©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Strand Transfer Mediated by Human Immunodeficiency Virus Reverse Transcriptase in Vitro Is Promoted by Pausing and Results in Misincorporation (*)

(Received for publication, July 25, 1994; and in revised form, October 25, 1994)

Weimin Wu (1) (5) Benjamin M. Blumberg (3) (4) Philip J. Fay (2) Robert A. Bambara (1) (5)(§)

From the  (1)Departments of Biochemistry, (2)Medicine, (3)Neurology, and (4)Microbiology and Immunology and the (5)Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human immunodeficiency virus (HIV-1) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to another. The strand transfer reaction, catalyzed by HIV-1 reverse transcriptase (RT), was conducted in vitro between donor and acceptor RNA templates that were derived from natural HIV-1 nef genes. The donor and acceptor templates shared a nearly homologous region where strand transfer could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the donor. We sequenced elongated primers that underwent transfer. The position of transfer was revealed by the change of sequence from that of the donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancement of strand transfer. Elimination of a pause site, with a minimal change in sequence, decreased the frequency of strand transfer in the immediate area.

Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. Remarkably, approximately 30% of the products contained mutations. Base substitutions, short additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The identity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These results indicated that the detected mutations were related to the process of strand transfer.


INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) (^1)is the causative agent of acquired immunodeficiency syndrome (AIDS) (Fauci, 1988). HIV replicates through reverse transcription, during which the single-stranded (+) RNA genome is converted into double-stranded DNA prior to its integration into the host genomic DNA (Coffin, 1979). This process is carried out by the virally encoded, multifunctional enzyme reverse transcriptase (RT), which catalyzes RNA- and DNA-dependent DNA polymerization and ribonuclease H activity (Skalka and Goff, 1993; Starnes and Cheng, 1989; Hansen et al., 1987).

One unique feature of retroviruses is the packaging of two copies of the RNA genome in each virion (Bender et al., 1978; Murti et al., 1981; Weiss et al., 1985). The dimer of RNA molecules participates in efficient recombination during the process of replication. A high frequency of recombination has been observed from within internal regions of the viral genome (Srinivasan et al., 1989; Clavel et al., 1989; Hu and Temin, 1990a, 1990b; Goodrich and Duesberg, 1990; Howell et al., 1991). During reverse transcription, the nascent DNA strand can transfer to a similar or identical sequence on another template to continue primer extension. This results in homologous recombination. If the two parental RNA genomes are different, a unique recombinant molecule is generated. Frequent recombination enables viruses to recover from damage (Coffin, 1990a). It also increases the genetic diversity in the virus population (Katz and Skalka, 1990; Temin, 1991), allowing some viruses to evade host immune response and escape antiviral drug therapy.

It is not known whether recombination occurs with equal probability everywhere on the viral genome or whether features of genome structure promote recombination at particular sites. According to the forced-copy choice model proposed by Coffin (1990b), breaks in the viral RNA genome should force the RT and growing primer to switch to another genome copy. However, recent work (Xu and Boeke, 1987) suggests that breaks in the RNA genome are not necessary for strand transfer during minus strand DNA synthesis. Stalling of RT during synthesis might also increase the probability of template switching. When reverse transcriptase advances along the RNA template, it encounters some sequences or structures at which continued synthesis is difficult. These positions are called pause sites. While it pauses, RT may be equivalent, with respect to the transfer reaction, to an RT stopped at a break site in the template. Under those circumstances, it is likely that a copy choice will be made that results in transfer of the primer.

To investigate the role of pausing in the process of strand transfer, we measured recombination catalyzed by HIV-RT in vitro between two natural nef gene RNA templates. In this system, a primer initiated on one template, designated the donor, can transfer to the homologous region of the other template, designated the acceptor. Allelic markers distributed along the homologous region allowed us to determine the position of transfer by sequencing the recombinant DNA molecule. The results showed a very positive correlation between the positions of pausing during DNA synthesis and an enhanced level of strand transfer. We also examined the sequences of two recombination junctions where frequent transfer occurred. At one recombination junction, no mutations were observed. At the other recombination junction, however, we found that over 30% of the recombinant molecules examined had incorrectly encoded nucleotides inserted at the crossover junction. Finally, we determined that the high rate of mutation was caused by the process of strand transfer.


EXPERIMENTAL PROCEDURES

Materials

Recombinant HIV-RT having native structure (p66/p51 heterodimer) was generously provided to us by Genetics Institute (Cambridge, MA). The enzyme had a specific activity of about 40,000 units/mg. One unit of RT 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 primer-template. As starting material to make DNA templates for primer extension and strand transfer reactions, we used nef clones J14 and T17, prepared as described previously (Blumberg et al., 1992) from DNA extracted directly from pathogenic brain tissue of two HIV-1-infected children. The sequences of clones J14 and T17 are identical to KFJ3 and AGT7, respectively, in that publication. These clones displayed substantial sequence differences in the highly variable ``duplication region.'' Restriction enzymes XhoI, HindIII, BglII, placental RNase inhibitor, DNase I (RNase-free), T4 DNA ligase, rNTPs, dNTPs, T7 RNA polymerase, and Taq DNA polymerase were from Boehringer Mannheim. T4 polynucleotide kinase and the DNA sequencing kit were obtained from U. S. Biochemical Corp. Plasmid vectors pGEM7zf(-), pGEM4z, and SP6 RNA polymerase were from Promega. The 20-nucleotide DNA primer complementary to the donor template was synthesized by Genosys. [-P]ATP was from DuPont NEN. S-alpha-dATP was from Amersham Corp. All other chemicals were from Sigma.

Methods

Generation of Donor and Acceptor RNA Templates by Run-off Transcription in Vitro

Two donor and two acceptor RNA templates were used in the reactions. To make the donor A template (Fig. 1A), the nef J14 insert, 649 nucleotides in length, was excised from the pCR1000 vector (Invitrogen) by digestion with EcoRI and HindIII and subcloned into pBSM13(+) (pBSM13(nef J14)). The plasmid was then linearized by BglII, which makes a unique cut in the nef J14 coding region, and then transcribed in vitro by T7 RNA polymerase. This generated the donor A RNA template, 265 nucleotides in length. Donor B (Fig. 6A) was derived from the same nef J14 insert subcloned into pGEM4z (pGEM4z(nef J14)). The plasmid was cleaved by BglII in the nef J14 coding region and then transcribed by SP6 RNA polymerase. The final RNA transcript donor B was 267 nucleotides long. Two acceptor templates were also generated by run-off transcription. To make acceptor A (Fig. 1A), the nef T17 insert of 685 nucleotides, cloned into the pCR1000 vector (pCR1000(nef T17)), was cut with XhoI in the nef T17 coding region and transcribed by T7 RNA polymerase. This generated acceptor A, 203 nucleotides in length. Acceptor A* was derived from the original pCR1000(nefJ14) plasmid. The plasmid was cleaved by XhoI in the nef J14 coding region. T7 RNA polymerase was used to produce acceptor A*, 168 nucleotides long.


Figure 1: Templates used to measure the positions of strand transfer. A, generation of the donor A and acceptor A templates used in the strand transfer assay. To generate donor A template, nef J14 was subcloned into the pBSM13(+) vector. The plasmid was then linearized by BglII, which cuts in the nef coding region. This DNA was used for run-off transcription, resulting in the donor A RNA template 265 nucleotides long. Donor A contained a 10-nucleotide plasmid-derived sequence at the 5` end. The subsequent downstream sequence was derived from nef J14. Likewise, the acceptor A template was produced by run-off transcription of the pCR1000 (T17) vector after cleavage by XhoI. The resultant RNA template was 203 nucleotides long, containing a 68-nucleotide plasmid-derived sequence at its 5` end. Homologous regions shared by donor A and acceptor A templates are bracketed. Also shown is the position where the 20 nucleotide DNA primer is annealed to the donor A template. DNA synthesis initiates from this primer. B, sequence alignment of homologous regions shared by donor A and acceptor A templates. The top and bottom lines represent the homologous regions in donor and acceptor A templates respectively, starting from the nef initiation coden ATG to the XhoI cleavage site. The nucleotide sequence is from 5` to 3`. The numbers above the nucleotide sequence indicate the nucleotide position. The dashed line indicates the absence of a 36-nucleotide segment in the donor A template. Nucleotides that are different in the donor versus acceptor templates are underlined. The 5` end sequence of acceptor A, upstream of the homologous region, is 5`-CCCGGUACCGAGCUCACUAGUUUAAUUAAAAGCUUAUCGGCCGAGGUGAGAAGGGUUUUU-3`. The underlined sequence indicates the HindIII site, which was used during the cloning of all the recombinant products.




Figure 6: Elimination of pausing at position 151 on donor B template. A, construction of the donor B template. The nef J14 segment was subcloned into pGEM4z, generating the pGEM4z (nef J14) plasmid. The plasmid was then linearized by BglII, which cuts in the nef coding region. Subsequent transcription by T7 RNA polymerase resulted in the donor B template of 267 nucleotides. The presence of a 12-nucleotide plasmid sequence at the 5` end of the donor B template distinguishes it from donor A. The 12-nucleotide sequence is given in the figure and differs in both length and sequence from the 10 nucleotide plasmid sequence at the 5` end of donor A. B, comparison of the time course of primer extension on the donor A and donor B templates. HIV-RT (4 units) was preincubated with primed donors A and B, respectively. Primer extension was initiated by the addition of MgCl(2) and dNTPs and terminated at the indicated times. The full-length primer extension products (F) directed by donors A and B are 170 and 172 nucleotides, respectively. The positions of pausing are marked on the left side of the figure.



Run-off transcription was conducted according to the Promega Protocols and Applications Guide. The DNA template was digested with DNase I (RNase-free) after transcription. The RNA transcript was then extracted once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 3 volumes of ethanol and 1.25 M (final concentration) ammonium acetate. This RNA sample was subjected to 8% denaturing polyacrylamide gel electrophoresis. The full-length RNA was located by UV shadowing, excised, and eluted from the gel slice.

5` End Labeling of the DNA Primer

A DNA primer (5`-CAGCATTGTTAGCTGCTGTA-3`) was designed to anneal to positions 1-20 on the donor template (Fig. 1A). The dephosphorylated primer was labeled at 5` end using T4 polynucleotide kinase and [-P]ATP (3000 Ci/mmol), as described in the protocol provided by U. S. Biochemical Corp. Unincorporated [-P]ATP was removed by passing the labeling reaction mixture through a Nensorb(TM) (DuPont NEN) column according to manufacturer's specification.

Hybridization of the DNA Primer to the RNA Template

The labeled DNA primer was mixed with the RNA template at a 4:1 (primer/template) molar ratio of 3` termini in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 80 mM KCl. The mixture was heated to 65 °C for 10 min and then slowly cooled to room temperature.

Primer Extension and Internal Strand Transfer Assay

Primer extension was performed in a final volume of 25 µl. Four units of reverse transcriptase was preincubated with 2 nM (in template termini) of primer-template at 37 °C for 3 min in 50 mM Tris-HCl, pH 8.0, 80 mM KCl, and 1 mM dithiothreitol. The reaction was started by the addition of MgCl(2) and dNTPs to final concentrations of 6 mM and 50 µM each, respectively. It was terminated at the indicated times by addition of 1 volume of gel-loading buffer (90% formamide, 10 mM EDTA, pH 8.0, and 0.1% each of xylene cyanole and bromphenol blue).

The strand transfer reaction was carried out in a final volume of 15 µl. Four units of HIV-RT were preincubated with 2 nM (in template termini) of primer-template and various amounts of acceptor template for 3 min at 37 °C in 50 mM Tris-HCl, pH 8.0, 80 mM KCl, and 1 mM dithiothreitol. The reaction was started by the addition of MgCl(2) and dNTPs to final concentrations of 6 mM and 50 µM each, respectively, and terminated after 1-h incubation at 37 °C by adding 1 volume of gel-loading buffer.

Analysis of Reaction Products by Polymerase Chain Reaction (PCR) and DNA Sequencing

The transfer product to be analyzed was isolated by 8% polyacrylamide gel electrophoresis. It was cut from the gel and amplified by PCR. One set of primers specific for the amplification of all recombinant molecules was used. One primer (5`-CAGCATTGTTAGCTGCTGTA-3`) was complementary to positions 1-20 on donor A. The other (5`-CCCGGTACCGAGCTCACTAG-3`) had the sequence of the first 20 nucleotides at the very 5` end of acceptor A. The isolated transfer product was incubated in a total volume of 100 µl, containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl(2), 0.1% (w/v) gelatin, 50 µM dNTP, 15 pmol of each primer, and 2.5 units of Thermus aquaticus (Taq) DNA polymerase. The reaction mixture was denatured at 94 °C for 2 min and then amplified through 30 cycles (1 min at 94 °C, 1 min at 50 °C, 1 min at 72 °C). The PCR products were digested with XhoI and HindIII, which cleave in the positions described in the legend to Fig. 1B, and then subcloned into pGEM7zf(-). The inserts were sequenced by the dideoxynucleotide chain termination method, as described in the sequencing manual from U. S. Biochemical Corp.

Similarly, the full-length primer extension products were isolated and amplified by PCR, using two primers, with one primer (5`-CAGCATTGTTAGCTGCTGTA-3`) annealed to positions 1-20 on donor A and the other one (5`-GTCAGAATTCATGGGTGGCAAGTGGTCA-3`) having the sequence of the 5` end of donor A. The amplified products were linearized by EcoRI and XhoI and subcloned into pGEM4z. The inserts were analyzed as described above.


RESULTS

Construction of Templates Used to Measure the Positions of Strand Transfer

Nef RNA donor and acceptor templates were used in our strand transfer system in vitro, to simulate internal homologous recombination that could occur during minus strand DNA synthesis in vivo. As shown in Fig. 1A, the donor A RNA template contained a 10-nucleotide plasmid-derived sequence at the 5` end. The subsequent downstream sequence was from the nef J14 coding region. The acceptor A RNA template contained a 68-nucleotide plasmid-derived sequence at its 5` end followed by downstream sequence from the nef T17 coding region. The donor A and acceptor A templates shared a long homologous sequence of the nef coding region. Since the homologous region in acceptor A contained a 36 nucleotide insertion absent in donor A and 6 base substitutions that were different from donor A (Fig. 1B), these sequence differences served as allelic markers allowing us to determine where specific strand transfers occurred.

Template switching between donor A and acceptor A was expected to generate transfer products of two distinct lengths, 227 and 263 nucleotides (Fig. 2). If the growing point of the nascent DNA strand left the donor template before position 91 and transferred to the homologous region of acceptor A, continued synthesis on the acceptor template should have included the 36-nucleotide insertion present only in the acceptor template, resulting in a recombinant molecule 263 nucleotides long (T1). Otherwise, if the growing point of the nascent DNA transferred after position 91, then continued synthesis on the acceptor A template could bypass the insertion, generating a recombinant product 227 nucleotides long (T2). The polyacrylamide gel electrophoresis system used could readily resolve the two potential transfer products from the 170-nucleotide full-length primer extension product and also from each other.


Figure 2: Homologous recombination makes products of two lengths. Transfer products of two distinct lengths are expected to be generated by homologous recombination between donor A and acceptor A. If the growing point of the nascent DNA strand leaves the donor template before position 91 and transfers to the homologous region of acceptor A, continued synthesis on the acceptor template should include the 36-nucleotide insertion that exists solely in the acceptor A template, resulting in a recombinant molecule of 263 nucleotides long (T1). Otherwise, if the growing point of the nascent DNA transfers after position 91, then continued synthesis on the acceptor A template can bypass the insertion, generating a recombinant product of 227 nucleotides long.



Time Course of Primer Extension Catalyzed by HIV-RT to Locate the Pause Sites on the Donor A Template

The DNA synthesis pattern on the donor A template was examined by a time course of primer extension catalyzed by HIV-RT (Fig. 3). A 20-nucleotide-long DNA primer was labeled at its 5` end and annealed to the positions 1-20 of the donor A template (Fig. 1). Primer extension was initiated by addition of HIV-RT and terminated at the times indicated. The reaction products at the different time points were resolved by electrophoresis and visualized by autoradiography. As observed in Fig. 3, there were several positions having prominent accumulations of partially extended primers. Sites were located at 68, 81, 119, 125, and 151 nucleotides downstream from the 5` end of the primer. These represent positions on the template where the enzyme has a relatively high probability stalling or dissociating and will be referred to as pause sites.


Figure 3: Pausing during primer extension catalyzed by HIV-RT on the donor A template. HIV-RT (4 units) was preincubated with donor A template with 5` end-labeled primer. Primer extensions were initiated by the addition of MgCl(2) (6 mM) and dNTPs (50 µM). Reactions were terminated by adding 1 volume of gel-loading buffer at the times indicated. The full-length primer extension product is 170 nucleotides long. Positions of the prominent pause sites during the primer extension are shown by the markers on the left side of the figure. The number also corresponds to the length of the nascent DNA strand at the marked sites. As shown in the figure, the prominent pause sites were located at positions 68, 81, 119, 125, and 151.



Strand Transfer from Donor A to Acceptor A Templates

The strand transfer reaction was carried out in our assay system and included donor A, acceptor A, and purified HIV-RT (Fig. 4). In the absence of acceptor A template, the enzyme extended the primer to the end of the donor A template, resulting in the full-length primer extension product of 170 nucleotides. In the presence of acceptor A, three other products longer than the 170-nucleotide full-length primer extension product were generated, designated as T1, T2, and T3, respectively. The amount of each product, as judged by its intensity on the gel, increased as more acceptor templates were added to the reaction, indicating that all three products were generated by strand transfer events. This was surprising in that only two products were expected. According to their migration positions on the gel, we could determine that T1 and T2 were the expected homologous recombination products. However, the origin of the T3 product was not clear.


Figure 4: Products of strand transfer from donor A to acceptor A templates. HIV-RT (4 units) was first preincubated with primed donor A template (2 nM) in the absence or presence of increasing amounts of acceptor A template (1, 10, and 50 nM). The reaction was initiated by the addition of MgCl(2) and dNTPs and terminated after 1 h of incubation. The full-length donor A-directed primer extension product is 170 nucleotides long, indicated as ``F.'' Three transfer synthesis products, designated T1, T2, and T3, were generated by template switching of the primer and HIV-RT from the donor A to the acceptor A template.



Analysis of the Structures of T1 and T2 Products Generated by Homologous Recombination between Donor A and Acceptor A

To examine the nature of the recombinant molecules, we purified T1, T2, and T3 from the polyacrylamide gel. The purified products were subjected to PCR with a set of primers (see ``Methods'') that allowed only recombinant molecules to be amplified. The PCR products were then digested with XhoI and HindIII and subcloned into pGEM7zf(-) plasmid vectors. The cloned products were sequenced. Results confirmed that T1 and T2 were generated by homologous recombination events.

Twenty-seven clones of T1 transfer products were sequenced. To describe the positions of recombination, we divide the donor template into several regions, R1, R2, R3, R4, R5, and R6, which lie between the insertion and base substitutions that serve as markers to distinguish the template sequences. The site of transfer was localized to the region designated by the switching of markers from those of the donor to those of the acceptor (Fig. 5). Twenty clones of T1 represented the transfer products generated by template switching from the R2 region. Six clones of T1 resulted from template switching from the R3 region. Only one clone of T1 was generated by template switching from the R1 region. This result indicated that R2 was the most frequently used transfer region.


Figure 5: Structures of the T1 and T2 products generated by homologous recombination between donor A and acceptor A. The homologous sequences of the donor A and acceptor A templates are shown. The donor template is divided into the regions R1, R2, R3, R4, R5, and R6, which lie between the insertion and base substitutions that distinguish the template sequences. The base substitution markers on the donor A and acceptor A templates are represented as open and closed triangles, respectively. The 36-nucleotide insertion in the acceptor A template is also shown. The locations of synthesis pause sites within the homologous region of the donor A template are marked with arrows. All possible transfer product primary sequences are depicted. Each is distinguished by the particular distribution of sequence markers shown. The region where the recombination junction had to have occurred is indicated by the bracket and region number under each transfer product. Numbers in parentheses indicate the number of each particular transfer product sequence detected.



We also examined the distribution of pause sites in the R1, R2, and R3 regions. One prominent pause site at position 81 was located in the R2 region. A series of minor pause sites were observed in R3. Virtually no pausing was seen in R1. The number of detected transfer products was greatest from R2, less from R3, and least from R1. At least in this case, the presence of paused extension products in a region correlated with a relatively higher frequency of strand transfer, compared with other regions.

Likewise, 27 clones of T2 transfer products were analyzed (Fig. 5). Results revealed that T2 was mainly composed of two groups of transfer products. Twenty-four clones of T2 were generated by template switching from the R6 region. The other three clones of T2 represented template switching from the R5 region. No transfer event was detected from the R4 region, since there was no corresponding transfer product. In the detected transfer regions R5 and R6, several pause sites were also observed, with pause sites 119 and 125 in the R5 region and pause site 151 in the R6 region. There was virtually no observed pausing in the R4 region, again correlating with low efficiency of strand transfer in this region.

Elimination of Pause Site 151 Resulted in a Lower Level of Strand Transfer in the Immediate Area

To directly test whether a pause site could facilitate strand transfer, we eliminated one pause site without changing the local sequence and then compared the efficiency of nearby strand transfer in the presence or absence of synthesis pausing. To do this, we constructed another template, donor B. As shown in Fig. 6A, the sequences of donor B and donor A are identical except for their 5` end plasmid-derived sequence. When comparing the time course of primer extension on donor A and donor B (Fig. 6B), it was observed that all the prominent pause sites remained unchanged on donor B except for the disappearance of pause site 151, indicating that pausing at position 151 has been eliminated without any change of local template sequence.

We compared HIV-RT-mediated strand transfer products using donor A versus donor B. Various amounts of acceptor A were added to each reaction. Both transfer reactions generated two homologous recombination products T1 and T2 as expected (Fig. 7), since both donor A and donor B share the same homologous region with acceptor A. The full-length primer extension product (F) and the transfer products T1 and T2 were quantitated by scanning the autoradiogram with a densitometer. The efficiency of strand transfer, defined as the ratio of transfer product (T) to the sum of transfer and full-length donor-directed products (T + F), was evaluated at the highest acceptor concentration. For transfer on either donor A or donor B, the ratio T1/(T + F) remained unchanged. However, the ratio T2/(T + F) decreased by 40% (±3%) when donor B was used in the transfer reaction. As mentioned above, T2 was mainly generated by strand transfer from the R5 and R6 regions (Fig. 5). The only difference between donor A and donor B in these two regions is the disappearance of pause site 151 in the R6 region of donor B, which suggests that elimination of pause site 151 was the primary cause of the lower level of T2 transfer product.


Figure 7: Strand transfer from donor A and donor B templates showed that elimination of pausing at position 151 lowers the efficiency of nearby strand transfer. HIV-RT (4 units) was incubated with 2 nM of primed donor A or donor B, in the absence or presence of increasing amounts of acceptor A (1, 10, and 50 nM). The reaction was run for 1 h. Markers on the left side of the figure indicate the reaction products of strand transfer from donor A. F represents the 170-nucleotide-long full-length primer extension product directed by donor A. Three transfer products from donor A are labeled T1, T2, and T3. Markers on the right side of the figure indicate the reaction products of strand transfer from donor B. F indicates the 172-nucleotide full-length primer extension product directed by donor B. Two transfer products from donor B are labeled T1 and T2.



Detection of Nucleotide Misincorporation at One Frequently Used Recombination Junction

To determine the fidelity of the strand transfer process, we examined the progeny DNA sequences at two frequently used recombination junctions, R6 and R2. First, we inspected 24 clones of T2 transfer products generated by template switching from the R6 region of donor A. Remarkably, about 30% of the recombinant molecules contained misincorporated nucleotides near pause site 151 in the R6 region (Fig. 8). In contrast, none of 28 clones of primer extension products directed by the donor A template without transfer displayed any misincorporation in the R6 region. This demonstrates that the mutations detected around pause site 151 in the R6 region were not caused by HIV-RT polymerization errors during donor template-directed primer elongation. Also, they were not caused by errors introduced by the PCR amplification. These observations show that a mutagenic event takes place at or near position 151, presumably accompanying transfer of the primer. Consequently, they suggest a direct relationship between pausing, the position of transfer, and transfer-related mutagenesis.


Figure 8: Detection of mutations generated in one frequent recombination region. DNA sequences of 24 recombinant molecules generated by strand transfer synthesis in region R6 were examined. The primer strand DNA sequences surrounding pause site 151 in R6 are shown. The numbers of recombinants isolated are given in parentheses. Underlined letters correspond to incorrectly encoded bases at the recombination junction. &cjs1219; represents the position where the nucleotide was deleted.



We also directly sequenced 24 clones of the T2 product generated by transfer in the presence of the donor B template. The result showed that 18 clones represented transfer from the R6 region. Only one clone displayed a mutation near pause site 151, altering the wild type sequence shown in Fig. 8to 5`-GACCACTTT&cjs1219;&cjs1219;CACCC-3`. Since the mutations near pause site 151 were likely to be caused by the transfer event at position 151, the apparent decreased level of mutation in this area indicated much lower level of transfer right from position 151. Thus, the sequencing data further demonstrated that pause site 151 promoted the transfer from position 151.

Misincorporation may occur at recombination junctions by at least two different mechanisms. Peliska and Benkovic(1992) observed non-template-directed nucleotide addition by HIV-RT at the 5` end of an RNA template prior to strand transfer. The large majority of nucleotides added were deoxyadenosine residues. After strand transfer the extra 1 or 2 nucleotides would not necessarily be complementary to nucleotides on the acceptor template. However, they could be extended by the RT, resulting in misincorporation. The second mechanism involves incorrect copying of the acceptor template. Homologous recombination is carried out by transferring of the 3` end of the nascent DNA strand to an identical sequence on the other RNA template. Unsuccessful annealing attempts could occur between the transferring DNA strand and the acceptor RNA template prior to completion of the transfer. These could lead to duplicate copying of the same nucleotide or skipping of a template nucleotide. We refer to this as slippage synthesis.

The sequences of the mutations (Fig. 8) suggests that some were caused by slippage synthesis while others derived from non-template-directed addition. Evidence for slippage synthesis was the repetition in the newly synthesized DNA of nucleotides complementary to the sequence near position 151. Evidence for non-template-directed nucleotide addition was the presence of dA residues in the newly made primer, although no corresponding dT residues were present in the template.

Examination of DNA sequences of 20 clones of T1 transfer products generated from another frequently used recombination site, R2, revealed no mutagenesis. This result indicates that mutagenesis can be much more frequent at some sites than others. The frequency must be strongly influenced by local sequence or secondary structure.

Detection of Nonhomologous Recombination Products

Sequence analysis of the T3 product indicated that it was the result of nonhomologous recombination. We use the term nonhomologous recombination to describe transfer at positions that does not lead to exact copying of the long homology shared by the donor and acceptor templates. In this case, the actual point of transfer could have an incidental homology of several nucleotides between the two templates, that could promote the transfer reaction.

To improve our detection of nonhomologous recombination products, we measured strand transfer with another acceptor template (acceptor A*) derived from nef J14 instead of nef T17. The plasmid pCR1000(nef J14) was linearized by XhoI in the nef J14 coding region and transcribed using T7 RNA polymerase. Acceptor A* shared exactly the same homologous region with donor A template. Therefore, strand transfer between donor A and acceptor A* should produce only one single homologous recombination product, allowing easier detection of all nonhomologous recombination products.

As shown in Fig. 9A, two nonhomologous recombination products, T3a and T3b, were detected, in addition to the expected homologous recombination product ``T.'' Both of these products were generated when HIV-RT carried out the primer extension to the end of the donor template where there were three Gs. The three incorporated Cs apparently guided the growing point of the nascent DNA strand to the two positions of GGG on acceptor A*, generating T3a and T3b, respectively (Fig. 9B). The sequence of T3a showed that it was the same as the T3 product observed in Fig. 4.


Figure 9: Detection of nonhomologous recombination products. A, strand transfer between donor A and acceptor A* templates. F indicates the full-length primer extension product directed by the donor A template. Three transfer products, marked as T3a, T, and T3b, were generated in the presence of acceptor A* template. T is the homologous recombination product. T3a and T3b are major nonhomologous recombination products. B, schematic representation of the generation of T3a and T3b. Only the template sequences are shown. The sequences in bold are part of the homologous region. The plain text shows the plasmid sequence at the 5` end of each template. T3a was generated by template switching of RT and primer from the GGG at the 5` end of donor A to the GGG located in the 5` end plasmid sequence of acceptor A*. T3b was generated by template switching from the same 5` end GGG of donor A to another GGG of acceptor A* residing in the homologous region.



Interestingly, the efficiency of the nonhomologous recombination resulting in T3a was especially high, comparable with the level of homologous recombination. Furthermore, although the recombination events generating T3a and T3b all made use of a short three nucleotide sequence identity d(GGG), the level of T3a product was much higher than that of T3b. The results indicate that arrival of the primer at the end of the template greatly promotes strand transfer, compared with transfer from internal positions on the template. The existence of 100 bases of homology behind the recombination junction might also cause the high yield of nonhomologous recombination product. The base pairing between the donor and acceptor templates brought the recombination sites on both templates to juxtaposition, which could favor the nonhomologous recombination process. This suggests that for the nonhomologous recombination process in vivo any local base pairing between the nascent DNA strand and the acceptor template may mediate the selection of a downstream recombination site. We also observed in Fig. 7that when the 5`-GGG-3` sequence of donor A was replaced by 5`-GAATA-3` of donor B, the level of T3 product decreased so much that it could not be detected on the gel, even in the presence of the 100-base homology. This indicates that the strength of hydrogen bonding at the point of transfer is important for the efficiency of nonhomologous recombination.


DISCUSSION

We have evaluated the role of pausing in the process of HIV-RT-mediated strand transfer in vitro. Both the donor and the acceptor templates were natural viral RNA templates that share a relatively long homologous region. Allelic markers distributed along the template allowed us to determine where the transfer events took place. Results demonstrated a positive correlation between pause sites and an elevated level of strand transfer in their immediate area. Transfer at one location was accompanied by a very high frequency of mutagenesis (about 30%), whereas in other locations no mutations were observed. Mutations appeared to be caused by both non-templatedirected nucleotide addition and slippage synthesis. A high frequency of nonhomologous recombination was also observed.

We observed a number of positions where synthesis paused on template A. Examination of sequences at these points revealed a generally higher level of C and G residues in the template, but no exact sequence appeared at all sites. This suggests that a combination of sequence and secondary structure features create sites that pause synthesis. This interpretation is consistent with previous results (DeStefano et al., 1992; Klarmann et al., 1993; Abbotts et al., 1993), demonstrating that sequence context independent of secondary structure can influence pausing. The apparent effects of secondary structure could be seen in the case of the pause site at position 151. As shown in Fig. 6, an alteraton in the downstream plasmid-derived sequence totally eliminated the pausing at position 151. One likely explanation is that DNA synthesis was interrupted by physical interaction of the region near position 151 with sequences near the 5` end of donor A template. Change of the downstream sequence would then have disrupted this interaction such that the enzyme no longer paused at that position.

Although positions of transfer correlated with pause sites, other factors also appeared to influence the total level of transfer. We observed that transfer occurred more frequently from the R6 region than the nearby R5 region, in spite of a similar quantity of paused primers in both regions. The especially high level of transfer from the R6 region could have resulted from a local sequence feature, for example the stretch of A residues in the template upstream of pause site 151. The strength of the hybrid between the 3` region of the nascent DNA strand and the donor template should be weak immediately after synthesis over this region, because of the low number of hydrogen bonds, facilitating transfer to the acceptor template.

There is a strong pause site at position 68. However, we did not detect any promotion of strand transfer by this site in the R1 region. The result is consistent with the observation that the nascent DNA strand had acquired only an 8-nucleotide homology with the acceptor template by the time its growing point reached position 68. As reported by Luo and Taylor(1990), such a short homology is not adequate for efficient strand transfer. Apparently, the length of homology between the primer and acceptor template is also an important influence on the frequency of transfer. Recognizing the need for some homologous nucleotides to be incorporated into the nascent DNA strand before transfer can occur, we designate the possible region of transfer R1 as beginning just beyond position 68 (Fig. 5).

Elimination of the pause site at position 151 decreased the quantity of the T2 transfer product by about 40%. Examination of allelic markers by DNA sequence analysis revealed that transfer from within the R6 region, as opposed to the R5 region which also contributed to the T2 product, was specifically lowered. The sequences further showed that only 1 out of 18 clones of T2 products contained mutations around position 151, much lower than the 30% misincorporation rate we measured in the presence of pause site 151. This reduced misincorporation suggests that the strand transfer events specifically promoted by pausing at position 151 were particularly mutagenic. This result clearly supports a relationship between pausing, induction of strand transfer, and mutagenesis.

The proposition that pausing facilitates strand transfer can be used to interpret the recent results obtained by Zhang and Temin(1994) in a Moloney murine leukemia virus system. They evaluated the effect of the length of sequence identity on recombination efficiency. Their data indicate that two similar sequences of the same length did not recombine at the same rate. Examination of both sequences did not reveal any unique features, such as potential stem loops or runs of A or T. Since sequence determines positions of pausing (Klarmann et al., 1993; Abbotts et al., 1993), the sequence preferred for recombination might have resulted from the presence of a prominent DNA synthesis pause site that would stimulate strand transfer.

We hypothesize that pausing plays two roles in promoting strand transfer. First, pausing should favor the cleavage of the RNA template beneath the nascent DNA strand by the RNase H activity of HIV-RT. According to our previous results (DeStefano et al., 1991), the polymerase and RNase H activities of HIV-RT are not strictly coupled. During processive DNA synthesis, the enzyme does not degrade the RNA template extensively. When RT pauses, the RNase H activity has a greater opportunity to cleave at the preferred position 18 or 19 bases upstream of the polymerase active site (DeStefano et al., 1991a; DeStefano et al., 1994). Furthermore, the 3` to 5` directional endonucleolytic activity of RNase H would have a greater potential to enlarge the gap in the RNA (DeStefano et al., 1991b). This process exposes the nascent DNA strand, facilitating hybridization between the nascent DNA and the new RNA template. Second, pausing allows the accumulation of nascent DNA products that have not completed synthesis on the donor template. These are available to serve as substrates for internal template switching to another RNA template.

Peliska and Benkovic(1992, 1994) and Patel and Preston(1994) presented a model for misincorporation by retroviral RTs. They proposed that when the RT encounters a break in the RNA template, it completes synthesis to make a blunt end with the template. It is then likely to add one or more non-template-encoded nucleotides extending the primer beyond the RNA template. These are added with the preference A > G T C. Transfer of the nascent DNA strand to the acceptor template, driven by the forced-copy choice mechanism, results in the fixation of the mutation at the recombination junction. It is not clear whether this model is applicable to our experimental conditions. When the RT pauses, it should degrade the RNA initially about 18-19 nucleotides behind the primer terminus, with further 3` to 5` endonuclease action as described above. When template RNA ahead of the primer finally dissociates from the primer terminus, the primer would extend well beyond any template polymer. The blunt end precursor to non-template nucleotide addition would not be expected to form. This explanation is consistent with the observation that pause sites are generally not promutagenic except in the vicinity of homopolymeric nucleotides (Bebenek et al., 1991). Therefore, it is understandable that we did not detect any mutation at position 151 in primer extension products without transfer.

Nevertheless, we have observed mutations that result from unexpected additions of dA during transfer, indicative of non-template-directed nucleotide addition or other similar mechanisms (Clark et al., 1987). This could mean that non-template-directed nucleotides can be added by mechanisms other than the mechanism proposed above. Alternatively, some blunt-ended intermediates may form during internal strand transfer.

Most mutations detected in our system in the process of homologous recombination could be generated by a slippage mechanism. When strand transfer occurs, hybridization between the nascent DNA strand and the new RNA template may go through a period of instability, during which hydrogen bonds are made and broken. It is likely that such a process would favor misincorporation, copying of the same template nucleotide twice, or skipping of a template nucleotide. Since HIV-RT does not have an editing activity and is unusually capable of extending a mismatched template-primer (Perrino et al., 1989), misincorporated nucleotides at the recombination junction are readily retained. Since it involves the template, misincorporation by this mechanism is expected to be very dependent on local sequence at the point of transfer. A stretch of A-T-rich sequence in the template-primer stem can decrease the strength of hybridization between the nascent DNA strand and the new RNA template, thereby favoring misincorporation at the template-primer termini. This can account for the high rate of mutation detected at position 151, where the upstream sequence was A-T-rich.

Using their Moloney murine leukemia virus system in vivo, Zhang and Temin(1994) examined the sequence at one recombination junction with 60-nucleotide sequence homology. No misincorporation was detected in any of 22 recombinants. Several factors may account for the absence of misincorporation. The recombination junction sequence they examined simply might not have promoted misincorporation. Hence, the mutation rate at that specific location was so low that no misincorporation could be detected within 22 recombinants. Also, compared with HIV-RT, Moloney murine leukemia virus RT is more faithful at copying complementary nucleotides and has a lower capacity to extend a mismatched terminus. (^2)This could have resulted in increased fidelity of strand transfer. Alternatively, the cell culture system they utilized may have had some intrinsic difference from the cell free system used here.

In summary, our results demonstrate that pausing correlates positively with an enhanced level of strand transfer in vitro. Elimination of a pause site was shown to decrease the efficiency of strand transfer in the immediate area. Examination of two frequently used recombination regions showed that misincorporation occurred in our system, but did not accompany every strand transfer. The rate of misincorporation may be highly affected by local sequence context or secondary structure. We have previously speculated that recombination in this region of nef is modulated by protein factors (Blumberg et al., 1992). Further study is required to investigate this presumption.

Suppression of viral recombination is likely to be an important step in the current effort to develop a stable live attenuated HIV-1 vaccine for acquired immune deficiency syndrome (Desrosiers, 1992). To define viral sequences and mutations that are likely to be stable, it is essential to learn the mechanism of this recombination.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM 49573 and in part by NS28754 and Core Grant CA11198 to the University of Rochester Cancer Center. 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: HIV, human immunodeficiency virus; RT, reverse transcriptase; RNase H, ribonuclease H; PCR, polymerase chain reaction.

(^2)
B. D. Preston, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Jasbir Seehra and John McCoy, representing the Genetics Institute, for the generous gift of HIV-RT.


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