©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage Specificities of Moloney Murine Leukemia Virus RNase H Implicated in the Second Strand Transfer During Reverse Transcription (*)

(Received for publication, June 22, 1995; and in revised form, August 9, 1995)

Sharon J. Schultz (§) Samuel H. Whiting (¶) James J. Champoux (**)

From the Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3` and 5` ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5` of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5` of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5` end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.


INTRODUCTION

The single-stranded plus sense RNA genome of a retrovirus is converted into double-stranded linear DNA by the process of reverse transcription (for review, see (1) and (2) ). At the two ends of the viral genome, there is a direct repeat (R) sequence that is bordered by an internal unique 5` (U5) or 3` (U3) sequence. Immediately downstream of the 5` R-U5 sequence is the primer binding site (PBS), (^1)which positions the primer tRNA used to initiate minus strand synthesis. During reverse transcription, these sequences are duplicated through two template jumps to generate long terminal repeats (LTRs), which are present at both ends of the unintegrated viral DNA and have the structure U3-R-U5 (1, 2, 3) . Upon integration into the host cell chromosome, two base pairs are lost from the termini such that the left LTR begins with the sequence 5`-TG . . . , and the right LTR ends with the sequence . . . CA-3` ( (4, 5, 6) and for review, see (7) ).

The enzyme responsible for reverse transcription is the viral-encoded reverse transcriptase (RT), which contains an RNA- and DNA-dependent DNA polymerase and an RNase H activity(1, 2) . The RNase H activity serves three distinct roles during reverse transcription (for review, see (8) ). First, RNase H degrades the viral RNA genome in the RNA-DNA hybrids generated during minus strand DNA synthesis (see Fig. 1). This degradation appears to be relatively nonspecific and produces fragments ranging from 2 to 20 bases in length (9, 10, 11, 12, 13, 14, 15, 16, 17) . While concomitant DNA synthesis is not required for RNase H activity(18, 19) , the positioning of the RNase H active site is strongly influenced by binding of the polymerase to a 3` DNA primer terminus(16, 18, 20, 21) . The second role of RNase H is to cleave the viral template at the polypurine tract (PPT), a purine-rich region immediately upstream of U3, to generate the primer for plus strand DNA synthesis. Unlike genome degradation, the PPT cleavage is sequence-specific and highly precise(22, 23, 24, 25, 26) . Third, RNase H removes the RNA primers used to initiate plus and minus strand DNA synthesis. Removal of the plus strand PPT primer uniformly occurs at the junction between the RNA primer and the first deoxynucleotide of plus strand DNA (25, 26) . In contrast, removal of the tRNA primer from minus strand DNA appears to depend on the type of virus examined. For avian myeloblastosis virus, the primer is cleaved precisely between the last ribonucleotide of the tRNA and the first deoxynucleotide of the minus strand(27, 28) . However, the tRNA primer of human immunodeficiency virus type 1 (HIV-1) is cleaved between the last and the penultimate ribonucleotides to leave a ribo A residue at the 5` end of the minus strand DNA(18, 29, 30) .


Figure 1: RNase H cleavage sites important for second jump in reverse transcription. The current model of reverse transcription is depicted in sequential steps (stages i-v) through the second template jump, with RNA strands as light lines and DNA strands as dark lines. Viral sequences that serve as landmarks for replication are shown in capital letters, and the complementary sequences are indicated with a prime. The 3` end of each strand is indicated by an arrowhead. For the second jump, RNase H cleavages are required in the regions marked I and II. RNase H cleavage of the genome template in region I (stage i) determines the maximal position to which the minus strand can be extended prior to the second jump (stage iv). RNase H cleavage in region II (stage iii) removes the tRNA primer to free the PBS sequences for the second jump and determines the 5` end of the minus strand (stage iv). The second jump intermediate is depicted in stage v.



The model of reverse transcription predicts that RNase H activity is necessary for both the first and second template jumps to occur (3) (see Fig. 1). After minus strand synthesis has extended the tRNA primer to the end of the genome (minus strong stop DNA; stage i), RNase H facilitates the first jump by degrading some portion of R at the 5` end of the viral RNA. This frees minus strand R` for transfer to R at the 3` end of the genome and continuation of synthesis (stage ii). Consistent with this model, RNase H cleaves the template RNA up to 14-24 nucleotides from its 5` end in DNA-RNA hybrids, including those which contain R(20, 31, 32, 33) . While some in vitro studies using model substrates and RNase H-deficient RT have found that RNase H activity is necessary for a template switch(15, 34) , others have reported that RNase H activity is not required(32, 35) . However, viral mutants lacking RNase H activity do not complete reverse transcription in vivo and only synthesize minus strong stop DNA in endogenous reactions (36).

For the second jump, cleavages by RNase H are important for three different stages of reverse transcription (see Fig. 1). The first cleavage occurs on the genome 5` of the PBS after minus strand synthesis has begun (designated ``region I,'' stage i). The 3`-most cleavage in this region determines the maximum point to which the minus strand DNA can be extended prior to the second jump (stage iv). Whether this RNase H cleavage is random or highly specific has not been previously addressed. The second RNase H cleavage removes the tRNA primer after plus strand synthesis has extended from the PPT primer through to the end of PBS (designated ``region II,'' stage iii). This cleavage determines the 5` end of the minus strand and presumably frees the 3` end of plus strand DNA for the second jump (stage iv). Finally, the PBS` sequence of the extended minus strand DNA is exposed by RNase H degradation of the genomic template (stage iv). Together, these RNase H cleavages permit the PBS sequence of the plus strand to pair with the PBS` sequence of the minus strand for the second jump (stage v). The specificity of RNase H for the first two cleavages is an intriguing issue, as any duplication in sequence between the 3` and 5` ends of minus strand DNA could generate a problematic branched intermediate subsequent to the second jump (stage v).

Using Moloney murine leukemia virus (M-MuLV) as a model system, we have examined the specificities of RNase H that define the two termini of minus strand DNA prior to the second jump. These results allow us to predict the predominant structure that would result from the second jump according to the current model of reverse transcription.


MATERIALS AND METHODS

Nucleic Acids

To generate single-stranded DNAs containing the R-U5-PBS region in both the plus and minus sense orientations, the 280-bp SfaNI-XmaIII fragment (positions 66-346 in the M-MuLV genome(37) ) was inserted into the HincII site of M13mp7 and clones of both the plus polarity (M13mp7/PBS) and minus polarity (M13mp7/PBS) were isolated. Clone pGMPBS was constructed by introducing the 1529-bp KpnI-XhoI fragment (positions 32-1560 in the M-MuLV genome) into the pGEM vector (Promega Corp.). Clone pGMPBSR was created by inserting the 165-bp PvuII-SfaNI fragment of pGMPBS into the SphI site of pGMPBS, digesting with PstI to release the 176- and 821-bp fragments in the M-MuLV sequence, and religating the DNA. A 738-bp region of the M-MuLV genome containing the entire LTR region was amplified by polymerase chain reaction and cloned into pBluescript II KS(+) at the BamHI and EcoRI restriction sites to generate a phagemid that produced single-stranded DNA corresponding to the plus sense LTR (pBSMOLTR+). The various recombinant M13 phages and DNAs and the M13 phagemid DNA were isolated by established procedures(38) .

Enzymes

Recombinant wild-type M-MuLV RT, T7 RNA polymerase, and Sequenase version 2.0 were purchased from U. S. Biochemical Corp., RNase H RTs (Superscript and Superscript II) were obtained from Life Technologies, Inc., and M-MuLV virion RT was purified as described previously(22) . Recombinant heterodimeric HIV-1 RT was kindly provided by Lawrence Loeb (University of Washington). Restriction enzymes and T4 DNA polymerase were purchased from New England Biolabs, Inc. T4 polynucleotide kinase was purchased from both U. S. Biochemical Corp. and Life Technologies, Inc.

Oligonucleotides and 5` End Labeling

DNA oligo 1 (5`-TGGCCAGCTTACCTCCCGG-3`) was purchased from Oligos Etc. DNA oligo 2 is represented by two oligonucleotides sharing the same 3` end but differing at the 5` end; oligo 2A (5`-TGGTCTCGCTGTTCCTTGGG-3`) was synthesized with a Biosearch model 8600 DNA synthesizer, and oligo 2B (5`-TGTGGTCTCGCTGTTCCTTGGG-3`) as well as the other DNA oligonucleotides described below were purchased from DNA Express. Results were identical using oligo 2A or oligo 2B. RNA oligo Mol15R (5`-CCGGACGAGCCCCCA-3`), which matches the 15 3`-most nucleotides of the tRNA primer, was kindly provided by Brian Reid (University of Washington). The first 30 bases of DNA oligo U5mis (5`-AAATGAAAGACCCCCGCTGACGGGTAGTCAGAGTG-3`) are complementary to the M-MuLV sequence from positions 146 to 117 with the five 3`-most nucleotides deliberately chosen to prevent base pairing with the genomic sequence. DNA oligo PB (5`-ATCCCGGACGAGCCCCC-3`) is complementary to the M-MuLV sequence from positions 163 to 147, which corresponds to the PBS without the 3`-terminal A residue. Oligo PBA is identical to oligo PB with the addition of three A residues on the 3` end.

Oligonucleotides were 5` end-labeled in a 15-µl reaction containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl(2), 5 mM dithiothreitol (DTT), 50 µg/ml of bovine serum albumin, 50 µCi of [-P]ATP (DuPont NEN), 17 pmol of oligonucleotide, and 2-30 units of T4 polynucleotide kinase. The reaction was incubated at 37 °C for 60 min and stopped with 10-50 mM EDTA. In most cases, the labeled oligonucleotides were separated from labeled ATP using a spin column containing Sephadex G-25 (Pharmacia Biotech Inc.)(38) .

M-MuLV Endogenous Reaction

M-MuLV virions were isolated as described previously (25) (final concentration 2 µg/ml of protein) and were incubated in a 250-µl reaction containing 50 mM Tris-Cl (pH 8.0), 50 mM KCl, 10 mM MgCl(2), 0.4 mM DTT, 0.01% Nonidet P-40, and 0.25 mM deoxynucleoside triphosphates (dNTPs) at 40 °C for the times indicated. After termination with 15 mM EDTA, SDS and proteinase K were added to final concentrations of 0.25% and 100 µg/ml, respectively, and the reactions were incubated at 37 °C for 60 min. Samples were extracted twice with phenol and twice with chloroform and precipitated with 20 µg/ml of glycogen (Boehringer Mannheim) in the presence of 0.3 M sodium acetate (pH 5.2) with two volumes of ethanol. After resuspension in 20 µl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), 1-4 µl of each sample was treated with 0.3 M NaOH at 65 °C for 20 min, neutralized with acetic acid, and ethanol precipitated prior to analysis by primer extension. For the zero time control, the sample was prepared identically except that virions were added to a 250-µl reaction that lacked any dNTPs, and the sample was directly purified without incubation.

Analysis of Plus Strand RNA Cleavages in U5 Proximal to the PBS

Production of RNA Template

To prepare RNA containing plus sense R-U5-PBS sequences, 2.5 µg of HindIII-linearized pGMPBSR was transcribed in a 100-µl reaction containing 40 mM Tris-HCl (pH 7.5), 10 mM MgCl(2), 5 mM DTT, 50 µg/ml of bovine serum albumin, 0.4 mM ribonucleoside triphosphates, 100 units of RNasin (Promega), and 40 units of T7 RNA polymerase at 37 °C for 90 min, and the DNA template was digested with 2.5 units of DNase I (Promega) at 37 °C for 15 min. The reaction was brought to a final volume of 200 µl with TE containing 0.1% SDS, and extracted once with phenol and twice with chloroform. The RNA was precipitated in the presence of 2.5 M ammonium acetate with 2.5 volumes of ethanol and resuspended in 15 µl of TE.

Preparation of Hybrid Substrates

A 50-µl reaction containing 120 ng of RNA oligo Mol15R and 2 µg of RNA template in 50 mM Tris-HCl (pH 8.0), 40 mM KCl, 6 mM MgCl(2), and 1 mM DTT was heated at 65 °C for 3 min and cooled at room temperature for 3 min. dNTPs were added to a final concentration of 0.5 mM each followed by 200 units of RNase H RT (Superscript II). The reaction was incubated at 37 °C for 30 min and terminated by the addition of EDTA to 10 mM. In addition to the desired extension of the RNA oligonucleotide, some snap-back self-priming resulted in the formation of an RNA-DNA hybrid region 3` of the PBS(39) . This led to some cleavage of the RNA in this region and reduced the overall signal in the primer extension assay but otherwise had no affect on the results. Hybrids were ethanol precipitated twice in the presence of 2.5 M ammonium acetate and resuspended in 10 µl of TE. Substrates in which the RNA primer was not extended were prepared by annealing the RNA primer to the RNA template followed by direct precipitation.

Treatment with RT

1.5 µl of the hybrids prepared above were treated with 250 units of recombinant M-MuLV RT in a 10-µl reaction containing 1 times RT buffer (50 mM Tris-HCl (pH 8.0), 10 mM MgCl(2), and 0.8 mM DTT) at 37 °C for 15 min and terminated by addition of EDTA to a final concentration of 10 mM. Sodium acetate was added to 0.3 M and the samples were ethanol precipitated. The pellet was resuspended in 7.5 µl of TE and analyzed by primer extension using oligo 1 (see Fig. 2A).


Figure 2: Assays for RNase H cleavage sites. The drawings show the different assays used to map RNase H cleavages in the genome 5` of the PBS (A) and the RNase H cleavage event that removes the tRNA primer (B). RNA strands are depicted as light lines, DNA strands as dark lines, and relevant viral sequences in capital letters. The asterisks indicate P-labeled 5` ends of RNA or DNA oligonucleotides as specified in the text, and the 3` end of each strand is indicated by an arrowhead. Depending on the experiment, the RNA paired with the PBS (PBS`) is either the RNA oligo Mol15R or the primer tRNA. A, primer extension from labeled oligo 1 maps cleavages on the plus strand genome RNA. B, extension from labeled oligo 2 was used to map the 5`-end of the minus strand DNA after primer removal and 5`-end-labeled Mol15R was used to determine the fate of the RNA primer.



Analysis of the 5` End of Minus Sense DNA

The 322-base insert of M13mp7/PBS was released from the single-stranded recombinant using EcoRI as described previously(22) . The resulting mixture of single-stranded insert and vector DNA is referred to as ``EcoRI-cut M13mp7/PBS DNA.''

Preparation of Substrates

490 ng of RNA oligo Mol15R was annealed to 3.5 µg of EcoRI-cut M13mp7/PBS DNA, and the RNA was extended with Sequenase essentially as described previously(29) .

Treatment with RT

One-fourth of the substrates prepared above were treated with 100-250 units of recombinant RT or 40 units of purified virion RT in a 10-µl reaction containing 1 times RT buffer at 37 °C for the indicated times. After termination with 10 mM EDTA, one-half of each reaction was treated with alkali as described above, brought to a final volume of 20 µl, and ethanol precipitated. The remaining half of each reaction was diluted to 20 µl with TE and ethanol precipitated in the presence of 0.3 M sodium acetate. All precipitates were resuspended in 7.5 µl of TE and analyzed by primer extension using oligo 2A or 2B as indicated in the figure legends (see Fig. 2B).

Primer Extension Analyses

One-half of each RT-treated sample and 0.4 pmol of the specified 5` end-labeled DNA oligonucleotide were used in primer extension reactions with RNase H RT (Superscript) essentially as described (29) or with 1 unit of T4 DNA polymerase in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl(2), 1 mM DTT, 0.2 mM dNTPs and analyzed in an 8% polyacrylamide gel containing 8 M urea.

Analysis of RNA Primer Fate

Preparation of Substrates

25 ng of 5` end-labeled RNA oligo Mol15R was annealed to 21 µg of EcoRI-cut M13mp7/PBS DNA in 1 times Sequenase buffer and extended with Sequenase as described above. The reaction was stopped by the addition of 25 µl of 5 times DNA sample buffer (25% Ficoll, 100 mM EDTA, 5% xylene cyanole, 5% bromphenol blue) and subjected to electrophoresis in a 5% polyacrylamide gel in a Tris borate/EDTA buffer(38) . The extended products were eluted from a gel slice in 0.5 M ammonium acetate, 1 mM EDTA (pH 7.9) for a total of 16 h, precipitated with 2 volumes of ethanol, and then reprecipitated in the presence of 0.3 M sodium acetate with two volumes of ethanol. The products were resuspended in a small volume of TE, heated at 60 °C for 5 min, and purified a second time by 5% polyacrylamide gel electrophoresis. Alternatively, reactions containing the extended products were terminated by adding an equal volume of formamide stop buffer and heating at 85 °C for 3 min, and the single-stranded extension products were isolated from a 5% polyacrylamide gel as described above. The recovered extension products were annealed to 2.4 µg of cut M13mp7/PBS DNA in 20 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 1 mM EDTA at 65 °C for 10 min, cooled to room temperature for 20 min, and precipitated with ethanol. To generate substrates in which the RNA primer was not extended, RNA oligo Mol15R was annealed to EcoRI-cut M13mp7/PBS DNA and isolated directly from a 5% polyacrylamide gel as described above. In all cases the products were resuspended in TE after precipitation.

Treatment with RT

One-fourth to one-half of the purified substrates was treated with 10 units of recombinant M-MuLV or HIV-1 RT in a 10-µl reaction containing 1 times RT buffer at 37 °C for the indicated times. Samples were mixed with an equal volume of formamide stop buffer, denatured at 90 °C for 5 min, and analyzed by electrophoresis in a 20% polyacrylamide gel containing 8 M urea.

Size Markers

Sequencing ladders were prepared using a Sequenase kit essentially as described by the manufacturer (U. S. Biochemical Corp.). The plus sense ladder was generated using oligo 2A or 2B and M13mp7/PBS DNA, while the minus sense ladder was generated using oligo 1 and M13mp7/PBS DNA. A size ladder of the labeled RNA oligo Mol15R was produced by partial digestion with 0.2 µg/ml of nuclease P1 (P-L Biochemicals) in a 10-µl reaction containing 50 mM sodium acetate (pH 5.5) at 37 °C for 30 min.

Assay for Capacity of RT to Initiate Displacement Synthesis with a Mismatched Primer Terminus

5` end-labeled oligo U5mis (2 pmol) was annealed to 2.7 pmol of pBSMOLTR+ single-stranded circular template DNA in 30 µl of 100 mM Tris-HCl (pH 8.3), 100 mM KCl, and 12 mM MgCl(2). Control experiments showed that the five mismatched nucleotides at the 3` end of oligo U5 mis were sufficient to prevent its usage as a primer and that no displacement by RT occurred in the absence of added upstream primer (data not shown). The annealing reaction was divided in half and annealed with 1 pmol of unlabeled oligo U5mis and 7.5 pmol of oligo PB or PBA in 100 mM Tris-HCl (pH 8.3), 100 mM KCl, and 12 mM MgCl(2) (final volume 20 µl). To minimize the potential for resolution of the unpaired oligo PBA 3`-terminus by branch migration, the second annealing step was carried out for only 5 min at 37 °C. The subsequent extension reactions (20 µl final volume) contained 9 µl of the annealed mixture in 50 mM Tris-Cl (pH 8.3), 50 mM KCl, 6 mM MgCl(2), 5 mM DTT, and 0.2 mM dNTPs. A sample was removed for analysis as the zero time point, synthesis was initiated with 120 units of recombinant RT, and 3.9-µl aliquots were terminated at 30, 60, 90, 120, and 150 s time points by the addition to a final concentration of 13% Ficoll, 20 mM EDTA, and 0.05% SDS. The products were treated with 40 µg/µl of proteinase K for 30 min at 37 °C before electrophoresis in a 15% nondenaturing polyacrylamide gel. Quantitation of the reaction products was carried out after exposure of the wet gel to a PhosphorImager screen at 4 °C. The amounts of free (displaced) and total oligo U5mis were determined by the area integration function of ImageQuant software (Molecular Dynamics).


RESULTS

Sites of RNase H Cleavage 5` of the PBS after Initiation of Minus Strand DNA Synthesis

To define the template that determines the 3` end of minus strand DNA prior to the second jump (Fig. 1), we examined RNase H specificity using a model substrate in vitro. Mol15R, a 15-mer RNA oligonucleotide that corresponds to the last 15 bases of the tRNA primer for M-MuLV, was annealed to RNA containing plus sense R-U5-PBS sequences and extended using RNase H RT (Superscript II) (Fig. 2A). The resulting RNA-DNA hybrids were incubated with recombinant wild-type RT alongside controls in which the RNA primer had not been extended. To identify RNase H cleavage sites that occurred 5` of the PBS sequences on the plus strand RNA (Fig. 1, stage i, region I), the products were denatured, and a labeled DNA oligonucleotide complementary to the template RNA was used in a primer extension assay (Fig. 2A, oligo 1). As expected in control reactions without RT, a major primer extension product corresponding to the 5` end of the plus strand RNA template was observed (Fig. 3, lanes 1 and 3, arrow a). Products slightly smaller than full-length were also apparent; these species likely resulted from premature termination during extension on the full-length RNA. Treatment of the hybrid substrates with RT resulted in the loss of all full-length and near full-length products, indicating that most RNA molecules had been cleaved somewhere along their length (Fig. 3, lane 2). These cleavages did not result from nonspecific ribonuclease activity associated with the recombinant RT, as no degradation was observed when template RNA was treated with RT in the absence of RNA primer Mol15R (data not shown). A major primer extension product was detected (Fig. 3, lane 2, arrow b) corresponding to RNase H cleavage between the second and third ribonucleotides immediately 5` of the PBS. Cleavages one base upstream and both one and two bases downstream of this primary cleavage site were less abundant but clearly detectable (Fig. 3, lane 2). Longer exposures revealed minor products corresponding to cleavages at every position up to 10 nucleotides 5` of the PBS (data not shown). As expected, no cleavages were detected upstream of the U5/PBS boundary when the control substrate containing unextended RNA primer was examined (Fig. 3, lane 4). However, some cleavages were observed within the RNA-RNA duplex region at the PBS site (Fig. 3. compare lanes 3 and 4, arrow c). Since cleavage of RNA-RNA duplexes has been reported for RNase H(40) , these species might result from a low level of RNase H* activity.


Figure 3: Determination of the cleavage sites within the plus strand U5-PBS region. RNA oligo Mol15R was annealed to a template RNA containing R-U5-PBS sequences and either used directly as a substrate for RT (Primer only) or Mol15R was extended with RNase H RT (Superscript II) prior to use as a substrate (Extended primer) (see Fig. 2A). The 5` ends of the template RNAs were mapped either before (lanes 1 and 3) or after treatment with recombinant M-MuLV RT for 15 min (lanes 2 and 4) by primer extension using labeled oligo 1. Primer extension products were analyzed in an 8% polyacrylamide, 8 M urea gel adjacent to a dideoxy-sequencing ladder generated using the same labeled oligonucleotide (lanes T, G, C, and A). The lower signal in lane 2 resulted from cleavages in the RNA 3` of the PBS due to the presence of some snap-back priming during the preparation of the substrate (see ``Materials and Methods''). Arrow a indicates the mobility of products that extend to the 5` end of the RNA template; arrow b marks the predominant extension product that results from the RNase H cleavage site within the hybrid region just 5` of the PBS; and arrow c indicates the position of products resulting from cleavage within the PBS. The RNA sequence of the U5-PBS region is shown with the PBS boxed at the left.



We next examined the RNase H cleavage pattern on genomic RNA upstream of the PBS in endogenous reactions. Detergent-disrupted M-MuLV virions were incubated with dNTPs, and products synthesized over time were analyzed by primer extension using oligo 1 (Fig. 2A). In the absence of synthesis (zero time), the primer extension products were similar to those observed with substrates containing unextended RNA in the in vitro assay with model substrates (compare Fig. 4, lane 1, with Fig. 3, lane 4). The vast majority of products mapped to the 5` end of the genomic RNA (Fig. 4, lane 1, arrow a), with a small fraction mapping to the PBS region itself (arrow c). Cleavages within the PBS could have resulted from very low levels of RNase H* activity within the virions prior to DNA synthesis. It would appear that these minor cleavage products persisted throughout the time course, although the bands are fainter in the 4-24 h time points (Fig. 4, lanes 2-5) owing to a reduced recovery of products for these samples. After 4 h of synthesis, the amount of genomic length products decreased dramatically, and several products mapping immediately 5` of the PBS appeared. The major product was identical to that observed using the RNA-DNA hybrid substrate in the in vitro assay and corresponded to RNase H cleaving the viral genome between the second and third ribonucleotides 5` of the PBS (Fig. 4, lane 2, arrow b). Several minor cleavages were also detected, beginning with the first ribonucleotide 5` of the PBS and extending up to 10 ribonucleotides 5` of the PBS. The relative abundance of these secondary cleavage sites decreased with increasing lengths of time, reflecting further RNase H activity on the genomic RNA template as reverse transcription progressed (Fig. 4, lanes 3-5).


Figure 4: Cleavage sites on genomic RNA after minus strand synthesis in the endogenous reaction. Using detergent-disrupted M-MuLV virions, endogenous reverse transcription reactions were carried out for 4 h (lane 2), 8 h (lane 3), 16 h (lane 4), or 24 h (lane 5), and products were recovered by phenol extraction and ethanol precipitation. Products were similarly prepared from detergent-disrupted virions in the absence of any synthesis (0 h; lane 1). To map the 5` ends of genomic RNA upstream of the PBS, primer extensions were performed with labeled oligo 1, and samples were analyzed as described in Fig. 3. The designations for the arrows and RNA sequence are identical to Fig. 3.



tRNA Primer Removal by RNase H Leaves a Ribo A on the 5` End of Minus Strand DNA

The 5` end of the minus strand is defined by the RNase H cleavage that removes the tRNA primer in region II after the PPT primer has been extended through U5-PBS (Fig. 1, stage iii). To initially address the specificity of tRNA primer removal, we used an in vitro assay with a model substrate. The RNA oligonucleotide Mol15R was annealed to single-stranded DNA containing R-U5-PBS sequences and extended with Sequenase to generate a DNA-DNA duplex containing the RNA primer (Fig. 2B). These substrates were incubated with recombinant RT and analyzed by a primer extension assay using RNase H RT and labeled oligo 2. As shown in Fig. 5A, some of the starting substrate remained, as reflected by the presence of a primer extension product, which extended through to the end of the RNA primer and co-migrated with the single band present in the untreated material (arrow a, lanes 2 and 4). The major primer extension product migrated slower by the equivalent of one base (arrow b, lane 4) than the extension product in the control samples that had been treated with alkali prior to the assay (arrow c, lanes 1 and 3). Furthermore, this species co-migrated with the band in the sequencing ladder consistent with a single ribo A remaining on the 5` end of the minus DNA strand. Primer extension analyses on the same samples using T4 DNA polymerase gave similar results (Fig. 5A, lanes 5-8) except that the T4 enzyme is much less efficient at utilizing the RNA portion of the molecules as a template as compared with RNase H RT.


Figure 5: Determination of the 5` end of minus strand DNA at the U5`-PBS` junction after primer removal. A, RNA oligo Mol15R was annealed to single-stranded EcoRI-cut M13mp7/PBS DNA and extended with Sequenase (see Fig. 2B). The resulting substrate was treated with recombinant M-MuLV RT for 10 min (lanes 3, 4, 7, and 8) or not treated (lanes 1, 2, 5, and 6), and the 5` ends of the minus strand DNAs were mapped by primer extension using labeled oligo 2A and RNase H RT (Superscript) (lanes 1-4) or T4 DNA polymerase (lanes 5-8). To remove any remaining RNA oligonucleotide from the 5` ends of the minus strands, the products in the indicated lanes were treated with alkali before carrying out the primer extension reactions. Primer extension products were analyzed adjacent to a dideoxy sequencing ladder generated using labeled oligo 2A (lanes T, G, C, and A) as described in Fig. 3. B, same as for A except that the substrate was not treated (0 min, lanes 1 and 2) or treated with recombinant RT for 1 min (lane 3), 3 min (lane 4), 10 min (lane 5), 30 min (lane 6), or 60 min (lane 7), and primer extensions were performed only with RNase H RT. For each panel, the sequence of the RNA oligonucleotide is indicated at the left. Arrow a marks the position of extension products corresponding to the 5` end of the RNA primer; arrow b indicates the position of the major cleavage product containing one ribonucleotide A on the 5` end of the minus strand; and arrow c indicates the position of the 5` end of the DNA.



Since a portion of the molecules was not cleaved by RNase H in this experiment (Fig. 5A, lane 4, arrow a), we tested whether longer reaction times might facilitate removal of the ribo A. As shown in Fig. 5B, treatment with RT through 60 min greatly reduced the amount of 5` ends containing the full-length RNA primer (arrow a, lanes 3-7). Notably, 5` ends containing a single ribo A increased (arrow b), and in other experiments, the ribo A persisted after incubating the substrates with RT for as long as 120 min (data not shown).

We next addressed whether primer removal by RNase H during the endogenous reaction also leaves a ribo A at the 5` end of minus strand DNA. The minus strand products of a 16-h endogenous reaction were isolated and analyzed by primer extension reactions using oligo 2 before and after alkali treatment to determine whether any ribonucleotides remained on the 5` end of the DNA. Pretreatment with alkali yielded the expected T4 DNA polymerase primer extension product corresponding to the 5` end of the minus strand DNA (Fig. 6, lane 3). When the same alkali-treated sample was analyzed using RNase H RT as the primer-extension polymerase, a second band one nucleotide shorter than the expected product was observed (Fig. 6, lane 2). Since only a single product was observed with T4 DNA polymerase, we conclude that the shorter primer-extension product observed in lane 2 resulted from RNase H RT pausing one nucleotide short of the 5` end of the template. It is unclear why pausing at this site was consistently more pronounced with the products of the endogenous reaction as compared with those of the reconstituted reaction (see Fig. 5A, lane 4). The primer extension analysis on the sample that had not been treated with alkali revealed three bands in addition to the expected full-length product resulting from complete extension through the RNA primer (Fig. 6, lane 1). The upper of these three bands corresponds to a ribo A on the end of a significant fraction of the minus strands initiated in the endogenous reaction since this product disappears after alkali treatment (compare lanes 1 and 2). The middle band corresponds to cleavage precisely at the RNA-DNA junction, while the fastest migrating species results from premature termination by RNase H RT during the primer extension assay. Because premature termination could, in principle, occur on molecules either with or without the ribo A residue, it is not possible to accurately quantify the proportion of molecules in each class from the endogenous reaction. Since T4 DNA polymerase did not efficiently copy ribonucleotides in primer extensions carried out on the model substrates (see Fig. 5A, lanes 6 and 8), this enzyme could not be used to confirm that the extension product, which is two nucleotides shorter than ribo A species in Fig. 6, lane 1, indeed resulted from premature termination.


Figure 6: Determination of the 5` end of minus strand DNA synthesized in the endogenous reaction. An endogenous reaction was incubated for 16 h, and the 5` ends of the minus strand DNAs were mapped using labeled oligo 2A and RNase H RT (Superscript) (lanes 1 and 2) or T4 DNA polymerase (lane 3) as described in Fig. 5. Samples shown in lanes 2 and 3 were treated with alkali. The sequence of the tRNA primer is indicated at the left.



To ask whether the recombinant RT differs from the virion-derived RT in its ability to cleave at the RNA-DNA junction, we directly compared the RNase H activities of these enzymes on the model substrate described in Fig. 2B. The substrate was incubated with either RT purified from M-MuLV virions or the recombinant RT for 10 or 90 min, and the products were analyzed by primer extension using labeled oligo 2 and RNase H RT. For both enzymes, a ribo A remained at the 5` end of minus strand DNA (Fig. 7, lanes 4, 6, 8, and 10). Treatment of identical samples with alkali prior to the primer extension analysis confirmed the presence of the ribo A on the 5` ends of the DNAs (Fig. 7, lanes 3, 5, 7, and 9). Based on these results, it would appear that the virion-derived RT is indistinguishable from the recombinant enzyme.


Figure 7: Primer removal by RT purified from virions. Substrates were prepared as described in Fig. 5and treated with purified virion M-MuLV RT (Virion RT; lanes 3-6) or recombinant RT (Recomb. RT; lanes 7-10) for 10 min (lanes 3, 4, 7, and 8) or 90 min (lanes 5, 6, 9, and 10). Untreated samples are shown in lanes 1 and 2. Samples in lanes 1, 3, 5, 7, and 9 were treated with alkali prior to the primer extension analysis by RNase H RT using oligo 2B. The sequence of the RNA oligonucleotide is indicated at the left.



Although retroviral RNase H activity is maximal in the absence of KCl, typical RT conditions contain 40-50 mM KCl(31, 41) . We have varied the concentration of KCl with the model substrates and endogenous reactions to find that, under all conditions, the ribo A remained at the 5` end of minus sense DNA (data not shown).

Fate of the RNA Primer

To address the fate of the RNA primer in the cleavage reaction, 5` end-labeled RNA oligonucleotide Mol15R was annealed to single-stranded DNA containing R-U5-PBS sequences and extended with Sequenase to generate the model substrate described above (Fig. 2B). When analyzed by denaturing polyacrylamide gel electrophoresis, the labeled extended RNA migrated much more slowly than the original 15-mer RNA oligonucleotide (Fig. 8, compare lanes 2 and 1, arrows a and b). As early as 20 s after incubation with RT, the major product was clearly shorter than the starting 15-mer RNA oligonucleotide and by comparison with a sizing ladder migrated as a species 14 nucleotides in length (Fig. 8, lane 3). After 3 min, the amount of starting material decreased dramatically, but the major RNase H cleavage product was still a 14-mer RNA (Fig. 8, lane 5). With increasing incubation times, the 14-mer RNA was cleaved further into smaller products (Fig. 8, lanes 6 and 7). In these experiments, a 15-mer RNA corresponding to cleavage at the RNA-DNA junction was not observed. When the labeled RNA primer was annealed to the single-stranded DNA but not extended and subsequently incubated with RT for as long as 27 min, the primer was not cleaved by RNase H at all (data not shown). These data support the conclusion that the first RNase H cleavage on the RNA primer occurs between the last and penultimate ribonucleotides, and show that the primer is subsequently cleaved into smaller fragments.


Figure 8: Time course of RNA primer cleavage by RNase H. 5` end-labeled RNA oligo Mol15R was annealed to single-stranded EcoRI-cut M13mp7/PBS DNA and extended with Sequenase (see Fig. 2B). The resulting substrate was gel-purified, treated with recombinant RT for 0.3 min (lane 3), 1 min (lane 4), 3 min (lane 5), 9 min (lane 6), or 27 min (lane 7) and analyzed in a 20% polyacrylamide, 8 M urea gel. As controls, the untreated substrate (lane 2, arrow a) and 15-mer RNA oligo Mol15R without extension (lane 1, arrow b) are shown. Positions of size markers generated by nuclease P1 digestion of labeled Mol15R are indicated at the right.



Removal of M-MuLV RNA Primer by HIV-1 and Avian Myeloblastosis Virus RTs

Previous studies that examined RNA primer removal by HIV-1 RT using the homologous tRNA substrate showed that, similar to the case for M-MuLV described above, a ribo A remained at the 5` end of minus sense DNA and that the RNA primer is initially removed intact (18, 29, 30) . With the same HIV-1 substrate, both the M-MuLV and avian myeloblastosis virus RTs also left a ribo A at the 5` end of the minus sense DNA(29) . To test whether the RNase H activity of HIV-1 RT might similarly leave a ribo A on the 5` end of the M-MuLV substrate, a model substrate (Fig. 2B) containing the labeled Mol15R primer was incubated with either the HIV-1 or M-MuLV RTs for increasing lengths of time, and the products were examined as described above. In contrast to the studies with the M-MuLV enzyme, the HIV-1 RT cleaved the RNA primer to release a small amount of intact 15-mer as well as the 14-mer (Fig. 9, compare lanes 1-4 with lanes 5-8, respectively). Like the M-MuLV enzyme, HIV-1 RT cleaved the RNA primer into smaller fragments with longer incubation times (Fig. 9, compare lanes 3 and 4 with lanes 7 and 8). Primer extension analyses to map the 5` ends of the minus strand DNA after cleavage confirmed that the HIV-1 enzyme cleaved the M-MuLV substrate both at the DNA-RNA primer junction and after the first ribo A in the RNA primer (data not shown). Similar experiments carried out with avian myeloblastosis virus RT on the M-MuLV substrate also yielded heterogeneous cleavage products in which the RNA primer was either removed completely or as a 14-mer (data not shown).


Figure 9: Removal of the RNA primer by M-MuLV versus HIV-1 RT. Substrates prepared as described in Fig. 8were treated with recombinant M-MuLV (lanes 2-4) or purified HIV-1 (lanes 6-8) RT for 0.3 min (lanes 2 and 6), 3 min (lanes 3 and 7), or 27 min (lanes 4 and 8) and analyzed as described in Fig. 8. Substrates without treatment (0 min; lanes 1 and 5) and the labeled RNA oligonucleotide without extension (lane 9) are also shown. Positions of size markers are shown on the right as described in Fig. 8.



Initiation of Displacement Synthesis from the Primer Terminus Generated by the Second Jump

Based upon the foregoing results, which map the 5` and 3` limits of the minus strand, it is possible to predict the structure of the replicative intermediate generated by the second jump. Because the minus DNA strand defined in the above experiments is typically longer than genome length by three nucleotides (one 5` rA and two 3` dAs), the second jump would be expected to create a branched rather than a nicked replication intermediate (3) (Fig. 1, stage v; see ``Discussion'').

To determine whether such a structure would affect completion of the minus strand, we used a model system designed to compare initiation and early synthesis on templates representing either the branched or nicked product of the second jump. Oligonucleotides corresponding to the 5` end (oligo U5mis) and two possible 3` ends of the minus strand were annealed to single-stranded DNA containing the plus sense LTR (Fig. 10A). Oligo U5mis contained 5 unpaired 3`-terminal nucleotides to prevent its utilization as a primer, and the two 3` upstream oligonucleotides contained either the minus sense PBS sequence (oligo PB) or the extended PBS sequence (oligo PBA) to generate a nicked or three base-branched substrate, respectively. Shown in Fig. 10B is a graph depicting the time course of displacement of the 5` end-labeled downstream oligonucleotide (oligo U5mis) as determined by nondenaturing polyacrylamide gel electrophoresis. Of note is that RT utilized and extended oligo PBA nearly as well as fully base-paired oligo PB, although a slight but reproducible lag in displacement and an apparent decrease in the total amount of displaced product were associated with the branched substrate. In a separate experiment, it was found that incubation of the templates with RT for 3 min prior to the initiation of synthesis with dNTPs did not decrease the observed lag (data not shown).


Figure 10: Displacement synthesis on model primer-templates comparing an unpaired with a paired primer terminus. A, schematic diagram showing the structures of the model primer-templates constructed from a tandem pair of DNA oligonucleotides corresponding to the putative 5` and 3` ends of the completed minus strand annealed to plus sense LTR-containing single-stranded DNA. The arrow indicates the primer terminus that in structure I is base paired with the template and in II is unpaired and extends beyond the 5` end of the downstream oligonucleotide by three deoxyadenosine residues. The 3` terminus of the downstream oligonucleotide is not complementary so it cannot be used as a primer, and the 5` end is P-labeled (indicated with an asterisk). B, The release of the labeled oligonucleotide by RT-mediated displacement synthesis is shown as a function of time for the two primer-template combinations.




DISCUSSION

We have examined the RNase H cleavages that determine the length of minus strand DNA prior to the second jump during reverse transcription of M-MuLV. In both the endogenous reaction and on a hybrid RNA-DNA substrate, RNase H cleaved the genomic template in U5 very proximal to the PBS. The primary RNase H cleavage site was between the second and third ribonucleotides 5` of the PBS (Fig. 11A), but other cleavages extending through 10 nucleotides 5` of the PBS were also apparent. These cleavages define the 5` end of the template for minus strand synthesis and would allow the majority of the minus strands to extend only a few nucleotides beyond the PBS prior to the second jump (Fig. 11B). Interestingly, Götte et al.(42) have shown that HIV-1 RT also cleaves the RNA genome two bases upstream from the U5/PBS boundary during minus strand synthesis. Using model substrates, we also found that RNase H cleaves the tRNA primer between the last and penultimate ribonucleotides (Fig. 11C), leaving a single ribo A residue at the 5` end of minus strand DNA (Fig. 11D). Similar results were obtained from an analysis of the products of the endogenous reaction except in this case some of the molecules were apparently cleaved instead at the RNA-DNA junction. We cannot rule out the possibility that a ribonuclease other than RNase H was responsible for the cleavage at the DNA-RNA junction or that this difference results from a coupling of the cleavage to concomitant DNA synthesis. Subsequent to the initial cleavage, RNase H cleaved within the RNA primer to generate smaller fragments.


Figure 11: RNase H cleavages that generate the two ends of the minus DNA strand and the resulting structure after the second jump. The sequences at the U5-PBS border are indicated for a number of structures synthesized during reverse transcription with the RNA sequences shaded. A, the arrow above the RNA sequence indicates the major RNase H cleavage site 5` of the PBS that determines the 5` end of the template for minus strand DNA synthesis. B, predicted 3` sequence for the major minus strand product assuming synthesis proceeds to the end of the template diagrammed in A. C, the arrow indicates the site of the RNase H cleavage that removes the tRNA primer and determines the 5` end of the minus DNA strand shown in D. E, the predominant structure that is predicted after the second jump. Note that the three unpaired A residues, which extend from the site of the nick, can become paired with the opposing plus strand to varying extents by branch migration (see text).



The apparent 5` and 3` termini for M-MuLV minus strand DNA suggest a detailed structure for the intermediate after the second jump of reverse transcription (Fig. 11). Upon initiation at the polypurine tract, plus strand DNA synthesis extends through the PBS and terminates at the first modified base in the tRNA(43) . To continue synthesis, plus strand strong-stop DNA transfers to the 3` terminus of minus strand DNA (Fig. 1, stage v), with the complementary sequences (PBS/PBS`) at the ends of minus strand DNA facilitating the jump(3) . We predict that the first three bases at the 3` end of the minus strand (3`-AAA . . . 5`) are not initially base-paired to the plus strand DNA (Fig. 11E). As a consequence, branch migration is necessary to position the free 3` end of the minus strand for use as a primer, and then displacement synthesis is required for minus strand elongation. In contrast, the 3` end of plus strand DNA is situated for immediate extension after the second jump. Only the intermediate arising from the major RNase H cleavage between the second and third nucleotides upstream of PBS is shown in Fig. 11E, but others are also predicted by our results. These additional structures would possess unpaired 3` termini, which are even longer, resulting in a greater requirement for branch migration prior to the initiation of displacement synthesis.

For the intermediate structure proposed for the second jump, we inferred the structure of the 3` end of the minus strand by assuming that synthesis proceeds to the 5` end of the RNA template prior to the jump. Although we cannot rigorously exclude that the second jump occurs before completion of the minus strand, this possibility seems unlikely for the following reasons. First, given the displacement activity of RT (44) , there is no compelling reason to suppose that the known secondary structure in the template in the U5-PBS region (for review, see (45) ) would present a barrier to the completion of the minus strand. Second, it seems unlikely that strand transfer would precede the extension of minus strand DNA through to the end of the RNA template because degradation of the template by RNase H is required for the first jump (36) and is likely to be required for the second jump as well(3) . Based on footprinting and structural studies, the DNA polymerase and RNase H active sites of RT are separated by approximately 18 base pairs(46, 47, 48) . Since the PBS of M-MuLV is 18 nucleotides in length, the RT that is synthesizing the minus strand would be near the 5` end of the genomic template before the RNase H activity would be positioned for cleavages in the PBS. The properties of partial deletion and substitution mutants in the HIV-1 PBS further suggest that minus strand synthesis may typically traverse the entire PBS prior to the second jump(49, 50) . Notably, Luo and Taylor (32) found that 20 but not 10 nucleotides are sufficient for the M-MuLV strand transfer reaction in vitro.

An interesting question is whether the proposed branched intermediate created by the second jump slows minus strand DNA synthesis significantly (Fig. 11E). Extension of minus strands subsequent to the second jump would require resolution of the unpaired primer by branch migration, which in principle could be facilitated by RT. If such branch migration were to be an impediment to extension of the minus strand, resolution could result from displacement through the LTR from the 5` end by extension of the plus strand DNA. Using substrates that model the predicted second jump product, RT extended a 3` end requiring a three-base branch migration nearly as efficiently as a completely base-paired 3` primer terminus. The slight lag in extension associated with the unpaired primer terminus suggests that the structure was not resolved by branch migration during the 5-min annealing period prior to the initiation of synthesis. Additionally, the failure of RT to reduce the lag following an incubation with the template minus dNTPs suggests that, at least under nonsynthesis conditions, RT does not alter the equilibrium between the base paired and unpaired forms. Given that reverse transcription occurs over a period of hours(1) , these results suggest that, although an unpaired 3` terminus is not without consequence for RT, the magnitude of the delay is small enough that it is unlikely to have a significant impact on the overall course of retroviral replication. It is uncertain what effect longer extensions of the minus strand 3` end would have on minus strand synthesis following the second jump. We would anticipate that an increase in the size of the unpaired primer terminus would lead to an increase in the lag before synthesis and perhaps a decrease in the overall utilization of the 3` minus strand following the second jump.

The presence of a ribo A at the 5` terminus of the majority of M-MuLV minus strands may affect the structure of the right LTR end and is interesting to consider with regard to integration. During retroviral integration, two base pairs are generally lost from each end of the linear viral DNA such that the first and last two bases of the integrated virus are highly conserved and begin with 5`-TG . . . at the left terminus and end with . . . CA-3` at the right terminus (for review, see (7) ). The avian RT precisely cleaves its tRNA primer at the junction between minus strand DNA and RNA(27) , which generates symmetrical ends for integration. In contrast, the tRNA primer of HIV-1 is incompletely removed, and a ribo A remains at the 5` end of the minus strand on model substrates(18, 29, 30) . These biochemical studies are supported by the observation that most HIV-1 circle junctions contain an additional A, which is not present in the predicted sequence (51, 52, 53) and that viral double-stranded DNA contains the extra base pair in the right LTR(54) . However, the presence of a ribo A at the 5` end of the HIV-1 minus strand DNA still results in symmetrical LTRs from which two base pairs are removed from both ends during integration.

In the case of M-MuLV, if the ribo A residue is retained on the 5` end of minus strands, and synthesis of plus strands extends to the end of the minus strand template to copy the ribo A, then the right end of linear unintegrated DNA would end with the sequence 5` . . . CATTT-3`, and three base pairs would have to be removed from the right LTR end during integration. It seems unlikely that the additional base pair would affect viral integration as previous studies have demonstrated that extra sequences flanking the conserved 5`-TG . . . and . . . CA-3` bases at the termini of M-MuLV do not interfere with integration(55, 56) . The addition of two extra base pairs to the right LTR resulted in a viral mutant that replicated and retained the two base pair addition. Interestingly, two of the four circle junction sequences analyzed for this virus contained an additional T that might have arisen from incomplete removal of the tRNA(56) .

Alternatively, M-MuLV minus strand DNA may undergo some additional processing that specifically removes the ribo A prior to the completion of plus strand synthesis or integration. Although we did not observe removal of the ribo A residue in the reconstructed reactions in vitro, it appeared that the ribo A was removed from some minus sense DNA ends in the endogenous reaction. Thus we might expect a mixed population of molecules with respect to the structure of the 5` ends of minus strands in vivo. The sequence of the one reported M-MuLV circle junction (57) does not contain an extra A at the position predicted by our data, but given the small sample size, it is difficult to generalize from this observation. Several studies examining the M-MuLV double-stranded DNA product of reverse transcription failed to detect minus strand DNA with a length suggesting the presence of an extra A at the 5` end(43, 58, 59) , and one of these studies did not find an extra T at the 3` end of plus strand DNA(43) . While these observations might indicate that the ends of intracellular intermediates are different from those generated in this study, it remains equally likely that the ribo A is somehow removed from minus strand 5` ends after the second jump but before completion of the plus strand. Sequence analyses of additional M-MuLV circle junctions might reveal the relative frequency of occurrence of the two predicted structures for the 5` ends of minus strands.

It remains to be determined why the RNase H activities of both M-MuLV and HIV-1 can remove the plus strand primer precisely at the RNA-DNA junction (25, 26) yet prefer to cleave the tRNA primer one nucleotide away from the junction to leave a ribonucleotide on the DNA strand. Interestingly, Huang et al.(60) showed that the calf thymus RNase H1 invariably cleaves one base away from the RNA-DNA junction. Through the use of two-dimensional NMR studies, the structure of a duplex oligonucleotide that models the M-MuLV tRNA-minus DNA junction has been determined. (^2)Structural anomalies at the RNA-DNA junction, which include distortions in the base pairs, a bend in the helix axis, and unusual sugar conformations could influence the cleavage specificity of RNase H. Recently, it was suggested that the width of the minor groove could be an important feature recognized by RNase H(61) . Based on the NMR analysis of the Moloney structure, it is possible that narrowing effects of the junctional DNA base pair on the minor groove could preclude cleavage by RNase H at the junction. The determination of which, if any, of these structural features prevents removal of the ribo A by the viral RNase H awaits a structural analysis of a substrate that is cleaved precisely at the RNA-DNA junction.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R37 CA51605. 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.

§
Supported by the Graduate and Postdoctoral Training Grant in Viral Oncology (NIH T32 CA09229).

Supported by the Medical Scientist Training Program (NIH 5T32GM07266) and the Poncin Scholarship Fund.

**
To whom correspondence should be addressed: Dept. of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242; Tel.: 206-543-8574; Fax: 206-543-8297; champoux{at}u.washington.edu.

(^1)
The abbreviations used are: PBS, primer binding site; LTR, long terminal repeat; RT, reverse transcriptase; PPT, polypurine tract; HIV-1, human immunodeficiency virus, type 1; M-MuLV, Moloney murine leukemia virus; bp, base pair(s); DTT, dithiothreitol; dNTP, deoxynucleoside triphosphate.

(^2)
Salazar, M., Fedoroff, O. Y., and Reid, B. R., personal communication.


ACKNOWLEDGEMENTS

We thank Knut Madden, Leon Parker, Caterina Randolph, and Lance Stewart for helpful discussions during the course of these experiments.


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