©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Use of an Oligoribonucleotide Containing the Polypurine Tract Sequence as a Primer by HIV Reverse Transcriptase (*)

(Received for publication, July 24, 1995)

Gloria M. Fuentes (1) Lorna Rodríguez-Rodríguez (2) Philip J. Fay (2) (3) Robert A. Bambara (1) (2) (4)(§)

From the  (1)Departments of Microbiology & Immunology, (2)Biochemistry, and (3)Medicine, and the (4)Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A primary site for initiation of plus strand DNA synthesis in human immunodeficiency virus (HIV) corresponds to a 19-nucleotide-long purine rich sequence located just upstream of the U3 region, designated the polypurine tract (PPT). The HIV reverse transcriptase (RT) uses its RNase H activity to cut the genomic RNA after minus strand DNA synthesis. A plus strand PPT primer is formed, extended, and then removed. In vitro, the HIV-RT recognizes this primer specifically, using it much more efficiently than other RNA primers. However, the PPT still primes significantly less efficiently than DNA primers. The 19-nucleotide PPT primer is partially resistant to degradation when compared with other oligoribonucleotides. Prior to initiation of DNA synthesis, several nucleotides are removed by the RT from the 3` ends of some of the PPT primers. Cleavage is enhanced in the absence of dNTPs. We suggest that DNA synthesis suppresses primer degradation, so that primer extension and cleavage occur in proper sequence. As a result of 3` end degradation, PPT elongation products contain 5`-RNA segments from 16 to 19 nucleotides in length. These shorter segments are also generated from a longer transcript containing the PPT sequence, indicating that they are not created as a result of binding of the RT to the 5` end of the PPT oligoribonucleotide. Full-length and shorter versions of the PPT primers are cleaved from the extended DNA by RT. These experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT but that the ends of the primer are not well defined.


INTRODUCTION

Retroviruses convert their RNA genomes into double stranded DNA by the process of reverse transcription (see (1) and (2) for reviews). The whole process of reverse transcription can be carried out in vitro by the viral RT. (^1)Reverse transcription starts from a cellular tRNA that binds near the 5` end of the virus to the primer binding site. Synthesis proceeds to the end, forming the minus strong stop DNA. Transfer of this DNA to the 3` end of either co-packaged RNA molecule is necessary to complete the minus strand DNA. During elongation of the minus strand DNA, the RNase H activity leaves behind oligoribonucleotides that could serve as primers for the second, or plus, strand. Plus strand synthesis is initiated from a purine-rich sequence, the PPT, located just upstream of the U3 region. Synthesis from the PPT forms the plus strong stop DNA. This DNA is transferred to the 3` end of the minus strand, and DNA synthesis proceeds. The result of reverse transcription is a double stranded DNA copy of the viral RNA genome. During this process the unique sequences found at the 3` and 5` end (U3 and U5) and a direct repeat (R) found at both ends of the viral genome are duplicated forming the long terminal repeats. The specific generation and removal of plus and minus strand primers are important events in integration because they define the ends of the long terminal repeats, which need to have the appropriate size and sequence for integration to occur (reviewed in (2) and (3) ).

All retroviruses contain one or multiple PPT sequences used to initiate synthesis of the plus strand(4, 5) . For HIV, it is still not clear whether the PPT located just upstream of the U3 region is the sole point of initiation of plus strand synthesis, the earliest point, or the major point. In most reverse transcription models, synthesis of the plus strand of DNA starts solely at this site. An additional initiation site has been reported with a sequence similar to the U3 PPT, and it is found at the middle of the genome(4) . Whether the selection of the U3 PPT as the preferred or only primer for plus strand synthesis is due to the absence of other primers or the inability of the enzyme to extend other RNA primers remains to be determined.

It has been observed that HIV-RT releases the PPT completely and intact after it has been used(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . This specificity is curious because the retroviral RNase H does not generally exhibit a sequence specificity when cleaving an RNA-DNA hybrid. The enzyme usually cleaves 17-21 nucleotides from the 5` recessed ends of RNAs annealed to DNA(16) . This is the distance that separates the RNase H and polymerase domains of the HIV-RT(17) . The RNase H activity of the HIV-RT makes a specific cut one ribonucleotide upstream of the RNA-DNA junction to remove the tRNA primer for the minus strand(18) . It is unclear whether the enzyme shows yet a different specificity to remove the PPT primer.

The sequence of events of the PPT primer formation, elongation, and removal in HIV is not well understood. Some steps in this process have been reconstituted in vitro by incubating HIV-RT with a long RNA-DNA hybrid containing the PPT sequence(6, 19) . When this hybrid contains the wild type sequence, a 19-nucleotide primer is created, extended, and removed intact(6) . When HIV-RT is incubated with a hybrid containing sequence changes in the PPT, the specificity of cleavage is altered(19) .

In order to recreate the events occurring after the primer has been generated, we synthesized a 19-nucleotide-long ribonucleotide with the HIV PPT sequence and measured its ability to support synthesis and subsequent degradation. This oligoribonucleotide primes significantly less efficiently than a DNA primer of the same sequence but is extended much more efficiently than RNA primers of other sequences. The PPT sequence is partially resistant to cleavage by HIV-RT. However a significant number of smaller primers are created in the region of the PPT by RNase H action of the HIV-RT. These shorter primers can also be used efficiently by HIV-RT. Overall, these results show that HIV-RT has a specificity to recognize the region of the PPT and use it to generate a plus strand primer but that the ends of the primer are not unique.


EXPERIMENTAL PROCEDURES

Materials

HIV-RT with native primary structure was provided by the Genetics Institute (Cambridge, MA). The enzyme had a specific activity of 40,000 units/mg. One unit is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37 °C using poly(rA)-oligo(dT) as template primer. The enzyme was divided into aliquots and stored at -70 °C, and a fresh aliquot was used for each experiment. An RNase H mutant of HIV-RT (D498N) was prepared as described by DeStefano et al. in (20) . This enzyme had a specific activity of approximately 8000 units/mg. T4 polynucleotide kinase, calf intestinal phosphatase, XbaI, SalI, KpnI, and glycogen were obtained from Boehringer Mannheim. The dNTPs and the RNA sequencing kit were from Pharmacia Biotech Inc. E. coli RNase H, PstI, and T3 RNA polymerase were purchased from Life Technologies, Inc. The Sequenase version 2.0 DNA sequencing kit was obtained from U. S. Biochemical Corp. pBS+ plasmid was from Stratagene Cloning Systems. [-P]ATP (3000 Ci/mmol) and [alpha-P]dATP (800 Ci/mmol) were purchased from DuPont NEN. The RNA PPT primer was synthesized by Midland Certified Co. The DNA PPT primer, the oligodeoxyribonucleotides containing the T7 promoter, and the 80- and the 60-mer DNA template were synthesized by Genosys, Inc. The other DNA templates were kindly synthesized by Bristol-Myers Squibb. All other chemicals were from Sigma.

Methods

HIV-RT Reactions

The substrate, 2 nM of primer-template, was preincubated with 4 units of enzyme for 5 min in 50 mM Tris-HCl (pH 8.0), 80 mM KCl, 1 mM dithiothreitol, and 0.1 mM EDTA, and the reactions started with the addition of 6 mM MgCl(2) and 50 µM of each dNTP. The reactions were performed in a final volume of 12.5 µl at 37 °C. The incubation time used in each experiment is specified under each figure legend. The reactions were stopped by adding an equal volume of a 2 times loading buffer (90% formamide (v/v), 10 mM EDTA (pH 8), 0.1% xylene cyanol, and 0.1% bromphenol blue).

HIV-RT Reactions Using [alpha-P]dATP

These reactions were performed as indicated above except the substrate concentration was 20 nM primer-template, and the reactions contained 1.25 µM of [alpha-P]dATP (800 Ci/mmol) and 5 µM of the other three dNTPs.

Run-off Transcription

Alternative RNA primers were made by run-off transcription using the T3 promoter of the pBS+ plasmid linearized with PstI (21-mer) or with SalI (31-mer) or from the T7 promoter linearized with XbaI (36-mer). The 46-nucleotide-long RNA primer was generated from run-off transcription of two annealed oligodeoxyribonucleotides containing the T7 promoter sequences. To generate 5` end-labeled primers, the transcript was treated with calf intestinal phosphatase and labeled with [-P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase.

Hybridization

The RNA or DNA primers were gel purified, quantitated by absorbance at 260 nm, and labeled at the 5` end with [-P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. The hybrids were prepared by mixing the primers (RNA or DNA) with the template at a ratio of 1:5 in 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, and 80 mM KCl. This mixture was heated to 65 °C for 10 min and then slowly cooled to room temperature. These primers were shown to be fully annealed by native gel electrophoresis.

Alkaline Hydrolysis

RNA fragments were degraded by incubating in 0.33 M NaOH at 65 °C for 15 min. The samples were neutralized with 0.33 M acetic acid, and they were ethanol precipitated using 3-5 volumes of ethanol and glycogen as a carrier. After precipitation the samples were resuspended in 2 times loading buffer. The base hydrolysis ladders were prepared by employing protocols supplied with the Pharmacia RNA sequencing kit.

Gel Purification of Elongated Primers

Cold PPT RNA primer annealed to template (200 nM) was extended with 4 units of HIV-RT for 1 h under the conditions described above, using 12.5 µM of [alpha-P]dATP (800 Ci/mmol) and 50 µM each dTTP, dCTP, and dGTP. The extended substrate was separated in a 10% denaturing gel. The appropriate bands were located by wet gel autoradiography, excised, and eluted using the crush and soak method(20) .

Sequencing Reactions

Sequence ladders were generated using the labeled DNA PPT primer using a DNA sequencing kit from U. S. Biochemical Corp. according to the manufacturer's instructions.

Gel Electrophoresis

Denaturing sequencing gels containing 7 M urea were prepared and subjected to electrophoresis as described previously(21) . The percentages of acrylamide used in individual gels are indicated in the figure legends. The gels were dried and analyzed by autoradiography. Quantitation of labeled species was done by a PhosphorImager (Molecular Dynamics) using the ImageQuant program.

Substrate Sequences

The sequences of the PPT containing primers and templates are shown below. The underlined section of the 80-mer corresponds to the sequences of the 60-mer template. The 19-mer PPT was constructed as an RNA or a DNA. The sequences are 5`-UUUUAAAAGAAAAGGGGGG (19), 5`-GGAAGGCAGCUGUAGAUCUUAGCCACUUUUUAAAAGAAAAGGGGGG (46), and TGTTCCGTCGACATCTAGAATCGGTGAAAAATTTTCTTTTCCCCCCTGACCTTCCCGATTAAGTGAGGGTTTFTTCTGTT-5` (80).

The sequence of the 60-mer template containing the KpnI restriction site is shown below. The underlined section corresponds to the KpnI recognition site, and the bold part corresponds to the PPT annealing region. The sequence is TCGGTGAAAAATTTTCTTTTCCCCCCTGACCATGGGCTTTAAGTGAGGGTTTCTCTTAAG-5` (60). The sequences of the alternative RNA primers are 5`-GGGAACAAAAGCUUGCAUGCC (21), 5`-GGGAACAAAAGCUUGCAUGCCUGCAGGUCGA (31), and 5`-GGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGA (36). These primers were annealed to a chemically synthesized, 75-nucleotide-long DNA template.


RESULTS

The PPT Is Relatively Resistant to Internal Cleavage by HIV-RT, and Cleavage Is Enhanced in the Absence of dNTPs

The RNA primer with the PPT sequence was annealed to a 60-nucleotide template containing the viral sequences of the PPT region. The resulting primer-template was incubated with HIV-RT in the absence and the presence of dNTPs (Fig. 1). The products generated over a time course were separated by electrophoresis on a 15% denaturing gel and analyzed by autoradiography. Some of the PPT was cleaved internally during the primer extension reaction, resulting in labeled products that were shorter than the intact PPT.


Figure 1: Time course of the degradation of the PPT oligoribonucleotide in the presence and absence of dNTPs. The 19-residue PPT oligoribonucleotide was 5` end-labeled and annealed to a 60-nucleotide-long DNA template. Standard HIV-RT reactions were performed as described under ``Experimental Procedures,'' and the reaction products were separated in a 15% polyacrylamide denaturing gel. The lane labeled L is a base hydrolysis ladder of the PPT oligoribonucleotide. The lane labeled C is a substrate control lane, which included all the reaction components except HIV-RT. The top arrow A indicates the formation of a PPT aggregate. The band at position 53 represents full-length extension product from the PPT. The band between positions 19 and 53 represents a pause site. The bands below position 19 represent cleavage products made by HIV-RT. The full-length extension product looks indistinct in this figure. When the products were separated on a 5% gel, it acquired the sharp appearance of a single species (not shown).



In reactions with dNTPs, full-length DNA extension products were observed (Fig. 1). Because the PPT was 5` end-labeled, this indicates that a portion of the extended PPT primers were not cleaved at all during the time of the reaction.

The earliest and most prominent observed cleavages occurred at positions 18 and 17 (Fig. 1). Subsequently other cleavages appeared. Cleavage at all sites was enhanced in the absence of dNTPs, but the cleavage pattern was unchanged. Degradation was quantitated by PhosphorImager analysis as the percent of radioactivity below position 19. Over the time course we measured up to 54% degradation in the absence of dNTPs and up to 37% degradation in the presence of dNTPs. This was an unexpected observation because previous results indicated that the PPT was removed intact by cleavage at the RNA-DNA junction (5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 19) . We had anticipated that DNA synthesis would be necessary before any cleavage and that the only cleavage would be between positions 19 and 20.

The PPT primers did not resist degradation by the RNase H of the RT because they were dissociated from the template. In control experiments using the D498N RNase H defective mutant of HIV-RT(20) , nearly all of the primers could be extended (data not shown). Furthermore, nearly all of the PPT primers were susceptible to cleavage by E. coli RNase H (data not shown). We also point out that a portion of the primers observed at position 19 after initiation of DNA synthesis are likely to have been extended and then cleaved at the RNA-DNA junction. This process would return them to their original position on the gel. The amount of the primers that had undergone such a process could not be quantitated in this experiment.

The upper band (designated A in Fig. 1) represents an aggregate formed by the PPT sequence. This product was first observed after labeling of the PPT. It was gel purified, and after base hydrolysis and electrophoresis it produced the normal PPT ladder of 19 nucleotides. Apparently the runs of guanosines in the PPT sequence can form extensive ``self-structures'' in solution. These structures have been documented in the literature and are referred to as guanine quartet structures(22) . Based on PhosphorImager analysis, the proportion of the PPT in the aggregate form remained approximately constant throughout our reactions (data not shown).

In order to determine whether the enhancement of cleavage was a phenomenon specific for the PPT sequence, we performed the same reactions using a 21-nucleotide-long RNA primer annealed to a 75-nucleotide-long template (Fig. 2). Using this substrate we observed up to 90% internal degradation in the presence and the absence of dNTPs. These results suggest that the PPT is resistant to cleavage when compared with other oligoribonucleotides. Furthermore, cleavage of other oligoribonucleotides does not appear to be affected by the presence of dNTPs because the percentage of cleaved products do not change in the presence and the absence of dNTPs. This primer could not be extended by HIV-RT under the same reaction conditions that resulted in extension of the PPT but could be extended with Sequenase (data not shown). Additionally we were unable to extend two other RNA primers of different sequences with HIV-RT (data not shown). These results support the observations of others(6, 14, 15) that the RT preferentially uses the PPT for synthesis compared with primers of other sequences.


Figure 2: Time course of the degradation of a 21-nucleotide-long oligoribonucleotide in the presence and absence of dNTPs. The 5` end-labeled oligoribonucleotide was annealed to a 75-nucleotide-long DNA template. Standard HIV-RT reactions were performed, and the reaction products were separated in a 15% polyacrylamide gel. The lane labeled L is a base hydrolysis ladder of the 5` end-labeled oligoribonucleotide. The lane labeled C is a substrate control lane, which included all the reaction components except HIV-RT. The bands below position 21 represent cleavage products made by HIV-RT.



The RNA PPT Is Not as Efficient for Priming as the DNA PPT

The above results show that the HIV-RT has some specificity for the use of RNA primers because it could extend the PPT but not three alternative RNA primers used in the earlier experiment. Theoretically, the efficiency of priming at the PPT could be quite low, but it would still be used by default if no other available RNA primers could sustain synthesis. To further assess the capacity of the PPT for priming, we compared the priming efficiency of the RNA PPT primer with a DNA primer of the same sequence. Both primers were unlabeled and extended in the presence of [alpha-P]dATP (Fig. 3). The DNA version of the PPT primed much more efficiently than the RNA version (about 15-fold). Priming efficiency was measured using internal label incorporation because the use of 5` end-labeled RNA primer could underestimate the amount of primer that is extended. This is because 5` end-labeled primers that are extended and later removed would appear as unreacted primers after denaturing polyacrylamide gel electrophoresis.


Figure 3: Priming efficiency of the RNA versus the DNA PPT primer. The same amount of unlabeled primers were annealed to the 60-nucleotide-long template and extended in the presence of [alpha-P]dATP with HIV-RT. The reaction products were separated in a 10% polyacrylamide denaturing gel. The first three lanes contain extension of the DNA primer, and the last three lanes contain extension of the RNA primer at 1, 7, and 15 min, respectively. The band at position 53 shows the full-length extension product.



Full-length and Shorter Versions of the PPT Prime Synthesis and All Are Removed Completely by HIV-RT

Because the label used in Fig. 1was at the 5` end of the PPT, we could not determine whether there was cleavage at the RNA-DNA junction. Furthermore, we could not distinguish whether the products smaller than 19 nucleotides resulted from degradation of unextended versus extended primers. When primers were extended using [alpha-P]dATP, we observed that products shorter than the full-length extension accumulated with time. One of these bands was particularly intense (position indicated by the arrow in Fig. 3). This product was gel purified to determine whether it was the full-length extension from which the PPT primer had been excised. After purification, the product was subjected to mild alkaline hydrolysis. This treatment degrades RNA but leaves DNA intact (6, 14) . The product was 34 nucleotides long and resistant to mild alkaline hydrolysis (Fig. 4). This is the correct size for the DNA extension product from the 19-nucleotide PPT primer after cleavage exactly at the RNA-DNA junction. The presumed full-length extension product, 53 nucleotides long, was also gel purified and subjected to mild alkaline hydrolysis. After hydrolysis and then electrophoresis, bands appeared corresponding to lengths of 34, 35, 36, and 37 as determined using a sequence ladder of the template (Fig. 4). These results indicate that the smaller cleavage products seen in Fig. 1were used as primers for plus strand synthesis. This would have resulted in addition of the 35, 36, and 37 nucleotide long segments of DNA that were measured after hydrolysis.


Figure 4: Analysis of gel purified products from the labeled extension of the RNA PPT primer with HIV-RT. The gel purified products were obtained as described under ``Experimental Procedures'' and separated in a 10% polyacrylamide denaturing gel. Lanes 1 and 2 contain the accumulating middle product (arrow on Fig. 3) with or without alkaline hydrolysis. Lanes 3 and 4 contain the full-length product with or without alkaline hydrolysis. Lanes T, A, C, and G contain sequencing reactions from the labeled DNA PPT primer. Sequencing reactions were performed as described under ``Experimental Procedures'' and served to identify the sizes of the products.



DNA Extension Products Are All the Same Length before Alkaline Hydrolysis

The full-length product at position 53 looks indistinct in Fig. 1. This suggested that the primers were not all extended to the same length. If so, interpretation of the above results would be compromised. When the extended product was gel purified from a 10% denaturing gel, only one band was observed and subsequently excised. However, the resolution for this size of product on a 10% denaturing gel may not have been sufficient to separate a group of products differing in length by 2 to 3 nucleotides. One possible reason that the full-length product would be mixture of sizes, was that the template was a mixture of slightly different length species. However, we labeled the template at the 5` end and showed that it is a single species by denaturing gel electrophoresis (data not shown).

It was also possible that a significant amount of non-template directed nucleotides were added to the end of the primer after it had been extended to the 5` end of the template. In order to determine whether the alkaline hydrolysis products observed above position 34 resulted from extension products having different lengths, the same experiment was performed using a 60-nucleotide template containing a KpnI restriction site (Fig. 5). Digestion with KpnI would then eliminate any differences in the length of primer extension. Note that the 53-nucleotide-long extended product looks indistinct even after gel purification (Fig. 5A). The KpnI digestion products should be 26 and 27 nucleotides long (Fig. 5B). After alkaline hydrolysis, the band at position 27, which contained the RNA primers, disappeared and bands representing the DNA portions of the 27 nucleotide segments appeared at positions 8, 9, 10, and 11. This experiment verifies that the multiple bands seen after alkaline hydrolysis are a result of extended DNAs containing different sizes of RNA primers.


Figure 5: Analysis of the PPT labeled extension product after KpnI digestion. Unlabeled RNA PPT was extended over a 60-mer template containing a KpnI restriction site, and the extended product was gel purified. A shows a 10% polyacrylamide denaturing gel containing the gel purified extension product (lane 1) and the gel purified extension product after alkaline hydrolysis (lane 2). In B, the extended product was reannealed to the template and digested with KpnI, and the products were separated in a 20% polyacrylamide denaturing gel. Lane 1 contains the gel purified extended product, and lane 2 contains the KpnI digestion products. The product that is approximately 30 nucleotides long may result from the star activity of KpnI, or it may be an aggregate formed by the RNA-containing fragments. Lane 3 contains the KpnI digestion products after alkaline hydrolysis. The remaining 26-mer has a slightly lower than expected mobility because of the higher concentration of residual salt in the sample, left from the alkaline hydrolysis. DNA sizes were determined using a DNA ladder generated by phosphodiesterase (PDE) digestion of an unrelated 5` end-labeled DNA (not shown).



The fragment at position 26 representing the end of the extended fragment is clearly a distinct species. The unique length of the 26-nucleotide fragment also shows that all primers were extended to the same length. This suggests that the indistinct appearance of the 53-nucleotide product results because the strand can assume a number of three-dimensional conformations that affect its mobility under some separation conditions.

Shorter Versions of the PPT Primer Are Removed Less Efficiently

It is notable that the predominant cleavage product seen in Fig. 2was 34 nucleotides long and that only faint bands were observed above this position. This suggested that primers shorter than 19 nucleotides were not efficiently cleaved off after full-length extension by RT. To further investigate the relative abundance of the 34-mer DNA compared with the other DNA species, we sampled a labeled primer extension reaction over a time course, subjecting the samples first to alkaline hydrolysis and then to electrophoresis (Fig. 6). Before alkaline hydrolysis the most intense band corresponded to 34 nucleotides. PhosphorImager analysis of a similar experiment showed this band to be 44-47% of the elongated DNAs with removed primers. The bands observed at positions above 34 added up to 53-56% of all the radioactivity in this region, indicating that RT can elongate and remove the shorter primers. After alkaline hydrolysis we observed bands at positions 34 and above since the early time points. In this case the band at position 34 corresponded only to 30-35% of the elongated DNAs with removed primers, whereas the bands at positions above 34 added up to 65-70% of the radioactivity. This modest change in ratios before and after alkaline treatment indicates that RT shows at least some preferential cleavage of the full-length PPT after extension.


Figure 6: Time course of the extension of the RNA PPT annealed to the 60-nucleotide template. Unlabeled primer-template was extended with HIV-RT in the presence of [alpha-P]dATP as described under ``Experimental Procedures.'' At each time point, an aliquot of the reaction was subjected to alkaline hydrolysis. The reaction products were separated in a 10% polyacrylamide denaturing gel. The product sizes were determined using sequence ladders as shown in Fig. 4.



Creation of Short Versions of the PPT Is Not Directed by the 5` End of the PPT Oligoribonucleotide

Previous results showed that the preferred cleavage of an oligoribonucleotide annealed to a DNA template occurs 17-21 nucleotides from the 5` end of the RNA (16) . We considered that the positions of cleavages creating the short primers observed in our system were influenced by the position of the 5` end of the primer and its effect on the binding location of the RT. To test this hypothesis, we designed a 46-nucleotide RNA primer containing the PPT sequence at the 3` end. The template was 80 nucleotides long and contained the same viral sequences in the 5` end as the previous template. Unlabeled primer-template was extended by HIV-RT in the presence of [alpha-P]dATP for two different time periods (Fig. 7). This longer transcript was also cut in the same locations as the 19-mer PPT primer because after primer removal we still observed bands at positions 34 and above. Additionally, we observed multiple bands around position 53, corresponding to products containing RNA primers with diverse 5` ends. The bands around position 53 correspond to DNA products containing RNA primers because they disappear after alkaline hydrolysis (data not shown). Fully extended but not cleaved, 46-mer was nearly undetectable under these conditions. Overall, these experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT, but the ends of the primer are not well defined.


Figure 7: Internally labeled extension of a 46-nucleotide-long RNA containing the PPT sequence at the 3` end. Unlabeled transcript was annealed to the 80-nucleotide-long template and extended with HIV-RT in the presence of [alpha-P]dATP. The reaction products were separated in a 10% polyacrylamide denaturing gel. The product sizes were determined using sequence ladders as shown in Fig. 4. The bands around position 53 correspond to DNA products containing RNA primers because they disappear after alkaline hydrolysis (not shown). The band at position 80 represents extension from the unprocessed 46-nucleotide-long RNA primer.



Removal of the Shorter Versions of the PPT Primer Is Affected by Template Sequences beyond the 5` End of the Primer

Using the 46-nucleotide RNA primer and the 80-nucleotide template, we observed that the bands above position 34 were intense even without alkaline hydrolysis. This suggests that removal of the shorter primers (discussed above) was more efficient using this template. To determine whether this was a result of the use of a primer with a longer 5` end or a template with a longer 3` extension, the 19-mer PPT primer was annealed to the 80-nucleotide template and extended with HIV-RT in the presence of [alpha-P]dATP (Fig. 8). The primer extension reaction was sampled over a time course, subjecting the samples first to alkaline hydrolysis and then to electrophoresis. Interestingly, the shorter versions of the PPT primer were removed efficiently when the longer template was employed. PhosphorImager analysis showed that the band at position 34 represents approximately 30% of all the elongated DNAs with removed primers before and after alkaline hydrolysis. This suggests that the enzyme binds to a template region upstream of the PPT and facilitates cleavage to remove the shorter primers. Experiments shown in Fig. 1and Fig. 3were repeated with the 80-mer template to ensure that the template was not affecting any other parameter. Results similar to those with the shorter template were obtained.


Figure 8: Time course of the extension of the RNA PPT annealed to the 80-nucleotide template. Unlabeled primer-template was extended with HIV-RT in the presence of [alpha-P]dATP as described under ``Experimental Procedures.'' At each time point, an aliquot of the reaction was subjected to alkaline hydrolysis. The reaction products were separated in a 10% polyacrylamide denaturing gel. The product sizes were determined using sequence ladders as shown in Fig. 4.




DISCUSSION

Efficient retroviral replication requires integration of the double stranded viral DNA into the chromosome of the infected cell. The specific generation and removal of plus and minus strand primers are important events in integration because they define the ends of the long terminal repeats. HIV-RT is responsible for the specific generation and removal of the plus strand primer. Huber and Richardson (6) reported that HIV-RT makes three specific cuts to process the 19-nucleotide PPT primer: one upstream to generate the 5` end and two downstream to generate the PPT and then release it after extension with DNA. They also observed that HIV-RT cleaved with low frequency at sites surrounding the 3` end of the PPT. The second major primer formed in their system was a 17-mer that was two nucleotides shorter from the 5` end.

Results shown here using a 19-residue oligoribonucleotide with the PPT sequence show that a significant proportion of the PPT primers are cleaved before extension. These shorter segments of the PPT can also prime synthesis by HIV-RT. The smaller PPT primers are also created using a 46-nucleotide RNA transcript containing the PPT sequences at the 3` end, indicating that the cleavages were not directed by the 5` end of the PPT oligoribonucleotide. Using the longer transcript we observed that RT also creates primers with diverse 5` ends. The full-length PPT and the shorter versions are removed completely by cleavage at the RNA-DNA junction. Efficient removal of the shorter versions required the RT to interact with template sequences upstream of the 5` end of the PPT. These results show that the enzyme has the specificity to recognize the PPT region as the plus strand initiation site but that the primers created have diversely positioned 3` and 5` ends.

Initiation of the plus strand in avian and murine retroviruses also occurs from polypurine tract sequences, although the PPT sequences in these viruses are different in size and sequence from that of HIV (7, 8, 10, 11, 12, 13, 14, 15, 19) . Early studies showed a loose specificity, within 1 or 2 nucleotides, for cleavages producing the PPT in avian retroviruses (8, 10) . It was found that a great proportion of the plus strands of avian and murine retroviruses retained their primers(8, 10, 11, 12) . However, a recent study by Randolph and Champoux (15) showed that the PPT in MuLV is formed, extended, and removed very precisely and very efficiently. They also showed that extended products containing the attached primer never comprised more than a small percentage of the total products and that cleavage of the PPT sequence is not influenced by the presence of dNTPs. Results shown here for HIV differ from those presented recently for MuLV but agree with those results found earlier for other retroviruses. As seen in Fig. 1, a portion of the extended products accumulate with time, showing that they retain their primers, and cleavage is influenced by the presence of dNTPs. Furthermore, there appears to be a relaxed specificity, within 3 or 4 nucleotides, for the cleavages to produce the HIV PPT. HIV PPTs of various lengths are all released by cleavage at the RNA-DNA junction.

A study by Pullen et al.(19) showed that HIV-RT can use the MuLV PPT sequence as a primer for plus strand synthesis, although their PPT sequences differ in two nucleotides. In that study, a series of MuLV PPT mutants were tested to determine sequence features of the PPT required for correct plus strand priming by HIV-RT. They found that when the sequences were changed in some positions, the position of cleavage by HIV-RT changed. Interestingly, the nucleotides that determined the specificity for MuLV-RT were different from those influencing the specificity of HIV-RT. This work suggests that certain bases in the PPT are important for the specificity observed in the generation of the plus strand primer.

The work presented here focuses on the action of the HIV-RT on the 5` and 3` ends of the PPT, after the initial 3` end cleavage that forms the primer. Our work shows a loose specificity in the HIV-RT cleavage even when we start with a PPT containing the wild type sequence. This loose specificity is found at the 3` and 5` ends of the primer. Our results indicate that the terminal RNA sequences are not critical to effective priming. Instead the internal hybrid structure seems to make the primer effective. This is most likely accomplished by promoting a helical structure that is recognized by the RT.

The PPT primer in HIV interacts differently with the RT in two respects compared with other oligonucleotides; it is relatively resistant to cleavage, and cleavage is enhanced in the absence of dNTPs. A possible explanation for resistance to cleavage is that the helical structure of the PPT-DNA hybrid resembles that of a wholly DNA primer-template. The presence of dNTPs may promote the binding of the polymerase active site of the RT to the 3` end of the PPT and to the 3` ends of subsequently added deoxynucleotides. This could either block RT binding at positions for RNA cleavage or sequester RTs to the DNA terminus so that they are not available for RNA cleavage. Alternatively, the addition of nucleotides to the PPT may alter its helical structure, making it less susceptible to cleavage. These results show that the RT promotes a sequential process of first elongating and then degrading hybrids that contain the PPT sequence.

We wanted to investigate what made the PPT the sole or most efficient primer for plus strand synthesis. The obvious possibilities are that it is the only primer available at the time of plus strand synthesis or that it is a much better primer than the other primers present at this time. Results from DeStefano et al. (23) show that after passage of the RT carrying out RNA directed DNA synthesis, approximately 15% of the RNA template remains as oligomers annealed to the newly synthesized DNA in sizes that range from 13 to 45 nucleotides. Theoretically, all of these fragments could serve as primers for plus strand synthesis. We were unsuccessful in trying to extend three different RNA primers with HIV-RT under the same conditions that the PPT RNA primer is extended. Randolph and Champoux (15) reported a similar observation in MuLV. In their system, three RNA primers containing the PPT sequence were extended by MuLV-RT but an RNA primer devoid of this sequence was not.

The PPT RNA primer was not utilized by the HIV-RT as efficiently as the DNA primer of the same sequence (Fig. 3). We expected the RNA PPT primer to be at least as efficient as the DNA primer, because it is the natural primer used by the enzyme. The fact that not even the RNA PPT is an efficient primer for HIV-RT suggests that the unique use of the RNA PPT primer for second strand synthesis results from the inability of other RNA primers to sustain synthesis.

The three-dimensional conformation formed by these purine-rich RNA-DNA hybrids may distinguish them from the other potential primers for synthesis. There have been some conformational studies that show that RNA-DNA hybrids form neither an A nor a B type conformation and that the helical parameters of the hybrids are highly dependent on their sequences(23, 24, 25) . It is conceivable that the PPT sequence forms a unique conformation with the DNA template that can be recognized by HIV-RT as a primer. The PPT sequence may form a conformation that resembles the B conformation formed by DNA primers in DNA-DNA hybrids. It has been our experience that DNA primers of any sequence are generally good primers for HIV-RT(16, 20, 23) .

The generation, usage, and removal of the PPT primer for plus strand synthesis is likely an essential step in retroviral replication. This makes the PPT a potential target for antiviral agents, particularly oligonucleotides. Understanding the steps involved is important for the development of new drugs and agents that can target this process.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM 49573 and AI 01146, Minority Predoctoral Fellowship 1F 31 GM 17200-01, Cancer Center Core Grant CA11198, and the James P. Wilmot Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-275-3269; Fax: 716-271-2683.

(^1)
The abbreviations used are: RT, reverse transcriptase; PPT, polypurine tract; HIV, human immunodeficiency virus; MuLV, murine leukemia virus.


ACKNOWLEDGEMENTS

We thank Dr. Jasbir Seehra from the Genetics Institute for the HIV-RT used in these studies. We also thank Dr. C. Palaniappan and Dr. S. H. Hughes for helpful suggestions and discussions on this work.


REFERENCES

  1. Gilboa, E., Mitra, S. W., Goff, S., and Baltimore, D. (1979) Cell 18, 93-100 [Medline] [Order article via Infotrieve]
  2. Whitecomb, J. M., and Hughes, S. H. (1992) Annu. Rev. Cell Biol. 8, 275-306 [CrossRef]
  3. Goff, S. P. (1992) Annu. Rev. Genet. 26, 527-544 [CrossRef][Medline] [Order article via Infotrieve]
  4. Charneau, P., and Clavel, F. (1991) J. Virol. 65, 2415-2421 [Medline] [Order article via Infotrieve]
  5. Rattray, A. J., and Champoux, J. J. (1987) J. Virol. 61, 2843-2851 [Medline] [Order article via Infotrieve]
  6. Huber, H. E., and Richardson, C. C. (1990) J. Biol. Chem. 265, 10565-10563 [Abstract/Free Full Text]
  7. Luo, G., Sharmeen, L., and Taylor, J. (1990) J. Virol. 64, 592-597 [Medline] [Order article via Infotrieve]
  8. Champoux, J. J., Gilboa, E., and Baltimore, D. (1984) J. Virol. 49, 686-691 [Medline] [Order article via Infotrieve]
  9. Whörl, B., and Moelling, K. (1990) Biochemistry 29, 10141-10147 [Medline] [Order article via Infotrieve]
  10. Smith, J. K., Cywinski, A., and Taylor, J. M. (1984) J. Virol. 52, 314-319 [Medline] [Order article via Infotrieve]
  11. Resnick, R., Omer, C. A., and Faras, A. J. (1984) J. Virol. 51, 813-821 [Medline] [Order article via Infotrieve]
  12. Finston, W. I., and Champoux, J. J. (1984) J. Virol. 51, 26-33 [Medline] [Order article via Infotrieve]
  13. Pullen, K. A., and Champoux, J. J. (1990) J. Virol. 64, 6274-6277 [Medline] [Order article via Infotrieve]
  14. Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456 [Medline] [Order article via Infotrieve]
  15. Randolph, C. A., and Champoux, J. J. (1994) J. Biol. Chem. 269, 19207-19215 [Abstract/Free Full Text]
  16. DeStefano, J. J., Mallaber, L. M., Fay, P. J., and Bambara, R. A. (1993) Nuc. Acids Res. 20, 4330-4338
  17. Kohlstaedt, L. A., Wang J., Friedman, J. M., Rice, P. A., and Steiz, T. A. (1992) Science 256, 1783-1790 [Medline] [Order article via Infotrieve]
  18. Pullen, K. A., Ishimoto, L. K., and Champoux, J. J. (1992) J. Virol. 66, 367-373 [Abstract]
  19. Pullen, K. A., Rattray, A. J., and Champoux, J. J. (1993) J. Biol. Chem. 268, 6221-6227 [Abstract/Free Full Text]
  20. DeStefano, J. J., Wu, W., Seehra, J., McCoy, J., Latson, D., Albone, E., Fay, P. J., and Bambara, R. A. (1994) Biochim. Biophys. Acta 1219, 380-388 [Medline] [Order article via Infotrieve]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 5.68-5.69, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Sen, D., and Gilbert, W. (1992) Methods Enzymol. 211, 191-199 [Medline] [Order article via Infotrieve]
  23. DeStefano, J. J., Mallaber, L. M., Fay, P. J., and Bambara, R. A. (1994) Nuc. Acids Res. 22, 3793-3800 [Abstract]
  24. Federoff, O. Y., Salazar, M., and Reid, B. R. (1993) J. Mol. Biol. 23, 509-523
  25. Salazar, M., Champoux, J. J., and Reid, B. R. (1993) Biochemistry 32, 739-744 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.