGM p2 (gag) and gpl20 (env) (Fig. 3B, lane c). The monkey serum was also reactive with HV-1 p2. However, the p2s were distinguishable by a slight size difference (Fig. 3B, lanes b and c). The protein profiles expressed by prcns-1 (Fig. 3, lanes d) and prcns-2 (lanes e) were the same as those of pnl32 (HV-1) (lanes b) and ps212 (SVGM) (lanes c), respectively. Whereas prcns-3 and - did produce gpl20 of HV-1 (Fig. 3, lane g) and p2 of SVGM (Fig. 3B, lane g) (data not shown for prcns-), recombinants prcns-5 to -10 expressed both p2 and p55 of HV-1 (Fig. 3, lane f) and gpl20 of SVGM (Fig. 3B, lane f) (data not shown for prcns-6 to -10). Growth of the chimeric viruses in CD+ cells. The susceptibilities of several CD+ cell lines, C8166 (7), MT- (25), 3.01 (18), and M-8 (29), to infection with various viruses were determined. T-cell lines were infected with cell-free virus samples derived from transfections and monitored for RT production and the appearance of CPEs for 3 to weeks. C8166 cells supported the replication of most of the virus clones (Fig., Table 1). Severe CPEs with concordant RT production were observed within 2 days after infection with parental viruses (virus from pnl32 and ps212 in Fig. ). n C8166 cells infected with recombinants (except viruses from prcns-1, -2, and -), similar evidence of productive infection with somewhat delayed kinetics was obtained (Fig., Table 1). Of the viruses not proven to be infectious in Fig., rcns-1 grew very poorly in C8166 cells as judged by coculture experiments (Table 1). Weak CPEs in C8166 cells infected with the rcns-2 virus were apparent several days postinfection and persisted thereafter (Table 1). Some viruses behaved differently in MT- cells (Fig. C, D, and E). a b cd e f 9 Ba b c d e f 9 gpl2o(s) gpl2o1h),:3..k ~~~~~~~~~w p2(h) FG. 3. Expression of viral proteins in SW80 cells transfected with the chimeric clones. Cell lysates were prepared 8 h after transfection, resolved on 12% polyacrylamide gels, and analyzed by Western blotting with either anti-hv-1 human serum () or anti-svgm frican green monkey serum (B). env gpl20 and gag p2 of HV-1 (H) or SVGM (S) are indicated. Size markers in kilodaltons are shown. Lanes: a, mock transfection; b, pnl32; c, ps212; d, prcns-1; e, prcns-2, f, prcns-5; g, prcns e

4 586 SHBT ET L. J. VROL days 32 e o :: X C B C days * C C D 32 k 7 8 c E days 0*-8 *.*e* days 32 * * C., FG.. Growth kinetics of the chimeric viruses in human CD+ cell lines C8166 (), 3.01 (B), and MT- (C to E). Cells were infected with cell-free virus samples obtained from transfected SW80 cells, and RT production was monitored at intervals. nput viral RT units for infection of cells were as follows:, 6 x 105 cpm to 5 x 105 cells; B, x 105 cpm to 1 x 106 cells; C, 7 x 105 cpm to 5 x 106 cells; D, 7.5 x 105 cpm to x 106 cells; E, 2 x 105 cpm to x 106 cells. 32, virus from pnl32 (parental HV-1); 212, virus from ps212 (parental SVGM); 1 to 10, recombinant viruses from prcns-1 to -10, respectively; k, virus from pnl-kp (HV-1 env frameshift mutant used as a negative control [1]); c, mock infection. The parental SVGM (virus from ps212) and the recombinant rcns-2 to - viruses did not propagate in MT- cells. n addition, the infection kinetics of MT- cells by various viruses were not always correlated to those observed in C8166 cells. n 3.01 and M-8 cells, the growth capacities of various virus clones were very limited in contrast to those in C8166 cells (Fig. B, Table 1). Only the parental HV-1 (virus from pnl32) and recombinant rcns-1 virus replicated in 3.01 cells (Fig. B). Replication of most of the recombinants was barely supported, if at all, in M-8 cells (Table 1). n total, the data presented in this section demonstrate that various HV-SV chimeras were replication competent, and that cell lines varied in responsiveness to the virus clones. Expression of viral proteins in the infected cells. The viral protein profiles expressed in the leukemia cells productively infected with various chimeric viruses were monitored by Western blotting (Fig. 5 and 6). C8166 or MT- cells were infected with cell-free viruses; when virus-induced CPEs appeared, cell lysates were prepared for Western analysis to ascertain the general structures of the replicating viruses. Figure 5 shows the viral proteins detected in C8166 cells infected with various virus clones. The human antiserum recognized a number of HV-1 proteins encoded by parental pnl32 in addition to gag products p55, p2, and env gpl20 (Fig. 5, lanes a and g), which were detected in the transfection experiments (Fig. 3). Most of these additional bands probably represented gag-related viral proteins because of their high reactivity with the human serum and cross-reaction with the monkey serum (Fig. SB, lanes a and g). The HV-1 pol product p66 was also observed (Fig. 5, lanes a and g). The frican green monkey serum recognized numerous viral proteins specified by parental ps212 (Fig. SB, lanes b and h) other than those observed in transient assays (gag p2 and env gpl20; see Fig. 3 for comparison), as did human serum. The very prominent bands, clearly observed also in Fig. SB, lanes d, j, k, and 1, were presumably glycoproteins encoded by the SVGM env gene. Of these viral proteins of SVGM, only gag p2 was easily

5 VOL. 6, 1990 TBLE 1. Viral clones Host range of the chimeric viruses between HV-1 and SVGM Transient RT productiona nfectivityb C8166 MT M-8 pnl ps prcns C + + _d prcns _ prcns prcns- + prcns d prcns d prcns d prcns d prcns prcns d a RT production in SW80 cells 8 h after transfection of viral DNs. RT activity relative to that directed by ps212 (the average of three to five independent experiments) was scored as + (30 to 120%), + + (130 to 20%), and (250 to 50%). b Virus growth was judged by RT production (C8166, MT-, and 3.01) or spreading, persistent CPE induction (M-8) after inoculation with cell-free supernatants of transfected SW80 cells. c Clear rise in RT production after cocultivation with infected MT- cells. d Persistent CPEs were observed (+) after infection of high-titer virus stocks (culture fluids of infected MT- or C8166 cells) or after cocultivation with infected MT- or C8166 cells. e Persistent weak CPEs with no clear RT production. detected by the human serum (Fig. 5, lanes b and h). n general, the protein profiles of recombinant viruses met the hybrid nature of the clones. The rcns-3 virus, which carried HV-1 env and SVGM gag, directed the synthesis of HV-1 gpl20 and SVGM p2 (Fig. 5, lanes c; note the size difference of p2s). The rcns-5 to -10 viruses carrying HV-1 gag-pol and SVGM env expressed HV-1 gag-pol and SVGM env products (Fig. 5, lanes d, e, j, k, 1, and m). The bands comigrating with HV-1 gp120 in Fig. S, lanes d, e, j, k, and 1, would be the HV-1 gag-pol precursor (the absence of the band in lanes b and h of part and the strong reactivities with frican green monkey serum in the regions in Fig. SB, lanes d, e, j, k, and 1). The recombinant rcns-2 virus, which induced CPEs (Table 1), did not express detectable viral proteins (Fig. 5, lanes i), in agreement with the lack of RT production (Fig. ). Western analysis of MT- cells infected with various virus clones gave data consistent with those from the infected C8166 cells (Fig. 6). n MT- cells, only the recombinants carrying the 5' half of the HV-1 genome grew (Fig., Table 1). HV-1 gag-pol products were expressed by all recombinants (Fig. 6, lanes c to e and j to 1). SVGM env proteins were produced by the same clones, except for the rcns-1 virus bearing HV-1 env (Fig. 6B, lanes c to e and j to 1). Monkey serum, which contains anti-simian T-lymphotropic virus antibody (our unpublished observation), reacted strongly with some proteins (indicated by asterisks in Fig. 6B) possibly originated from human T-lymphotropic virus (25). DSCUSSON major conclusion in this report is that the chimeric viruses of HV-1 and SVGM can grow in and induce CPEs in CD+ cell lines. series of recombinants generated in vitro all expressed viral proteins after transfection (Fig. 2B and 3), and some were clearly replication competent in the infected cells as determined by RT assays (Fig. ) and NFECTOUS CHMERC CLONES BETWEEN HV-1 ND SVGM 5865 a bc d e f g h j k mn B a b c d e f S:i.:... M tw. " _S!. 3 1-! :" m, _;. -; 0 --a - a- k - fi 0 _ _ 8 )0 --w 8 -J"; 2- --a if" 1 :3 3- NoQpw -- 0,.- 0 tw.m mwlo 9 h k mn,, s i r w S, FG. 5. Expression of viral proteins in C8166 cells infected with the chimeric viruses. Cell lysates were prepared, resolved on 10% polyacrylamide gels, and analyzed by Western blotting with human serum () or monkey serum (B). The same amount of cell lysates was applied to the corresponding lanes in panels and B. Size markers in kilodaltons are shown. The cells were infected with viruses derived from the following (lanes): a and g, pnl32; b and h, ps212; c, prcns-3; d, prcns-5; e, prcns-6; i, prcns-2; j, prcns-7; k, prcns-8; 1, prcns-9; m, prcns-10; f and n, negative controls (lysates from uninfected cells). Western blotting analyses (Fig. 5 and 6). The infectious recombinants also caused CPEs typical of HV and SV (Table 1). lthough predicted from previous genome analyses (6, 19, 20, 2, 26, 27, 33, 35, 0, 2-, 52, 5), no reports describing the viability of lentivirus recombinants have been formally presented to the best of our knowledge. The growth capacity and host range of the recombinant viruses can be summarized as follows. (i) ll chimeric clones grew less efficiently in CD+ cells than the parental viruses (Fig., Table 1). n particular, no evidence of infection was obtained for the rcns- virus. Clones that contained the recombination site at Nar (Fig. 1) tended to replicate poorly in CD+ cells (Table 1). There was no significant correlation between transient viral gene expression and the growth rate of recombinants (Fig. 2B and ; Table 1). (ii) Some recombinant clones could propagate in cell lines that were resistant to infection with one of the parental viruses, SVGM. Viral clones carrying the 5' half of the HV-1 genome (prcns-1 and prcns-5 to -10; Fig. 1) productively infected the MT- cells, and the recombinant rcns-1 virus (HV-1 with SVGM LTRs) grew in 3.01 cells (Fig. B, Table 1). (iii) Of the recombinant viruses that replicated relatively well in C8166 and MT- cells, those bearing the 3' portion of the SVGM genome infected M-8 cells (Table 1). (iv) mong chimeric clones consisting of the 5' half of HV-1 and the 3' half of SVGM (Fig. 1), the sites of recombination (and thus disrupting various genes) had little effect, if any, on the growth potentials of chimeras (Fig. ). t present, the molecular basis for observations i to iv is not known. Each recombinant showed distinct growth kinetics depending on the cell line used, and therefore many determinants may be

6 WJX 5866 SHBT ET L. a nc de fg r-nn d r)c f 9 h k rn n FG. 6. Expression of viral proteins in MT- cells infected with the chimeric viruses. Cell lysates obtained from infected cells were analyzed by Western blotting as in Fig. 5. n anti-hv-1 human serum () and an anti-svgm frican green monkey serum (B) were used for the detection of viral proteins. The same amount of cell lysates was applied to the corresponding lanes of panels and B. sterisks in panel B indicate the bands present in uninfected MT- cells (see the text). Size markers in kilodaltons are shown. MT- cells were infected with viruses derived from the following (lanes): b and i, pnl32; c, prcns-1; d, prcns-6; e, prcns-7; j, prcns-8; k, prcns-9; 1, prcns-10; a and h, negative controls (lysates from uninfected MT- cells). Control lysates prepared from M-8 cells infected with virus from ps212 (lanes f and m) and uninfected M-8 cells (lanes g and n) were also analyzed (see the text). responsible for the phenomena presented in this report. The expression of human T-lymphotropic virus would be one of the factors. MT- cells are known to be very susceptible to HV-1 infection and are producers of human T-lymphotropic virus (25). Human T-lymphotropic virus tax, but not gag, poi, or env, is expressed in C8166 cells, another exquisitely sensitive cell line (7). However, the growth kinetics of the recombinant viruses in C8166 and MT- cells were not parallel; some grew in human T-lymphotropic virus-negative 3.01 and M-8 cells (Fig. and Table 1). t is also possible that some mutation and/or recombination occurred in some of the chimeric clones to result in the phenotype observed here. These results also suggest the possible use of HV-SV chimeric viruses for animal experiments. Our final goal is the generation of recombinant HVs that can efficiently infect monkeys. By introducing the smallest amount of SV DN possible, we may be able to obtain chimeras mostly consisting of the HV genome. Toward this objective, construction and characterization of more recombinant clones are in progress in our laboratory. CKNOWLEDGMENTS We thank R.. Weiss for C8166 cells. This work was supported in part by a grant-in-aid for acquired immunodeficiency syndrome research from the Ministry of Education, Science, and Culture of Japan. J. VROL. LTERTURE CTED 1. dachi,., H. E. Gendelman, S. Koenig, T. Folks, R. Willey,. Rabson, and M.. Martin Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59: rya, S. K., and R. C. Gallo Human immunodeficiency virus type 2: analysis of regulatory elements. Proc. Natl. cad. Sci. US 85: Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. 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