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Originally published In Press as doi:10.1074/jbc.M610056200 on January 3, 2007

J. Biol. Chem., Vol. 282, Issue 9, 6222-6231, March 2, 2007
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Insights into the Multiple Roles of Pausing in HIV-1 Reverse Transcriptase-promoted Strand Transfers*

Lu Gao{ddagger}, Mini Balakrishnan{ddagger}1, Bernard P. Roques§, and Robert A. Bambara{ddagger}2

From the {ddagger}Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester, Rochester, New York 14642 and the §Department de Pharmacochimie Moleculaire et Structurale, INSERM U266, CNRS UMR 8600, Faculte de Pharmacie 4, Avenue de l'Observatoire 75270 Paris Cedex 06, France

Received for publication, October 27, 2006 , and in revised form, December 19, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously analyzed the role of pausing induced by hairpin structures within RNA templates in facilitating strand transfer by HIV-1 RT (reverse transcriptase). We proposed a multistep transfer mechanism in which pause-induced RNase H cuts within the initial RNA template (donor) expose regions of cDNA. A second homologous RNA template (acceptor) can interact with the cDNA at such sites, initiating transfer. The acceptor-cDNA hybrid is thought to then propagate by branch-migration, eventually catching up with the primer terminus and completing the transfer. The prominent pause site in the template system facilitated acceptor invasion; however, very few of the transfers terminated at this pause. To examine the effects of homology on pause-promoted transfer, we increased template homology before the pause site, from 19 nucleotides (nt) in the initial template system to 52 nt in the new system. Significantly, the increased homology enhanced transfers 3-fold, with 32% of the transfers now terminating at the pause site. Additionally, the acceptor cleavage profile indicated the creation of a new invasion site in the added region of homology. NC (nucleocapsid) increased the strand transfer throughout the whole template. However, the prominent hot spot for internal transfer remained, which was still at the pause site. We interpret the new results to mean that pause sites can also serve to stall DNA synthesis, allowing acceptor invasions initiated earlier in the template to catch up with the primer terminus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus-1 (HIV-1)3 is the cause of AIDS (Acquired Immunodeficiency Syndrome). A big challenge for HIV-1 treatment is drug resistance, which the virus develops quickly after antiviral drug treatment. HIV-1 is a highly mutagenic retrovirus because of the low fidelity of reverse transcriptase (RT), which lacks 3'-5' proofreading ability (1, 2). Recombination can disperse mutations to generate viruses that better evade the host immune system and are resistant to multiple antivirus drugs (3, 4). Recombination can also restore defective viruses (5) as well as generate novel mosaic viral strains (6, 7).

HIV-1 packages two copies of RNA as its genome. Reverse transcription generates a linear DNA duplex from the viral RNA genome, which later integrates into the host genome. Strand transfers, in which an elongating primer switches from one template (donor) to another (acceptor), are part of the replication process (8). Two template switch events are required to complete reverse transcription. They are referred to as the minus strong stop DNA transfer and the plus strong stop DNA transfer. RT can also transfer within internal regions of the viral genome (9–11). Internal strand transfer occurs primarily during minus strand synthesis from the RNA genomes (12–15). Genetic recombination occurs when such template switching occurs between two heterozygous genomic RNAs copackaged within the virion. Heterozygous virions are produced by double infection, a process which has been shown to occur during HIV-1 infection (16). Reverse transcription in HIV-1 is highly recombinogenic, with about three crossover events per genome per replication cycle (12, 17).

The ribonuclease H (RNase H) activity of RT (18–20) and template homology (21–26) are essential for strand transfer. RNase H cleavages on the donor template can lead to removal of the RNA template from the nascent DNA making it available for the homologous acceptor to anneal, which was proposed to facilitate strand transfer (18, 27, 28). Template degradation by RT-RNase H cleavage is not coupled with DNA synthesis (29), allowing for clustering of RNase H cleavages when DNA synthesis is slowed (30, 31). RT pausing during synthesis is proposed to promote strand transfer (18, 28, 32–34), because template degradation by the RNase H activity of RT is increased by RT. On the other hand, a pause-independent mechanism of strand transfer is also suggested, because weakly structured templates lacking strong pause sites also demonstrate efficient transfer (34–36).

Reverse transcription initiates in the viral core where nucleocapsid protein (NC) is associated with the RNA genome. NC, a nucleic acid chaperone, can rearrange substrate structures to induce the most thermodynamically stable structure (37–39). NC has been shown to strongly enhance strand transfer in highly structured templates, with less of an effect seen in weakly structured templates (36). NC can facilitate transfer by promoting strand exchange and annealing of the nascent DNA from the degraded donor to the acceptor template (27, 36). NC can also affect RT, enhancing its RNase H activity (40–42). NC has also been shown to destabilize secondary structures to facilitate DNA synthesis through the template (33, 43–45).

Previously, we observed that when a stable hairpin structure on the RNA template stalled RT synthesis at the hairpin base, extensive cleavages occurred on the RNA template around the pause site. However, most primer terminus transfers occurred within the hairpin. This leads to the proposal of the "Dock and Lock" mechanism for transfer (28, 42). RT pausing leads to clustering of RNase H cleavages exposing the nascent DNA, allowing the acceptor to invade at the hairpin base (Dock step). RT continues to synthesize the cDNA on the donor template. The acceptor-cDNA interaction propagates and transfer of the primer terminus (Lock step) occurs later in the stem and loop regions of the hairpin. Results suggest that invasion is most effective when the acceptor is weakly structured within the region of invasion, contributing to efficient transfer (28, 46, 47). In the current work, we modified the previously described system, containing the primer binding site (PBS) of Equine Infectious Anemia Virus (EIAV) that forms a stable hairpin structure (28), by increasing the homology before the hairpin. We found that the extended homology significantly increased strand transfer while also promoting a peak of primer terminus transfer at the pause site at the hairpin base. Results showed that stalling of synthesis at a pause site allows acceptor invasions initiated earlier in the template to catch up with the primer terminus. The combined data suggest that while pause-promoted RNase H cuts facilitate acceptor invasion, stalling of synthesis at a pause site can also promote primer terminus transfer of invasions occurring earlier.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Expression plasmids (HXB2 strain) pKK-p66(His)6 and pKK-p51(His)6 (48) were kindly provided by Dr. Neerja Kaushik. HIV-1 reverse transcriptase (p66/p51 heterodimer) was purified as described previously (42, 49). HIV-1 nucleocapsid protein NCp7-(1–72) was chemically synthesized (50). DNA oligonucleotides were obtained from Integrated DNA technologies (Coralville, IA). DH5{alpha} competent cells, NotI, HindIII, and Taq polymerase were from Invitrogen (Carlsbad, CA). BsrI was from New England Biolabs (Ipswich, MA). Templiphi Amplification kit was obtained from GE Healthcare (Piscataway, NJ). Poly oligo(dA) and oligo(dT) were purchased from the W. M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT). [{gamma}-32P]ATP (6000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Escherichia coli RNase H (10 units/µl) and the T7 MEGA shortscript were purchased from Ambion (Austin, TX). All other reagents were obtained from Roche Applied Science (Indianapolis, IN).

Generation of RNA Templates—The donor construct pEIAV-Donor and acceptor constructs pEIAV-A2 and pEIAV-A1 have been previously described (28). Donor RNA DI, DN, and acceptor RNA A19 were generated by run-off transcription in vitro from pEIAV-Donor linearized with NotI, pEIAV-Donor linearized with BsrI and pEIAV-A2 linearized with HindIII, respectively, using T7 RNA polymerase as previously described (28). The long homology acceptor RNA templates A52 and mA52 were generated by run-off transcription in vitro using the Ambion T7 MEGA shortscript kit, from PCR products derived from pEIAV-A2 and pEIAV-A1, respectively. The forward PCR primer (5'-CCTCTAATACGACTCACTATAGG) and reverse primer (5'-TTAGGTGACATTATAGAATATGTATCAC), with underscored nucleotides that vary by plasmid to determine transfer distribution, were used. All RNAs were purified by 8% denaturing PAGE. RNAs were quantitated using the Ribogreen assay (Molecular Probes, Eugene, OR) and UV absorption measured on a GeneQuant II from Amersham Biosciences.

Preparation of Substrates—To 5' end-label RNA template, the purified RNA was first treated with calf intestinal phosphatase to remove the 5'-phosphate. Then RNA templates and DNA primers were 5' end-labeled using polynucleotide kinase and [{gamma}-32P]ATP. Unincorporated ATP was removed using a Bio-Rad P-30 Micro BioSpin size exclusion column. Donor RNA templates and the DNA primers mixed in the appropriate ratios were incubated at 95 °C for 5 min and slowly cooled to room temperature. DNA primers SP6 (5'-TACGATTTAGGTGACACTATAG) and lg17 (5'-CAGTGCCAAGCTGAC) primed synthesis on donor DI and DN, respectively.

Primer Extension and Strand Transfer Assay—Reactions were performed as previously described with slight modifications (28). The donor template and radiolabeled DNA primer were annealed in a 1:2 ratio for primer extension experiments. For the strand transfer assay, the donor:acceptor:primer ratio was 1:2:2. RT was preincubated with the substrate mixture at room temperature for 5 min prior to initiation of reactions by addition of MgCl2 and dNTPs. The final reaction contained 4 nM primer, 2 nM donor, 4 nM acceptor, 35 nM RT, 50 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 50 µM dNTPs, 50 mM KCl, 1 mM dithiothreitol, and 1 mM EDTA. For reactions performed in the presence of NC, 200% NC, unless indicated otherwise, was added to the mixture prior to addition of RT and incubated at room temperature for 5 min. Reactions were incubated at 37 °C and were terminated at appropriate time points by addition of 2x termination dye (20 mM EDTA (pH 8.0), 90% formamide, and 0.1% each of bromphenol blue and xylene cyanole). Reaction products were separated by 6% denaturing PAGE and visualized by Storm PhosphorImager (GE Healthcare) and Image-Quant software v 1.2 (GE Healthcare).

Analysis of Transfer Products—To determine the location of primer terminus transfer among the transfer products generated, transfer products from the short homology templates DI/A19 were isolated, amplified, cloned, and sequenced as previously described (28). Transfer products from the long homology templates DN/A52 and DN/mA52 were purified similarly and PCR-amplified using primers lg11 (5'-ACACCGTCAGCCACCAGAGCTCACTAGGGATAAATATAA) and lg12 (5'-ACTGGGATCCTGCAGTGCCAAGCTGAC). The amplified DNA products were double-digested by SacI and BamHI, and cloned into pBluescript II SK(+) (51). Individual colonies were picked, and the plasmid was amplified for DNA sequencing. Each clone represented an individual transfer product. To address the possibility of PCR recombination, primer extension products isolated from separate donor and acceptor extension reactions were mixed in equal amounts and PCR-amplified using primers lg22 (5'-ACCAGAGCTCACTAGGGTCTACTCAGTCCC) and lg23 (5'-ACTGGGATCCTGTTAGGTGACATTATAGAATATG). The amplified products were cloned and sequenced to determine the percentage of recombinants generated during PCR.


Figure 1
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  		FIGURE 1.
Substrates used in the study. A, schematic of substrates used in the study. The short homology templates, the 140-nt RNA donor DI and the 110-nt RNA acceptor A19, share a homology of 97 nt comprised of a 78-nt hairpin and a 19-nt region before the hairpin. DNA synthesis is initiated from DNA primer SP6. In the long homology templates, the homology before the hairpin is 52 nt, giving a total homology of 130 nt. DNA synthesis is initiated from DNA primer lg17. X indicates that the last nucleotide at the 5'-end of the region of homology on the acceptor is different from the donor. B, illustration of the lowest potential energy structure of acceptor A52 predicted by M-fold. Bases circled are different from the donor sequence and are labeled from –2 to 8.

 
RNase H Cleavage Assays—For donor cleavage assays, transfer reactions were performed as described above using 5' end-labeled donor template and unlabeled primer. A 1:2 ratio of donor:acceptor was used. For acceptor cleavage assays, transfer reactions were set up as described above with 5' end-labeled acceptor template. A 1:3 ratio of donor:acceptor was used. For experiments using E. coli RNase H to detect acceptor:cDNA hybrids, transfer reactions were terminated with a final concentration of 1 nM poly oligo (dA):oligo (dT) and 50 µM ddNTPs at appropriate time points. The terminated reaction mixture was then incubated with 7 units of E. coli RNase H at 37 °C for 3 min, followed by addition of 2x termination dye, as described above, to terminate the reactions. Reaction products were separated by 10% denaturing PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In this study we designed substrates to examine how template homology before a pause site influences strand transfer.

Templates with Increased Homology before the Pause Site—Our previous work describing the pause-promoted two-step transfer mechanism was performed using stable hairpin structure containing donor DI and acceptor A19 RNA templates. The templates shared a homology of 97 nt, comprised of a 78-nt hairpin and a 19-nt region before the hairpin (Fig. 1A). The prominent pause site at the hairpin base in the template system facilitated acceptor invasion; however, very few of the transfers terminated at this pause site. To examine the effects of homology on pause-promoted transfer, the homology before the hairpin was increased from 19 to 52 nt, increasing the total homology to 130 nt (Fig. 1A). The last nucleotide at the 5'-end of the homology between the donor and acceptor RNA templates is different. This difference is designed to inhibit end transfer, the transfer after completion of synthesis to the end of donor. MFold (52, 53) structure prediction analysis of the acceptor template suggested that the original hairpin structure was undisturbed, and no additional stable structures were formed within the extended homology (Fig. 1B).

Increased Homology before the Pause Site Increased Transfer—Primer extension was performed by HIV-1 RT using a 5' end-labeled DNA primer annealed to the donor template. Donor extension product (DE) was formed when the DNA primer synthesized to the 5'-end of the donor. Because the 3'-end of the donor extension product can form a hairpin, it can use itself as a template to continue synthesis, producing a longer DNA fold-back product (FB) (Fig. 2A). For the short homology substrates (Fig. 2B), DE was 136-nt long and FB was 194-nt long. RT paused early in the reaction at the hairpin base +58, and that pause product was observed throughout the reaction, although decreasing in intensity. At 1 min, 41% of extended products in the donor extension system were paused at +58, while 5% were full-length products (DE and FB). At 30 min, 22% of the extended products remained at pause site +58 while 31% were full-length products. Other pausing was also observed at +80, +93, +120, and numerous minor sites. With the long homology substrate (Fig. 2C), a 164-nt DE and a 250-nt FB were formed. The extended homology did not significantly disturb the stable hairpin structure as indicated by little effect on RT pausing at the hairpin base +86. RT pausing at +86 was prominent until 5 min, but then efficiently chased away with time. At 1 min, 21% of extended products on the donor were paused at +86 while 5% were full-length products (DE and FB). At 30 min, 7% extended products remained at pause site +86, while 20% were full-length products. The other pause products at +108, +121, and +148 were equivalent to those at +80, +93, and +120 in the short homology substrates. Within the extended homology region in DN, a new pause site at position +42 was seen early in the reaction, but efficiently chased away.


Figure 2
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  		FIGURE 2.
HIV-1 RT catalyzed donor extension and transfer reactions. A, schematic of donor extension product, fold-back product, and transfer product formed during transfer reactions in vitro. B and C, donor extension and transfer reactions of DI/A19 and DN/A52, respectively. The DNA primers were 5'-end labeled. Reactions were performed in the absence (right) or presence (left) of acceptor. Reactions were sampled at different time points indicated on the top of each lane and separated on a denaturing 8 or 6% polyacrylamide gel. Donor extension product (DE), fold-back product (FB), transfer product (TP), and pause sites in each system are indicated on the left of each gel. L, 10-bp DNA ladder.

 
In the presence of acceptor template, HIV-1 RT could switch from donor RNA to acceptor RNA to make the transfer product (TP) (Fig. 2A). For the short and long homology substrates, TP lengths were 149-nt long (Fig. 2B) and 177-nt long (Fig. 2C) respectively. In both systems, pausing at the hairpin base decreased more quickly in the transfer reaction than in donor extension reactions. Especially for the long homology substrate, the prominent pause site almost disappeared with the accumulation of TP.

Transfer efficiency is indicative of how frequently the primer terminus switches from donor template to acceptor template and was calculated as TE = TP/(TP + DE + FB) x 100%. The TE showed that the extended homology played an important role in strand transfer. Compared with the short homology system DI/A19, the transfer efficiency of the long homology system, DN/A52, was greater, increasing from 6 to 20% at 30 min (Fig. 3A).

NC Decreased RT Pausing and Enhanced Transfer—Viral NC was shown to play a significant role in transfer with the short homology templates, DI/A19 (42). We performed the transfer reaction with the long homology substrate DN/A52 at different NC concentrations to determine the optimum for transfer. We considered 100% NC coating as one NC molecule bound per seven nucleotides, and performed the transfer reaction without NC, or with 50, 100, 200, or 400% NC coating (Fig. 4A). The transfer efficiency without NC was set to 100%, and the relative transfer efficiencies with different levels of NC coating were plotted (Fig. 4B). The highest transfer efficiency was achieved with 200% NC, which was more than double that without NC. Therefore, we used 200% NC for the rest of our NC studies.

In the presence of NC, the pause sites were similar in the donor extension reaction (data not shown) and the transfer reaction (Fig. 3B). NC decreased the intensity of pause products with both substrates, but the decrease was more significant with the long homology substrate (compare Figs. 2, B and C and 3B). Unlike the pause at the hairpin base, the other pause bands faded with NC (Fig. 4A). The inhibition of the fold-back product by NC was more distinct with the long homology substrate, than the short homology substrate (Fig. 3B).

NC enhanced TE in both systems (Fig. 3A). Similar to previous results (42), the TE of the short homology substrate at 30 min increased from 6 to 18%. For the long homology substrate, NC increased the TE from 20 to 46% at 30 min. In the presence of NC, the long homology substrate still had a higher TE than the short homology substrate, 46 versus 18%.


Figure 3
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  		FIGURE 3.
Transfer efficiency and transfer reactions in the presence of NC. A, transfer efficiencies were plotted for the short homology templates DI/A19 and the long homology templates DN/A52 in the absence or presence of NC. Transfer efficiency was calculated using TE = TP/(FL + TP + FB) x 100%. B, transfer reactions catalyzed by HIV-1 RT in the presence of 200% NC. Reactions were sampled at different time points indicated on the top of each lane and separated on a denaturing 6% polyacrylamide gel. Substrates in each reaction are shown in the schematic to the right of each gel. Donor extension product (DE), fold back product (FB), transfer product (TP), and pause sites in each system are indicated on the left of each gel. L, 10-bp DNA ladder.

 
The Extended Homology before the Pause Site Increased Terminus Transfer at the Pause Site—To identify the positions of primer terminus crossover in the transfer product, we introduced single nucleotide substitutions, which serve as markers, in the acceptor at periodic intervals. They are labeled 1–8 (28), starting at the 3'-end. Within the DN/A52 template system, two additional markers -1 and -2 were included in the extended homology to allow examination of the influence of this region on the transfer distribution (Fig. 5). Transfer products were purified, cloned, and sequenced to obtain the terminus transfer distribution profiles. Only products of odd crossover events result in a transfer product, while even crossover events return the primer to the donor template. Transfer products resulting from triple crossover events represented only 3% of the total transfer products analyzed.

First, we generated raw transfer distribution data to get the transfer frequency in each segment. To obtain transfer frequency, the number of crossover events in the segment was divided by the total crossover events. Multiplying the transfer frequency within each segment by the transfer efficiency produced the TE-corrected transfer distribution (Fig. 5A). It shows the absolute number of terminus transfers occurring within a given segment. In the TE-corrected data, the distribution of terminus transfers beyond the pause site (from marker 2 to the end) was quite similar in the long and short templates. A large portion, 4–6 terminus transfers, occurred between markers 4 and 6 while little occurred between markers 2 and 4, and between markers 6 and 8. However between markers 1 and 2 encompassing the pause site at the hairpin base, an average of 5 transfers occurred in the extended homology template compared with almost none in the short homology template. NC increased the transfer efficiency of the long homology substrate from 20 to 46%, and increased terminus transfer events relatively evenly throughout the whole template at least doubling the value in each segment (Fig. 5). NC also caused a shift of transfers toward the template 5'-end as indicated by the increase in transfers after marker 7.


Figure 4
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  		FIGURE 4.
NC titration with the long homology substrate. A, titration of NC with strand transfer using the long homology templates. The reactions were performed in duplicate with 0, 50, 100, 200, and 400% NC coating, indicated on top of the lanes. 100% NC coating is defined as 1 NC for every 7 nucleotides. Products were resolved on a 6% denaturing polyacrylamide gel. Pause sites at the hairpin base +86, plus sites +107, +120, and +148, and the donor extension product DE (164), transfer product TP (177) and fold-back product FB are labeled on the right of the gel. B, relative transfer efficiency with different percentages of NC coating are plotted.

 


Figure 5
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  		FIGURE 5.
Transfer distribution profiles of HIV-1 RT on the short and long homology substrates. Acceptor templates contain single nucleotide substitutions (open circles) at periodic intervals, indicated by the numbers –2 to 8, which serve as the markers to determine the location of primer terminus transfer. Transfer products were purified, cloned, and sequenced to determine the transfer distribution. Three independent experiments are shown, each including 35–50 recombinant clones. A, TE-corrected transfer distribution to determine the absolute number of primers that transferred within a given segment. The formula used is % transfers in each segment xTE x 100%. B, distance-corrected transfer distribution to account for the variation in marker distances. The formula used is (% transfers in each segment/the length of the segment) x 10. The distance-corrected distribution reveals preferred sites (hot spots) of transfer within the template. End transfer is excluded as distance correction cannot be applied.

 
Because the distance between markers is not identical, the transfer frequency within each segment was divided by the length of the segment to correct for variations in segment length (Fig. 5B). The distance-modified transfer frequency reflects the proportion of terminus transfer within each segment. For the short homology substrate, terminus transfer was most frequent between markers 4 and 5, consistent with previous observations (28). For the long homology substrate, the major hot spot was shifted to between markers 1 and 2 before the pause site. The overall impression is that with the longer homology, a second transfer profile centered on the hairpin base is superimposed on the first. With NC, the hot spot of terminus transfer was still at the pause site.

To exclude the possibility of PCR recombination, primer extension products isolated from separate donor and acceptor extension reactions were mixed in equal amounts and PCR-amplified. PCR products were cloned and analyzed similarly to the transfer products. 24 clones were sequenced. All represented faithfully amplified donor or acceptor cDNA, indicating that PCR recombination was less than 4%.

Evidence That Considerable Acceptor Invasion Occurred within the Extended Region of Homology—For the short homology substrate, DI/A19, the initial interaction of acceptor with cDNA was suggested to be at the base of the hairpin (28). RNase H cleavage accompanying RT pausing at the hairpin base facilitated donor and acceptor interaction, and terminus transfer occurred beyond the pause site. For the long homology substrate, DN/A52, the hot spot of terminus transfer shifted to the base of the hairpin. To locate the acceptor invasion sites, we examined RNase H cleavages on the donor template during synthesis (Fig. 6A). To detect donor cleavages, transfer reactions were carried out using a 5' end-labeled donor and unlabeled primer and acceptor. Cleavages on the donor are essential to facilitate exposure of the nascent DNA allowing for interaction with the acceptor (27, 51, 54, 55). Concentrations of RNase H cleavages on the donor would mark candidate regions for acceptor invasion. In the reactions without NC, RNase H cleavages always were detectable in the 52-nt homology region before the hairpin (+78 to +130). Some minor cleavages were detectable at 0.5 min, and then cleavages increased at 1 min, such as at + 112, +94, and +84. Some minor cleavage bands were chased away as synthesis progressed and others like +94 and +84 remained to the end of the reaction. In the presence of NC, a ladder of cleavages in the 52-nt homology region before the hairpin intensified from 3 to 5 min and was then chased away. The specificity of the cleavages in the presence of NC was somewhat different from that in the absence of NC. These results suggest that the entire region of homology before the hairpin is a candidate for the initial donor and acceptor interaction, both with and without NC. Also, in the hairpin stem loop, fewer cleavages were observed in the presence of NC, because NC decreased RT pausing in this region (Fig. 3B).


Figure 6
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  		FIGURE 6.
RNase H cleavage profile of HIV-1 RT in a transfer reaction. A, donor DN cleavage profile of HIV-1 RT in a transfer reaction. Reactions were performed in the absence or presence of 200% NC. DN was 5' end-labeled and annealed to primer lg17. B, acceptor A52 cleavage profile of HIV-1 RT in a transfer reaction and analysis of acceptor-cDNA interaction using E. coli RNase H. The reaction was performed in the presence of 200% NC. A52 was 5' end-labeled. The left part of the panel is only with HIV-1 RT, and the reaction is sampled at different times as indicated above each lane. To better visualize acceptor-cDNA hybrid formation, reaction aliquots terminated at various time points were treated with E. coli RNase H, and is shown in the right part of the panel. The reactions were sampled at different time points indicated above each lane and separated on a denaturing 10% polyacrylamide gel. Substrates and the location of the cleavage bands are shown in the schematic to the right of the gel. Lane C, reaction in the absence of RT incubated at 37 °C for 30 min. Lane L, 10-bp DNA ladder.

 
To define sites at which the acceptor first interacts with the cDNA, we performed the transfer reaction with 5'-end-labeled acceptor and unlabeled primer and donor. RT RNase H can cleave the acceptor RNA template only when it interacts with the cDNA to form an RNA/DNA hybrid (Fig. 6B). Therefore, the first sites of cleavage on the acceptor identify the position of cDNA acceptor initial interaction. In the absence of NC, the profile showed minimal cleavages, preventing the identification of an invasion site (data not shown). However, with 200% NC, which strongly promotes nucleic acid annealing and a high efficiency of transfer, there was a clear acceptor cleavage profile (Fig. 6B). A series of RNase H cleavages by RT were detectable at 3 min in the 52 nt of homology before the main pause. These cleavages increased until 15 min, and were then chased away, suggesting that the acceptor first interacted with the cDNA in the region upstream of the hairpin. Addition of E. coli RNase H to acceptor cleavage reactions increases the sensitivity of detection of the acceptor RNA and nascent DNA hybrid (56). With E. coli RNase H, most cleavages were concentrated in the 3'-end homology region (30 ~ 50 nt before the hairpin) at 1 min, later shifting in the 5' direction, and then chased away at 5 min. There were some cleavages that remained to the end of the reaction, in the same positions as those seen with only RT RNase H. The acceptor cleavage profile suggested that the initial interactions appeared 30 ~ 50 nt before the hairpin base, within the extended region of homology.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous analysis of HIV-1 RT-mediated strand transfer using RNA templates containing a stable hairpin revealed a major RT pause site at the hairpin base. However, transfer of the primer terminus mostly occurred at sites beyond the pause site (28, 32). About 90% of the template switching occurred after the pause site in the TAR (transactivation response region) hairpin template system (32) and about 80% in the EIAV genome-derived hairpin template system (28). Further analysis of the transfer products in the EIAV template system revealed a multistep transfer mechanism (28, 42). In this mechanism pausing of synthesis at the hairpin base facilitates RNase H cuts on the donor, leading to exposure of the cDNA and facilitating of cDNA-acceptor interactions. While primer extension continues on the donor, the acceptor-cDNA hybrid initiated in the region where the donor was cut expands by branch migration. Transfer is eventually completed with the switching of the primer terminus to the acceptor. In both experimental systems, the pause site promoted acceptor invasion, while transfer of the primer terminus occurred beyond the pause site.


Figure 7
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  		FIGURE 7.
Two proposed roles of pausing of HIV-1 RT in promoting strand transfer. Steps in strand transfer are diagrammed on a linear representation showing features of the donor RNA template. The multistep transfer mechanism involves invasion of the donor-cDNA by the acceptor, propagation of the acceptor-cDNA hybrid and the terminus transfer. A, analysis of the long homology template in this study suggests that pause sites promote recombination by serving as termination points for transfer. Stalling of synthesis at a pause site allows acceptor invasions initiated earlier in the template to catch up with the primer terminus. B, as previously shown (28), RT pausing can also initiate strand transfers that complete later on the template by creating an invasion site. The transfer profile on the long homology template represents both processes superimposed on each other.

 
Terminus transfers in other systems have been (18, 33, 34, 57, 58) observed to occur within regions of RT pausing, prompting us to ask why the major pause site in our hairpin templates did not serve as a termination point for the transfers. We considered the possibility that the limited template homology before the pause site (19 nt in the EIAV and 23 nt in the TAR template system) restricted transfers from being completed at the pause site using the invasion-induced or other mechanisms described to date. Indeed, 22% of total transfers terminated at the region of pausing when donor-acceptor homology before the TAR hairpin was increased (59). In the current study, we modified the EIAV-derived template system, by increasing homology before the pause site from 19 nt in the initial templates (DI/A19) to 52 nt in the new system (DN/A52), thereby increasing total donor-acceptor homology from 97 nt to 130 nt. We designed our new substrates to maintain the initial hairpin structure, while avoiding new structures within the extended region of homology. Increasing overall homology by 34% increased transfer efficiency from 6 to 20%, indicating that the added homology had a substantial impact on strand transfer on the template. The presence of NC significantly enhanced transfers, promoting 20 and 46% with the DI/A19 and DN/A52 systems, respectively. A striking feature of the transfer profile with the added homology was that a significant proportion of the transfers (31% in the absence of NC and 22% in the presence of NC) now occurred at the major pause site compared with almost none with the short substrate. In fact, the TE-corrected distribution profiles suggest that the increase in transfer efficiency with the DN/A52 system comes from a new population of transfers that complete at and around the pause site, super-imposed over those occurring in the short homology template (Fig. 5A, compare TE corrected distribution for DI/A19 and DN/A52).

The time course of RNase H cleavages on the donor and acceptor templates shows evidence for an acceptor invasion contributing to the transfers completed at the major pause site. In the presence of NC, RNase H cleavages were observed throughout the 52-nt region of homology before the major pause site on the donor template (Fig. 6A). The acceptor cleavage profile showed that the earliest cleavages occurred at least 30-nt upstream of the hairpin base within this extended region of homology (Fig. 6B). This suggests that acceptor-cDNA interactions were initiated very early in the region of homology. Derebail and DeStefano (34) have shown that conditions that slow down DNA synthesis on the donor, such as low dNTPs, allow acceptor-cDNA annealing to catch up with the primer terminus, promoting transfer. In our template system, the stable hairpin structure within the donor stalls synthesis, creating an opportunity for acceptor invasions initiated earlier in the template to catch up and terminate in transfers at the major pause site. Earlier studies with the EIAV hairpin template system revealed the role of major pause sites as facilitating acceptor invasion (28, 42). The results of this study suggest that pause sites also promote recombination by serving as a location for primer terminus transfer, with stalling of synthesis at a pause site allowing acceptor invasions initiated earlier in the template to catch up with the primer terminus. Combined, these data reveal that pause sites can serve two roles in promoting strand transfer, one as an initiator of acceptor invasion and a second as a site of primer terminus transfer (Fig. 7). The fact that the shorter homology template displayed the transfer distribution of one mechanism, but the longer homology showed a distribution of both mechanisms, has allowed us to readily distinguish them.

NC substantially increased the transfer efficiency from 20 to 46% for the long homology substrate. While NC produced only a minor effect on the pattern of RT pausing in the short homology substrate, the effects were greater in the long homology substrate (Fig. 3B and refer to Refs. 28 and 42). This suggested that the extended homology changed the overall structural stability of the template, weakening many of the structures that cause pausing, and allowing the NC to have a more substantial effect. NC has a minimal effect on RT pausing at the hairpin base (+86 nt) in the long homology templates (Fig. 3B) but decreased RT pausing at positions +108, +121, and +148. MFold predictions suggest pausing at these sites to be the result of hairpins formed transiently as the DNA primer synthesized through the original hairpin. NC generally increased the terminus transfer about 2-fold throughout the homology region, and even more between markers 2 and 4, and after marker 7. The peak of terminus transfers at the hairpin base was observed both in the presence and absence of NC.

The role of RT pausing in strand transfer has been a subject of extensive studies (18, 27, 28, 32, 42, 57, 60, 61). Secondary structures and specific sequence features within the RNA promote RT pausing (30, 31, 61–63). In the current research, we observed a hot spot for terminus transfer at the pause site, demonstrating that the paused DNA products can produce transfer products. Similarly, a hotspot for template switching was also observed at the synthesis pause site promoted by the TAR hairpin in a minus strand strong stop transfer system (59). DeStefano (57) have proposed that paused DNA products can transfer by two pathways. In the first, extensive RNase H cleavage of the donor frees the cDNA from the donor allowing it to anneal to the acceptor. This mechanism is reminiscent of the forced copy choice model of transfer (64), in which synthesis cannot continue on the donor, and the primer is salvaged upon transfer to the acceptor. In the second, the acceptor actively invades the donor-cDNA hybrid and displaces the donor at the primer terminus to complete the transfer. Detailed analyses of the transfer mechanism in different template systems have led to the proposed acceptor invasion process as initiating transfer (18, 27, 28, 41, 51, 57, 65). In fact, acceptor-cDNA interaction was proposed to be a rate-limiting step in the transfer process (19, 57). In the present study, data from acceptor cleavage provide evidence that at least a portion of transfers occur as a consequence of acceptor invasion early in the region of homology. Single-stranded regions within the acceptor are proposed to favor the acceptor-cDNA interaction or "docking" step (42, 46, 60, 66). In our template system, the absence of strong secondary structures within the proposed invasion site in the acceptor template correlated with efficient transfers compared with the poor transfers when the invasion site in the acceptor was part of a stable secondary structure (42).

With NC, the transfer distribution shifted toward the 5'-end of the template. NC has been shown to facilitate DNA synthesis on the donor, especially by reducing RT pausing caused by secondary structures (43, 45, 67, 68). NC also promotes annealing between the nascent DNA and acceptor and branch migration (Fig. 6 and Refs. 69 and 70). To shift the transfer profile toward the 5'-end of the template as observed, NC must have increased the rate of DNA synthesis more than it increased propagation of the hybrid between the nascent DNA and acceptor RNA. The combined effect would make the hybrid propagation catch the primer terminus later, resulting in the observed shift in terminus transfer, with a particular enhancement between markers 7 and 8. The single nucleotide mismatch at the end of the homology efficiently prevented end transfer in the absence of NC. However, with NC, end transfer, as indicated by a distinct peak after marker 8, is very evident (Fig. 5). To promote end transfer in this system NC must have enhanced the ability of RT to carry out mismatch extension (71).

It is interesting that even in the absence of NC, transfers were promoted efficiently in the long homology system (Fig. 3A). Without NC, RNase H cleavages on the donor were observed within the extended region of homology, with products representing cleavages at +112, +94, and +84, detectable as early as 1 min (Fig. 6A). Several of these cleavage products persisted throughout the reaction, consistent with the paused cDNA molecules dissociating from the donor (Fig. 6A). The acceptor template was cleaved minimally in the 3' region of homology (data not shown), preventing identification of the primary invasion sites. Comparing donor extension and transfer reactions in the presence of acceptor, pause products at the hairpin base (+86) were chased away with time, while the +113 pausing product was undetectable (Fig. 2C). A decrease in pause products corresponding to +108 and +121 was also evident. Sequencing of the transfer products revealed that ~31% of the transfers occurred at the hairpin base while ~25% occurred between markers 4 and 5, the region encompassing these pause sites (Fig. 5). Taken together, the data suggest that at least some transfers in the absence of NC occur because the paused cDNAs dissociate from the donor template and then transfer to the acceptor through a pathway not involving acceptor invasion, as previously described (57).

Our data provide clear evidence for NC-promoted invasion as a favorable pathway for strand transfer. Invasions initiated early in the region of homology could contribute to terminus transfers at the hairpin base (Fig. 7). Additionally invasions initiated both early and also at the hairpin base could contribute to the terminus transfers beyond the major pause site. However, we cannot exclude the possibility that a portion of transfers at the major pause site could also occur by an acceptor invasion-independent forced copy choice mode of transfer (57). Current analyses do not reveal what fraction of terminus transfers at the pause site and what portion of terminus transfers beyond the pause site were promoted by acceptor invasions initiated early in the region of homology.

Based on our findings we suggest that an RNA template structure that promotes creation of an invasion site followed by a downstream pause site is an environment particularly favorable for transfer. The completion of transfers initiated by invasion requires that the acceptor-cDNA hybrid first formed at the invasion site successfully catch up with the elongating DNA primer terminus. If this is not accomplished, transfer will not be completed. The downstream pause assures that the capture rate of the primer terminus by the acceptor will be high. The large transfer peak at the base of the hairpin, and the major increase in transfer efficiency with the extended template, support this idea.

Introduction of template sequences that cause progressively more pausing in vitro, also caused progressively more recombination in a retroviral context in vivo (72), indicating that pausing promotes recombination. We previously analyzed the pause profile for RT-mediated synthesis over a region of HIV1 gag RNA that showed a broad region of recombination in vivo and efficient strand transfer in vitro (58). It displayed a series of pauses spaced 20–30-nucleotides apart. Although recombination in vivo must derive from many of mechanisms, it is possible that when pauses are spaced favorably each can create invasion sites for downstream transfers and completion sites for upstream invasions.

Overall, our results combined with previous research suggest that RT pausing can promote strand transfer by two mechanisms: one is the creation of invasion sites by facilitating RNase H cleavages; the other is the promotion of terminus transfer, by allowing invasions that have occurred earlier on the template to catch the extending primer terminus.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM 49573. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Gilead Sciences, 333 Lakeside Dr., Foster City, CA 94404. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-3269; Fax: 585-275-6007; E-mail: robert_bambara{at}urmc.rochester.edu.

3 The abbreviations used are: HIV-1, human immunodeficiency virus-1; RT, reverse transcriptase; AIDS, acquired immunodeficiency syndrome; NC, nucleocapsid protein; EIAV, equine infectious anemia virus; DE, donor extension product; FB, fold-back product; TP, transfer product; TAR, transactivation response region; nt, nucleotide; RNase H, ribonuclease H. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mark Hanson, Dr. Vandana Purohit, and Sean Rigby for critical reading of the manuscript and useful discussions. We also thank Dr. Ricardo Roda for helpful suggestions and discussions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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