Specificity of Priming Reaction of H W 1 Reverse Transcriptase, 2”OH or 3’-0H*

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3'-OH group of the 3'-terminal adenosine residue of tRNA(Lys-3). In this report, we synthesized tRNA(Lys-3) of which the 3'-terminal adenosine residue lacks either a 2'-OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cell-free system consisting of a 141-base RNA template and purified HIV-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'-deoxyadenosine was able to initiate the cDNA synthesis, while the tRNA with the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3'-OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2'-OH group of an internal guanosine residue of a template RNA.

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3"OH group of the 3'4erminal adenosine residue of In this report, we synthesized tRNAun-3 of which the 3'4erminal adenosine residue lacks either a 2"OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cellfree system consisting of a 141-base RNA template and purified HW-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'deoxyadenosine was able to initiate the cDNA synthesis, while the tRNAwith the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3"OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2"OH group of an internal guanosine residue of a template RNA.
For the priming reaction of retroviral minus-strand DNA synthesis, tRNA molecules are known to be utilized (see Ref. 1 for a review). It is believed that the 3'4erminal adenosine residue of the tRNA molecules is used for the priming reaction forming a 3',5'-phosphodiester linkage between tRNA and the first nucleotide of cDNA. However, this has not been unambiguously demonstrated. In particular, two recent findings raised a serious question of whether retroviral reverse transcriptases may initiate cDNA synthesis from the 2"OH rather than the 3"OH group of their own individual primer tRNA molecules. The first finding is that seemingly primitive reverse transcriptases from bacterial retroelements exclusively prime cDNA synthesis from the 2"OH group of a n internal guanosine residue of the template RNA molecules forming a 2',5'-phosphodiester linkage (see Ref. 2 for a review). The second finding is that yeast retrotransposon Ty-1 requires a debranching enzyme for efficient transposition (3), raising the possibility that a 2',5'-phosphodiester linkage is formed during the cDNA syn-* This work was supported in part by a grant from the United States Public Health Service (to M. I. and S. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. thesis by its reverse transcriptase. In this report, we examined if HIV-l1 reverse transcriptase initiates its cDNA synthesis from either the 2"OH or the 3"OH group of the primer tR-  EXPERIMENTAL PROCEDURES Synthesis of the Mnucleotides, pCC (2'-Deoxy)ApThe cytidine and deoxyadenosine 2'-phosphoramidite unit was prepared from the reaction of 5'-dimethoxytrityl-N-protected-2'-0-l-(2-chloroethoxy~ethyl cytidine or 5'-dimethoxytrityl-N-protected-2'-deoxyadenosine with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (8). The oligonucleotides having 5',2'-terminal phosphates were synthesized by means of the phosphoramidite approach using the 2'-phosphates The unblocked oligomers were purified by use of reverse phase and ion-exchange HPLC. The product was used for determination of base composition by enzymatic degradation to nucleotides followed by HPLC analysis. Synthesis of (2'-0H) and tRNA"a-3 (3'-OH)-tRNA'-Ys-3 was synthesized in vitro by T7 RNA polymerase using pTL9, which contains the entire tRNAL.y"-3 gene under a T7 promoter (6). To amplify the tRNAL.ys-3 gene lacking the CCA sequence at the 3'-end, the following two PCR primers were used: primer 1,5'CGGCAG"TACG3'; and primer 2, 5'CGCCCGAACAGG3'. To amplify the full-size tRNAL.ys-3 gene, primer 1 and primer 3 (S'TGGCGCCCGAACB') were used. Primer 1 was designed to initiate PCR reactions immediately upstream of the T7 promoter. DNA fragments amplified were subsequently used to synthesize the tRNA in a cell-free system with T7 RNA polymerase (7). From the DNA fragment produced by primers I and 2, lacking the 3'-end CCAsequence (tRNALyn-3 (ACCA)) was synthesized. From the DNA fragment produced with primers 1 and 3, full-length tRNA'-rs-3 was synthesized. To synthesize tRNAL.y"-3 (2'-OH), 1 pg of tRNALy*-3 (ACCA) (37.5 pmol) was ligated with a trinucleotide, 5'-pCC (3'-deoxy)AZ'-OH (A,,, = 0.01; 375 pmol), by T4 RNA ligase (Pharmacia LKB Biotechnology Inc.) in 25 IMI HEPES buffer pH 7.5, containing 15 m MgCl,, 3.5 m dithiothreitol, 10 pg/ml bovine serum albumin, 15% dimethyl sulfoxide, and 50 ATP. The reaction was carried out in 20 pl at 16 "C for 16 h. tRNAL.yB-3 (3'-OH) was synthesized in the same manner as described above except that pCC (2"deoxy)Ap was used instead of pCC (3'-deoxy)A2'-OH. The 3"phosphate was removed by alkaline phosphatase after ligation. The product was analyzed by 7.5% polyacrylamide gel electrophoresis in 8 M urea.
H N cDNA Synthesis-The template was synthesized in vitro by T7 RNA polymerase using a 165-base pair DNA fragment. The DNA fragment was obtained by PCR using plasmid pNL4-3 (9) as a template and two appropriate primers. The RNA product synthesized by T7 RNA polymerase consisted of a 141-base nucleotide product encompassing a 75-base sequence consisting of a G residue plus 74 bases of U5 sequence, 18 bases of the primer binding site (PBS), a 43-base leader sequence downstream of the PBS, and an extra 5 bases. The sequence corresponds to the region from residue 562 to 696 of HIV-I NY5 from pNL4-3 (GenBank accession number M19921) (9). The 141-base RNA template contains a U5-leader stem structure, which is considered to enhance the retroviral cDNA initiation by reverse transcriptase in addition to the PBS sequence (5).
The cDNA synthesis was carried out as follows. 0.1 pg of tRNA and 0.1 pg of the template RNA were annealed in 40 m M Tris-HC1, pH 8.0, containing 50 IMI KC1 at 90 "C for 3 min followed by incubation at 37 "C for 30 min and at room temperature for 30 min. After annealing, dGTP  for 30 min in 20 pl of 50 rn Tris-HCI, pH 8.0, containing 8 n" MgC12, 50 m~ KCl, and 2 rn dithiothreitol. The reaction was stopped by adding 50 pl of the stop solution (20 rn EDTA, 0.5% SDS). The products were treated with ribonuclease A and analyzed by 7.5% polyacrylamide gel electrophoresis in 8 M urea, and the products were detected by autoradiography.

RESULTS AND DISCUSSION
T w o kinds of tRNALys-3 were synthesized; one tRNALys-3 contained only the 2'-OH group (3'-deoxy), designated tRNALys-' (2'-OH), and the other only a 3'-OH group (2'-deoxy), designated tRNALys-3 (3'-OH). First, tRNALys-3 lacking the 3"terminal CCA sequence was synthesized in vitro using a T7 expression system. To this 73-base tRNALys-3, synthetic pCC (3'deoxy)A2'-OH or pCC (2"deoxy)Ap was ligated with RNA ligase. In the latter case, the 3"phosphate was removed by alkaline phosphatase after ligation. The analysis of the resultant tRNALYs-3 is shown in Fig. 1. As a result  coded by a U5 sequence and one extra G residue at the 5'-end, which was added for efficient transcription from the T7 promoter. When the full-size tRNALys-' is used as a primer for cDNA synthesis by HIV-1 reverse transcriptase, 75 deoxyribonucleotides are added onto the tRNA. Digestion of this product with ribonuclease Ayields a 76-base product (75-base DNAplus one adenosine at the 5'-end; see reaction 1 in Fig. 2 A ) . On the other hand, the reaction with tRNALy8-' lacking CCA (ACCA) yields a 79-base product (78-base DNA plus one guanosine; see reaction 2 in Fig. 2 4 ) . These products are clearly observed in Fig. 2 B , lanes 1 and 2, for reactions 1 and 2, respectively. When the tRNAbS-' (T-OH) was used for the priming reaction, only a faint band at the 79-base position, but not at all at the 76-base position, was observed (Fig. 2 8 , lane 3 ) . This indicates that the full-length tRNALYs-' (Z'-OH) was not used as a primer, which would yield the 76-base band as illustrated in Fig. 2 A , reaction 3. The 79-base band in lane 3 is derived from the parental tRNALYa-3 (ACCA), which remained in the tR-NALY"-3 (2'-OH) preparation (see Fig. 1, lane 3). In contrast to tRNALys-3 (2'-OH), tRNALys-3 (3'-OH) was able to produce the 76-base band (Fig. 2 B , lane 4 ) as in the case of tRNALys-' (lane 1 ) . The 79-base band observed in lane 4 is again due to the unligated tRNALys-3 (ACCA) present in the reaction.
The present results now demonstrate that HIV-1 reverse transcriptase is able to prime cDNA synthesis from the 3'-OH group of the 3"terminal adenosine residue of tFtNALYs-3 but not from the 2'-OH group under the conditions used. However, in the present cell-free system, no other proteins other than reverse transcriptase were added, the tRNALYs-3 was not modified, and a shortened template RNA was used. Therefore, one may not completely rule out the possibility that HIV-1 reverse transcriptase in vivo is capable of initiating cDNA synthesis from the 2"OH group of the 3'-end adenosine residue of tRNALyS-'.