Characterization of Active Reverse Transcriptase and Nucleoprotein Complexes of the Yeast Retrotransposon Ty3 in Vitro *

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively.In vitro binding of HIV-1 NCp7 to viral 5′ RNA and primer tRNA3 Lys catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNAi Met to a 5′-3′ bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5′ PBS (1). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5′-3′ PBS, primer tRNAi Met, NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Retrotransposons and retroviruses are members of a large family of mobile elements called long terminal repeat-contain-ing retroelements that share the same basic mechanisms responsible for their life cycles. During the early phases of replication, the single-stranded genomic RNA is converted into a double-stranded cDNA copy with two long terminal repeats by reverse transcriptase (RT), 1 followed by integration into the host genome by integrase (2)(3)(4). The RT and integrase enzymes, the RNA genome and primer tRNA that participate in this replication process are present within a nucleocore or nucleocapsid (5)(6)(7)(8)(9)(10)(11). For human immunodeficiency virus type 1 (HIV-1) and more generally for lenti-and oncoviruses, nucleocapsid protein (NCp) is the major structural protein of the nucleocore, in which about 2000 molecules coat the dimeric RNA genome (9 -11). HIV-1 NCp7 is a small basic protein possessing two CCHC motif zinc fingers (12)(13)(14) that appears to function as an RNA/DNA binding and annealing protein chaperone, promoting specific reverse transcription to generate a complete double-stranded cDNA copy with two long terminal repeats (15)(16)(17)(18)(19)(20)(21)(22)(23).
HIV-1 nucleoprotein complexes resembling the virion nucleocapsid are formed in vitro following binding of NCp7 to viral RNA and primer tRNA 3 Lys , resulting in the dimerization of viral RNA containing the packaging (psi or ⌿) sequence and annealing of primer tRNA to the PBS (15,21,24,25). Interestingly, interactions between NCp7 and RT appear to promote the recruitment of RT in these nucleoprotein complexes, resulting in the initiation of cDNA synthesis with subsequent elongation (26), corresponding to the early phases of viral DNA synthesis (3,16).
The yeast Ty3 retrotransposon also encodes an RT enzyme as well as a nucleocapsid protein, designated NCp9, with a unique zinc finger required for Ty3 transposition in yeast (27). Recently we have shown that Ty3 has an unexpected bipartite PBS composed of sequences located at opposite ends of the genome and that the Ty3 cDNA synthesis requires NCp9 and the 3Ј PBS for primer tRNA i Met annealing and the 5Ј PBS as the transcription start site (1). Dimerization of Ty3 RNA was also found to necessitate tRNA i Met , the 5Ј-3Ј PBS, and NCp9 (1). To compare nucleoprotein complex formation and the early phases of cDNA synthesis in retrotransposons to that in HIV-1, we have established a model system formed of Ty3 RNA composed of the 5Ј and 3Ј terminal domains with the 5Ј-3Ј PBS, primer tRNA, Ty3 NCp9, and for the first time, Ty3 RT. Our results show that Ty3 nucleoprotein complexes are formed in vitro, allowing Ty3 RT to synthesize strong stop cDNA (ss-cDNA). Interestingly, the N-terminal domain of NCp9, but not the zinc finger, was found to be necessary for the formation of active Ty3 nucleoprotein complexes in vitro.

RNA Substrates, NC Proteins, and Enzymes-Chimeric
Ty3 5Ј-3Ј RNA corresponding to nt 1-355 and 4724 -5011 with the repeat (R), the untranslated 5Ј region (U5), the 5Ј PBS, the polypurine tract (PPT), the 3Ј-untranslated region (U3), and the 3Ј PBSs was generated in vitro using pTy3-CG3 linearized by NheI and T7 RNA polymerase (1). Yeast tRNA i Met was kindly provided by G. Keith and B. Ehresmann (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France). Plasmid DNA HG300 encoding yeast tRNA i Met (28) was also used to generate synthetic tRNA in vitro using T7 RNA polymerase. All RNAs were purified by spin column chromatography (Amersham Pharmacia Biotech S-300 HR) and dissolved at 1 mg/ml in sterile water. [ 32 P]UMPlabeled tRNA i Met was synthesized in vitro using T7 RNA polymerase, purified by polyacrylamide gel electrophoresis (PAGE) in 7 M urea, recovered, and dissolved at 0.1 mg/ml in sterile water. Ty1 5Ј RNA (nt 1 to 587) was synthesized in vitro using T7 RNA polymerase (1). Ribosomal 28 S rRNA was extracted from mouse 3T3 cells and purified by agarose gel electrophoresis. Poly(rA):oligo(dT) was from Roche Inc.
Highly pure NCp7 (72 amino acids, containing 2 Zn 2ϩ ), Ty3 NCp9, and mutants of NCp9 were synthesized by the Fmoc/o-pentafluorophenyl ester chemical method and purified by HPLC as described for HIV-1 NCp7 (29). Wild type and mutant NCp9 as well as NCp7 stocks were reconstituted at 1 mg/ml in 20 mM Tris acetate, pH 6.5, 30 mM NaCl, and 1.5 eq of ZnCl 2 .
Ty3 RT was expressed from plasmid p6HTy3RT 2 as a 55-kDa protein containing a short polyhistidine extension at its N terminus. Recombinant protein was purified to near homogeneity by a combination of metal chelate and ion exchange chromatography as described previously (30) and shown to be free of contaminating nucleases. Recombinant HIV-1 RT was purified from Escherichia coli as described previously (30). Moloney MLV RT purified from E. coli was from Life Technologies, Inc.
Nucleic Acid Annealing Assay-Reactions with Ty3 RNA, in vitro synthesized 32 P-labeled tRNA, or natural primer 5Ј [ 32 P]tRNA and NC were incubated for 10 min at 28°C in 10 l containing 20 mM Tris-Cl, pH 7.5, 30 mM NaCl, 0.2 mM MgCl 2 , 5 mM dithiothreitol, 0.01 mM ZnCl 2 , 5 units of RNasin (Promega), 1.5 pmol of RNA, 3 pmol of in vitro synthesized tRNA (or natural tRNA) and NCp9, or NCp9 mutant at the indicated molar protein-to-nt ratios. Reactions were stopped by 0.5% SDS, 5 mM EDTA, treated with proteinase K (2 g) for 10 min at room temperature, and phenol-chloroform-extracted, and RNA was analyzed by 1.3% agarose gel electrophoresis in 50 mM Tris borate, pH 8.3, and visualized by ethidium bromide staining. Thereafter, gels were fixed in 5% trichloroacetic acid, dried, and subjected to autoradiography. A 0.16 -1.77-kilobase RNA ladder was used for size determination. The percentage of primer tRNA annealing to HIV-1 or Ty3 RNA was determined by scanning densitometry.
Reverse Transcription Assay-The reactions were performed basically as described previously (15,16). After 5 min at 30°C for the nucleic acid binding assay in 10 l (see above), the reaction volume was increased to 25 l by the addition of 1 pmol of Moloney MLV RT (Life Technologies, Inc.) or 0.5-1 pmol of Ty3 RT, dNTPs (dATP, dGTP, dTTP at 0.25 mM each, and dCTP at either 0.25 mM in assays with [ 32 P]tRNA or 0.030 mM with 2 Ci of [ 32 P]dCTP (Amersham Pharmacia Biotech) per reaction, 60 mM NaCl, and 2.5 mM MgCl 2 . Incubation was for 30 min at 30°C, after which the reaction was stopped and processed as for the analysis of tRNA annealing (see above), except that after phenol extraction, nucleic acid was ethanol-precipitated, recovered by centrifugation, dissolved in 15 l of formamide, and denatured at 95°C for 2 min, and 2 to 6 l was analyzed by 8% PAGE in 7 M urea and 50 mM Tris borate, 1 mM EDTA, pH 8.3. 5Ј 32 P-labeled ⌽X174 DNA HinfI markers (Pro-mega) were used for size determination (not shown). The levels of cDNA synthesized by RT were quantified by scanning densitometry.

Characterization of the Retrotransposon Ty3 RT-Recombi-
nant Ty3 RT was expressed in E. coli as a 55-kDa protein containing a short polyhistidine extension at its N terminus and purified to near homogeneity as described before (30). Using the synthetic poly(rA):oligo(dT) template-primer system, Ty3 RT was found to be as active as HIV-1 and MLV and HIV-1 RTs (Fig. 1). Interestingly, the poly(dT) products were found to have similar sizes with all three RTs assayed (from about 100 to 900 nt in length; data not shown).
One general feature of retroviral RTs is their potential to copy an RNA or a DNA template by means of a self-priming mechanism (20,21). A number of different RNAs were used as templates in reverse transcription reactions (Fig. 2, A and B). In all cases examined Ty3 RT was found to be completely inactive, whereas HIV-1 and MLV RT were found to be very active. Reverse transcription by self-initiation of templates such as 28 S rRNA, Ty1 and Ty3 RNAs is shown in Fig. 2, A and B. HIV-1 and MLV RTs were able to reverse-transcribe 28 S rRNA ( Fig. 2A, lanes 4 to 7), and Ty1 or Ty3 RNAs (Fig. 2B,  lanes 3, 4, 7 and 8), but retrotransposon Ty3 RT was not ( Fig.  2A, lanes 2 and 3; Fig. 2B, lanes 2 and 6). HIV-1 RT is also able to copy single-stranded DNA using a self-priming mechanism (21). Ty3 RT was found to be completely inactive on a singlestranded DNA in vitro (data not shown).

Characterization of Ty3 Nucleoprotein Complexes Formed with Wild Type Nucleocapsid Protein NCp9 and Deletion Mutants of NCp9 -Ty3
NCp9 is a basic protein with a single canonical CCHC zinc finger and a long N-terminal domain, whereas the C terminus is short (Fig. 3A). Enzymes were assayed at 30°C with poly(rA):oligo(dT) (160 ng/assay) in the presence of [ 32 P]dTTP as described under "Materials and Methods." Ty3, MLV, and HIV-1 RT were added at 1, 0.5, and 3 pmol per assay, respectively. Poly(dT) products were recovered by filtration through DEAE-cellulose membranes. 32 P-Labeled poly(dT) was quantitated by phosphorimaging and expressed as arbitrary units. Poly(dT) products were also analyzed by 8% PAGE in 7 M urea, and results show that they were from about 100 to 900 nt in length (data not shown).
prompted us to progressively delete part or all of the N-terminal domain as well as the zinc finger of Ty3 NCp9. Fig. 3 reports sequences of NCp9 deletion mutants together with the Ty3 5Ј-3Ј RNA used.
Complexes were formed by incubating 32 P-labeled Ty3 5Ј-3Ј RNA with NCp9 at increasing protein:nt stoichiometries at 30°C under conditions reported under "Materials and Methods." Equivalent ratios were used with deletion mutants ⌬1, ⌬2, ⌬3, NCp9 dd, and ⌬2-NCp9 dd. Nucleoprotein complexes were subsequently analyzed by PAGE in presence of 50 mM Tris borate but in the absence of a strong denaturing agent. Clearly wild type NCp9 was most effective in generating nucleoprotein complexes at a NCp9:nt ratio of 1:20 (Fig. 4, A and  B, compare lanes 2, 6, 10, 14 in A and lanes 2, 6, and 10 in B). Completion of the assembly process was obtained upon increasing molar NCp9-to-nt ratios from 1:10 to 1:5 for ⌬1, ⌬2, and NCp9 dd (lanes 7-8 and 11-12 in Fig. 4A and 7-8 in Fig. 4B). For ⌬3-NCp9, reactions were never complete (Fig. 4A, lane 16), whereas with ⌬2-NCp9 dd, complexes appeared to be unstable (Fig. 4B, lane 12). These results indicate that the N-terminal domain of NCp9 is an important determinant for nucleoprotein complex formation.
Effects of NCp9 Deletions on Primer tRNA i Met Annealing and 5Ј-3Ј RNA:tRNA Dimerization-As recently reported, dimerization of Ty3 RNA is mediated by NCp9-promoted annealing of primer tRNA i Met to the 5Ј-3Ј PBS. The palindromic sequence at the 5Ј end of tRNA i Met most probably directs dimerization since deletion of the 14 5Ј nucleotides of tRNA i Met abolishes tRNA and Ty3 RNA:tRNA dimerization (data not shown; see Ref. 1). Primer tRNA annealing and Ty3 RNA dimerization assays were carried out with 32 P-labeled tRNA i Met , Ty3 5Ј-3Ј RNA, and either wild type NCp9 or deletion mutants using NCp9:nt ratios varying from 1:10 to 1:2 required for full complex formation (see Fig. 4). Subsequently, reaction mixtures were treated with proteinase K, phenol-extracted to remove NC protein, and analyzed by agarose gel electrophoresis (see "Materials and Methods"). Fig. 5A shows Ty3 RNA:tRNA dimerization, whereas Fig. 5B reports [ 32 P]tRNA annealing. Clearly, NCp9 dd was as efficient as WT NCp9 in promoting tRNA annealing and Ty3 RNA:tRNA dimerization (Fig. 5, compare lanes 2-3  and 11-12). On the other hand ⌬1, ⌬2, and ⌬2-NCp9 dd were much less efficient (lanes 5, 7, and 13-14). ⌬3-NCp9 was found to be very poorly active in these processes (lanes 8 -9). As previously shown, HIV-1 NCp7 was also able to promote primer tRNA annealing and Ty3 RNA dimerization (1).
Ty3 RT Is Active on Ty3 Nucleoprotein Complexes Formed in Vitro-We have previously shown that the addition of MuLV RT and dNTPs to Ty3 RNA:tRNA:NCp9 complexes resulted in the synthesis of ss-cDNA, the initial product of reverse transcription. It was also shown that Ty3 NCp9, and HIV-1 NCp7 were interchangeable using Ty3 and HIV-1 template/primer systems (1). In an attempt to reconstitute a complete homologous Ty3 reverse transcription system, we used Ty3 RT expressed as a 55-kDa protein containing a short polyhistidine extension at its N terminus (see "Materials and Methods" and Fig. 1). Nucleoprotein complexes were formed at NCp9:nt ratios of 1:20 to 1:4 to ensure that all RNA template has been recruited into nucleoprotein complexes (Figs. 4 and 5). Next, Ty3 RT was added at a molar RT to template/primer ratio of 1:3, and reverse transcription was allowed to proceed for 30 min at 30°C. Reaction mixtures were treated twice with phenol:chloroform to remove all proteins, and cDNA products were heat-denatured and analyzed by 8% PAGE in 7 M urea. As a control without NCp9, primer tRNA i Met was heat-annealed to Ty3 5Ј-3Ј RNA for 30 min at 60°C.
Without prior annealing of [ 32 P]tRNA i Met and no NCp9, ss-cDNA could not be detected (Fig. 6, lane 1), while upon heat-annealing of primer tRNA a faint band corresponding in length to ss-cDNA was visualized (lane 2). The addition of NCp9 or NCp9 dd resulted in high levels of ss-cDNA synthesis (lanes 3-7). It should be noted that at an NCp:nt ratio of 1:10, the level of ss-cDNA in the presence of NCp9 dd was consistently 30 to 40% less than that with wild type NCp9 (lanes  4 -7). On the other hand, ss-cDNA synthesis was 10 to 15 times lower in the presence of ⌬1-NCp9 (lanes 8 and 9) and undetectable with all other NCp9 deletion mutants (data not shown). Interestingly, ss-cDNA synthesis was also much lower with HIV-1 NCp7 (lanes 10 and 11), in contrast to our previous report where NCp7 was as potent as NCp9 in promoting ss-cDNA synthesis but in conditions of MuLV RT excess (1). DISCUSSION The ubiquitous nature of the RT and NC protein among retroviruses and retrotransposons such as yeast Ty3 and Drosophila Copia prompted us to analyze the reverse transcription process in Ty3 and compare it with that in HIV-1, a distantly related long terminal repeat-containing retroelement belonging to the lentivirus family (12). Interestingly, Ty3 NCp9 and HIV-1 NCp7 are interchangeable in the Ty3 and HIV-1 template/primer systems, although Ty3 reverse transcription clearly differs from that of HIV-1 on the basis of NC protein and PBS sequences as well as on the mechanisms of RNA dimerization and initiation of minus-strand DNA synthesis (1). In addition, anti-NCp7 compounds (36) were found to inhibit Ty3 reverse transcription (data not shown), suggesting that yeast Ty3 can be used to screen new anti-NCp7 inhibitors capable of impairing HIV-1 replication.
To extensively analyze the reverse transcription process of a retrotransposon, namely yeast Ty3, and to compare it with that of HIV-1, we devised a functional in vitro Ty3 template-primer system consisting of a bipartite Ty3 5Ј-3Ј RNA template, the cognate tRNA primer (tRNA i Met ), and NCp9. A purified, recom-binant 55-kDa version of Ty3 RT was included in the system, since it was highly active on a synthetic poly(rA):oligo(dT) template-primer (Fig. 1). In addition Ty3 RT was found to be more specific than that of MLV or HIV-1, since it was unable to copy an RNA or a DNA template by self-priming ( Fig. 2 and data not shown). However, it is important to point out that Ty3 NCp9 (data not shown), like MLV NCp10 or HIV-1 NCp7, can extensively inhibit cDNA synthesis by self-priming using MLV or HIV-1 RT (21). Trivial explanations for this finding might have included either nuclease contamination and destruction of the RNA templates or an inability of the Ty3 RT to recognize and polymerize from an RNA 3Ј hydroxyl. The former notion appears unlikely, based on observations that incubation of Ty3 RT with radiolabeled RNA/DNA to evaluate its DNA polymerase and RNase H activities reveal no breakdown of the singlestranded RNA template. 3 Moreover, we have shown here that Ty3 RT will support synthesis of poly(dT) (Fig. 1) and of minusstrand strong-stop DNA from its cognate tRNA primer (Fig. 6).
FIG. 3. Mutants of Ty3 nucleocapsid protein NCp9. Panel A, NCp9, mutants, and HIV-1 (mal isolate) NCp7 were synthesized by the Fmoc chemistry and purified by HPLC, and their sequences were controlled. These NC proteins were used as described under "Materials and Methods." Sequences are shown using the one-letter code. Zn corresponds to the Zn 2ϩ ion coordinated by the CCHC residues. N-terminal deletion mutants are ⌬1-, ⌬2-, and ⌬3and represent deletion of residues 1-17, 1-28, and 1-34, respectively. NCp9 dd has a deletion of the N-terminal first 9 amino acids, and the zinc finger is replaced by a G residue, whereas in ⌬2-NCp9 dd the N-terminal first 28 residues and the zinc finger have been deleted. This unexpected property of Ty3 RT together with the chaperoning activity of NCp9 (Figs. 4 and 5; see also below) may have evolved to ensure that in intracellular virus-like particles, retrotransposon genomic RNA is selectively reverse-transcribed. This concept is presently under investigation.
To further examine the chaperoning properties of Ty3 NCp9 and compare them to HIV-1 NCp7, we synthesized NCp9 deletion mutants and examined their ability to (i) promote nucleoprotein complexes, (ii) direct primer tRNA i Met annealing to the PBSs, and (iii) catalyze Ty3 RNA:tRNA dimerization. We also examined the activity of Ty3 RT to specifically direct minusstrand strong-stop cDNA synthesis, the early product of the reverse transcription process, in Ty3 nucleoprotein complexes. Functions of the NCp9 deletion mutants in nucleoprotein complex formation, tRNA annealing and Ty3 RNA dimerization, and minus-strand cDNA synthesis are summarized in Table I. Clearly, only wild type NCp9 was optimal in all these functional assays. Nevertheless, deletion of the zinc finger (NCp9 dd) only had a moderate inhibitory impact on nucleoprotein complex formation, tRNA annealing, Ty3 RNA dimerization, and tRNA-primed reverse transcription (Figs. 4, 5, and 6). On the other hand, deleting N-terminal residues (⌬3-NCp9) resulted in an almost complete loss of activity in vitro. This is reminiscent of the findings with HIV-1 NCp7 where deletion of the two zinc fingers only had a moderate inhibitory effect on ϩ   nucleoprotein complexes ϩϩϩ, ϩϩ, and ϩ correspond to 70 -100, 30 -70, and 10 -30% WT activity, respectively. ⑀ means that activity was below 10%. ND is not determined. Note that only WT NCp9 and NCp9 dd were found to be highly active in tRNA annealing and dimerization. Also, only WT NCp9 was capable of completely inhibiting self-primed reverse transcription by MLV RT. RNA dimerization, tRNA 3 Lys annealing, and tRNA-primed cDNA synthesis, whereas N-terminal deletion resulted in a drastic inhibition of functions in vitro (29,31).
Retroviral NC protein has an important chaperoning function in reverse transcription in directing specific tRNA-primed cDNA synthesis. This appears to be achieved in two ways: first, by inhibiting nonspecific self-primed reverse transcription of the genome and of cellular RNAs and nonspecific replication of newly made DNA products (20,21); second, by promoting primer tRNA annealing and minus-strand DNA synthesis (15,29). Retrotransposon Ty3 NCp9 was also found to chaperone specific Ty3 RNA reverse transcription by a retroviral RT in a manner similar to HIV-1 NCp7 and MLV NCp10 (data not shown; Ref. 21). Using MLV RT, this takes place with maximal efficiency with WT NCp9 (Table I). As has been documented for HIV-1 NCp7, the N-terminal and zinc fingers domains are essential for nucleic acid binding (17,22,29,37,38), and this may well be the case for NCp9 due to the presence of basic residues in the N terminus and both basic and aromatic amino acids in the zinc finger, as for HIV-1 NCp7 (Fig. 3). Therefore, the zinc finger may well stabilize NCp9 oligomers bound to nucleic acids, causing destabilization of intramolecular structures (39,40), thus preventing self-primed cDNA synthesis while promoting formation of intermolecular duplexes in presence of primer tRNA and PBS-containing RNA.
The first reconstitution of active Ty3 NCp:RNA:tRNA:RT complexes will allow us to examine the relationships between RT and NC protein in a retrotransposon and compare them with the situation prevailing in HIV-1 (25,26,41) and to investigate the chaperoning functions of NCp9 in Ty3 reverse transcription more precisely at the level of minus-strand DNA transfer. It will also allow us to examine the contribution of the bipartite 5Ј-3Ј PBS, tRNA i Met , NCp9, and RT in the mechanism of plus-strand DNA transfer, most probably different from that in HIV-1 (42,43). Last, these data on NCp9 reinforce the notion that Ty3 is an ancestor of HIV-1, whereas Ty1 is probably more ancient (44), and characterization of an NC-like activity in Ty1 is presently under investigation.