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Originally published In Press as doi:10.1074/jbc.M304608200 on June 11, 2003

J. Biol. Chem., Vol. 278, Issue 34, 31536-31546, August 22, 2003
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Role of the Reverse Transcriptase, Nucleocapsid Protein, and Template Structure in the Two-step Transfer Mechanism in Retroviral Recombination*,

Ricardo H. Roda {ddagger}, Mini Balakrishnan {ddagger}, Mark N. Hanson {ddagger}, Birgitta M. Wöhrl ¶, Stuart F. J. Le Grice ||, Bernard P. Roques **, Robert J. Gorelick {ddagger}{ddagger} and Robert A. Bambara {ddagger} §§

From the {ddagger}Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany, ||HIV Drug Resistance Program and {ddagger}{ddagger}AIDS Vaccine Program, Science Applications International Corporation, NCI Frederick, Frederick, Maryland 21702, and the **Department de Pharmacochemie Moleculaire et Structurale, INSERM U266, CNRS UMR 8600, 4 Ave. de l'Observatoire, 75270 Paris Cedex 06, France

Received for publication, May 2, 2003 , and in revised form, June 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Template switching during reverse transcription promotes recombination in retroviruses. Efficient switches have been measured in vitro on hairpin-containing RNA templates by a two-step mechanism. Pausing of the reverse transcriptase (RT) at the hairpin base allowed enhanced cleavage of the initial donor RNA template, exposing regions of the cDNA and allowing the acceptor to base pair with the cDNA. This defines the first or docking step. The primer continued synthesis on the donor, transferring or locking in a second step. Here we determine the enzyme-dependent factors that influence template switching by comparing the RTs from human immunodeficiency virus, type 1 (HIV-1), and equine infectious anemia virus (EIAV). HIV-1 RT promoted transfers with higher efficiency than EIAV RT. We found that both RTs paused strongly at the base of the hairpin. While stalled, HIV-1 RT made closely spaced cuts, whereas EIAV RT made only a single cut. Docking occurred efficiently at the multiply cut but not at the singly cut site. HIV-1 nucleocapsid (NC) protein stimulated strand transfers. It improved RNase H activity of both RTs. It allowed the EIAV RT to make a distribution of cuts, greatly stimulating docking at the base of the hairpin. Most likely, it also promoted strand exchange, allowing transfers to be initiated from sites throughout the hairpin. Minor pause sites beyond the base of the hairpin correlated with the locking sites. The strand exchange properties of NC likely promote this step. We present a model that explains the roles of RNase H specificity, template structure, and properties of NC in the two-step transfer reaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombination occurs frequently in retroviruses (1–3). For a simple retrovirus such as spleen necrosis virus, recombination happens once every three replication cycles (4, 5), whereas in HIV-11 as many as three recombination events take place in a single replication cycle (6, 7). More importantly, recombination appears to be an essential mechanism for retroviral survival. It allows viruses to repair genomic errors (8–11) and to combine resistance markers against anti-retroviral drugs (12–14), and it promotes genomic shuffling that frustrates immune surveillance and allows for the emergence of new mosaic viral strains (15, 16).

Recombination takes place during reverse transcription when the virus-encoded reverse transcriptase (RT) copies the single-stranded viral RNA into double-stranded viral DNA. Synthesis of the integration-competent double-stranded viral DNA involves two obligatory strand transfers from the ends of the genome (17–19). In addition to these end transfers, template switching can also occur within internal sequences of the genome (4, 6, 20–23) and is driven by template homology (24–30). Strand transfer or template switching results when a primer being extended on one copy of the template (donor) is transferred to a homologous site on a second copy of the template (acceptor). Although it has been proposed to occur during plus (31) and minus strand synthesis (32), most template switching occurs during RNA-templated synthesis (7, 20, 23, 33, 34). As a result, most mechanistic studies have focused on recombination from RNA templates. Intra-molecular transfers within the same molecule lead to deletions (17, 25, 35). However, because retroviruses package two complete copies of their RNA genome, template switching can also take place inter-molecularly. When two nonidentical (heterogeneous) genomes are co-packaged, such inter-molecular transfers lead to recombinant progeny (4).

Pausing of RT during synthesis was found to correlate with the locations of template switching (36–38). Because RT has DNA polymerase and RNase H activities within the same molecule (39, 40), it was suggested that pausing would temporarily inhibit nucleotide addition, but it would not affect the nuclease function. This would result in degradation of the RNA donor template in the region of pausing and subsequent exposure of the cDNA. Base pairing between a homologous acceptor molecule and the cDNA would then result in a transfer event (36–38) and polymerization would now resume on the acceptor template. In support of this model, RNase H activity has been found to be necessary for strand transfers. Reducing or deleting the RNase H activity of RT leads to a significant reduction in its ability to switch between RNA templates in vitro (37, 41–44) and a reduction in direct repeat deletion in virions (26, 27, 29, 30, 45, 46). The transfer process has been described as a "dynamic copy choice," meaning that an appropriate balance between the RNase H and polymerase activities is required to obtain efficient direct repeat deletion (27, 46).

The role of RNA template structure in triggering transfers has also been studied (47–54). RNA hairpins facilitate transfers in a number of ways. The kissing hairpin of HIV-1, which contains a palindromic loop sequence that induces template dimerization, has been shown to facilitate strand transfer in vitro (55, 56), and to provide a hot spot for recombination rescue of defective murine leukemia virus genomes in vivo (11). Similarly, kissing interactions promoted between the TAR loop sequences of the cDNA intermediate and acceptor RNA are proposed to facilitate the minus strand transfer (47).

RNA hairpins may also promote transfer by creating a transient pause site (36, 47, 48, 54, 57, 58). Although our initial expectation was that most transfers would take place at the site of pausing at the hairpin base (48), sequence analysis of the transfer products revealed that the transfer of the primer terminus took place at a distance, after synthesis through the pause site (48, 54). Detailed analysis revealed that template switching occurred through a two-step process (54). Pausing increased RNase H activity at the base of the hairpin, allowing the acceptor to "dock" to the cDNA. This initial step was followed by continued synthesis through the structure of the hairpin on the donor. The transfer event is not finished or "locked" until the cDNA primer terminus had annealed to the acceptor beyond the pause site. These results presented clear evidence for a previously suggested two-step transfer mechanism (27, 55, 59). More recently, transfers taking place in the 5' end of the HIV-1 genome have also been shown to take place through such a "dock and lock" process (55).

Reverse transcription takes place in the presence of the accessory protein nucleocapsid (NC) that binds to the genome. NC is packaged within the virion, is processed from the Gag precursor poly-protein, and it has RNA chaperone activity, as well as a modulating activity on RT (60). NC has been shown to increase the efficiency of the strand transfer process, either by promoting nucleic acid annealing or by increasing the RNase H activity of RT (38, 48, 51, 54, 56, 59, 61–71).

Here we seek to explore the enzymatic factors that contribute to the pause-driven two-step transfer mechanism. We have done so by carrying out assays with the RTs from human immunodeficiency virus, type I (HIV-1), and equine infectious anemia virus (EIAV) on hairpin containing RNA substrates. EIAV and HIV-1 belong to the lentivirus subfamily of retroviruses. Both replicate in cells of the immune system and have high mutation rates (72, 73). The RTs share 40% of amino acid homology, are heterodimers of a catalytically active p66 subunit and an inactive p51 subunit, and appear to be structurally similar (74–80). The present study demonstrates that despite such similarities in their overall properties, HIV-1 RT was more efficient in carrying out strand transfers from hairpin structures than EIAV RT. An important contributor to this were differences in the RNase H cleavages carried out when the RTs are paused at the base of the hairpin. We have found that although both RTs paused at the base of the hairpin, HIV-1 RT degraded the template into smaller fragments that could easily dissociate from the cDNA and could therefore allow more efficient docking. HIV-1 NC, but not EIAV NC, caused a robust increase in transfers with both RTs. We believe this enhancement to be a result of HIV-1 NC's enhancement of RNase H activity, and by making previously inaccessible regions of the acceptor more amenable to the invasion or docking step.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Expression plasmids, pKK-p66(6his) and pKK-p51(6his) (81), for overexpression of the RT p66 and p51 subdomains were kindly provided by Dr. Neerja Kaushik. EIAV RT was generated and purified as described previously (76, 79). Nucleocapsid protein-(1–72)-NCp7 was chemically synthesized (82) and was stored at –80 °C in a buffer of 50 mM Tris-HCl (pH 7.5) and 5 mM dithiothreitol. DH5{alpha}-competent cells, XbaI, and EcoRI were from Invitrogen. Radiolabeled compounds were from PerkinElmer Life Sciences, and Micro Bio-Spin columns were from Bio-Rad. Plasmid purification kits were from Qiagen. All DNA primers were synthesized by Integrated DNA Technologies, Inc. All other products were purchased from Roche Applied Science.

Methods
HIV-1 RT Purification—Overexpression and purification of the RT heterodimer was done as described previously (83), with a slight modification. Briefly, JM109 cells harboring the expression plasmids were grown in 2x YT media containing ampicillin (100 µg/ml). Cell pellets of the p66 and p51 clones were mixed together at the appropriate ratio (84), lysed, sonicated, and centrifuged at 28,000 x g for 45 min. The supernatant was diluted to achieve 0.5 M NaCl, treated with streptomycin sulfate at a final concentration of 4%, and centrifuged again at 28,000 x g for 30 min. The supernatant was applied to a nickel affinity column (Amersham Biosciences), and the heterodimer was purified as described earlier (83). Eluted factions containing equal amounts of p66 and p51, as judged from SDS-PAGE, were pooled together and dialyzed. Purified protein was more than 95% pure as judged by Coomassie Blue-stained gels and was stored at –20 °C. Protein concentration was determined by Bio-Rad assay.

Generation of EIAV-derived Donor and Acceptor Templates—The donor construct pEIAV-Donor and the acceptor construct pEIAV-A2 have been described previously (54). RNA templates were generated by run-off transcription in vitro from linearized plasmids using T7 RNA polymerase, as per the manufacturer's protocol. The pEIAV-Donor was linearized with NotI to generate DI donor templates, and pEIAV-A2 was linearized with HindIII and HaeIII to generate the AI-2 and AS acceptor templates, respectively. RNA substrates were gel-purified and tested for integrity.

Labeling and Annealing of Substrates—DNA primers or RNA templates (RNA templates first calf intestine phosphatase-treated) were labeled at the 5' end as described previously (54). For annealing, donor RNA and DNA primer at a ratio of 1:2 were heated for 2 min at 95 °C and slowly cooled to room temperature in 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, and 1 mM EDTA.

RT Assays—Donor DI was primed for reverse transcription with the DNA oligonucleotide SP6 (5'-TACGATTTAGGTGACACTATAG-3'). Donor extension and strand transfer assays were carried out in a final volume of 12 µl. Two units of HIV-1 or EIAV reverse transcriptase (50 ng) were incubated for 5 min at room temperature with 25:50 fmol (donor/primer) of hybridized substrate. For reactions with NC, the protein was added to the reactions prior to the addition of RT and incubated at room temperature for 5 min. By assuming that each NC molecule would cover 7 nucleotides of RNA, the amount of NC required for 200% coating was determined based on the length and amount of the primers and RNA templates in the reaction. Reactions were started with the addition of dNTPs and MgCl2 at a final concentration of 50 µM and 6 mM, respectively, incubated at 37 °C, and stopped at the appropriate time by adding 1 volume of termination buffer (90% formamide, 10 mM EDTA (pH 8.0), and 0.1% each of xylene cyanol, and bromphenol blue). Strand transfer reactions were carried out under the same conditions as extension assays, except that acceptor was included in the annealing reactions. For blocking oligonucleotide (EIAV-3, 5'-CAGACCATACCTGAAGCTTAC-P-3') experiments the oligomer was added to the reaction mixture at the same time as the start mix. The oligomer has a 3'-phosphate modification that prevents priming of synthesis.

RNase H Assays—RNase H assays were performed with 5' end-labeled donor RNA and unlabeled SP6, under the same reaction conditions as the extension assays but in a final volume of 80 µl. For the time course analysis, 10 µl of reaction was sampled at each time point and mixed to 10 µl of termination buffer. Reaction products were resolved on 8% polyacrylamide-urea gels, and visualized and quantitated using a PhosphorImager and ImageQuant software (Amersham Biosciences).

Analysis of Transfer Products—To determine the transfer distribution, the transfer products were isolated, amplified, cloned, and sequenced as described previously (54). Each clone represented an individual transfer event, and its sequence was used to determine the point of crossover between donor and acceptor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We propose that the characteristics of the two-step transfer process are determined by a combination of qualities of the reverse transcriptase, the nucleocapsid protein, the structure of the substrates, and the transfer intermediates these three components form. To distinguish specifically the roles of the proteins from those of the substrate, we examined each step in the transfer mechanism in the presence and absence of NC, using HIV-1 and EIAV RTs. The system we used to model strand transfers in vitro consists of a hairpin containing donor RNA molecule, on which reverse transcription is initiated, and a homologous acceptor RNA molecule onto which the primer can transfer. Single base markers on the acceptor molecule are used to determine the location of the crossover (38, 48, 54, 55).

HIV-1 RT and EIAV RT Show Similar Pattern of Pausing— Donor RNA DI contains a strong hairpin, and when used in conjunction with acceptor AI-2 most transfers are completed before the last nucleotide on the donor is copied (54). The sequence of DI and the location of the markers on acceptor AI-2 used to locate the locking site are shown in Fig. 1. Pausing during reverse transcription was proposed to affect both docking and locking during strand transfer (54). To compare the pausing profiles of HIV-1 and EIAV RTs extension reactions were carried out using labeled primer on donor DI. Previous assays carried out with these substrates and HIV-1 RT were done at 80 mM KCl. However, to get efficient synthesis with EIAV RT, a lower salt concentration (50 mM KCl) was necessary. As a result, assays using the HIV-1 RT have been repeated at the lower salt condition and incorporated in the present report for appropriate comparison.



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  		FIG. 1.
Sequence and structure of EIAV primer binding site (PBS)-derived hairpin substrates. Schematic of the DI donor, AI-2 acceptor, and ssAcceptor templates. Donor and acceptor RNAs share a homology of 93 and 75 nucleotides, respectively. Synthesis is initiated from a DNA primer (dashed arrow) annealed to the donor 3' end. The acceptors are longer at their 5' end, than the donor. Base substitution markers are shown as filled circles on the acceptors with the marker number and sequence indicated on top. Structural regions of the template with respect to the marker are also highlighted. The sequence and structure of DI donor RNA shown. Donor and acceptor RNAs shared the same structure. The dark line represents regions present only on the donor, and the sequence represents the region of homology between donor and acceptors. Nucleotide positions on the donor RNA are indicated with a + prefix, starting with the 5' end as +1. Marker numbers are indicated with a # prefix, with the first marker closer to the 3' end of the RNA.

 

The sites of pausing for both RTs were similar but not identical (Fig. 2, A and B). Full-length extension products were not seen until 5 min with EIAV RT, as opposed to 3 min with HIV-1 RT. EIAV RT paused more prominently at +78 and +79, corresponding to stalling at the base of the hairpin between markers 1 and 2. We proposed previously (54) that pausing and associated RNase H activity at this point is responsible for creating a major acceptor invasion site. HIV-1 RT had a unique pause site at location +68 (between markers 3 and 4). The next two pause sites, +44 and +57, both of which fall between the region delineated by markers 4 and 5 on the acceptor, were present in extensions by both RTs. EIAV RT presented a unique pause site at +31 (between markers 5 and 6), whereas both polymerases paused at position +16 albeit with different intensities. EIAV paused more intensely at position +16 than at any site upstream of the base of the hairpin. HIV-1 RT, on the other hand, paused approximately the same at +16, +44, and +57. The overall similarity in pausing profiles between both enzymes, especially at the base of the hairpin, led us to hypothesize that strand transfer occurs through an analogous mechanism for both RTs.



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  		FIG. 2.
HIV-1- and EIAV RT-catalyzed transfer reactions. Transfer reactions catalyzed by HIV-1 (A) and EIAV RT (B). The 5' end-labeled SP6 primer was annealed to donor DI, and extension reactions were carried out in the presence of acceptor AI-2 and in either the absence (lanes 1–6) or presence of HIV-1 NC (lanes 8–13). Enough NC for 200% coating of the templates (100% = enough NC to cover all nucleotides in the reaction once, assuming each NC molecule covers 7 nucleotides) was added to the reaction and incubated for 5 min prior to the addition of RT. The reaction was sampled at the times indicated on top of the lanes, and products were separated in an 8% denaturing polyacrylamide gel. Lane L was loaded with a 10-nucleotide DNA ladder. Products observed over the time of the assay correspond to products resulting from full-length synthesis to the end of the donor (FL), transfer product (TP) resulting from switching and synthesis to the end of the acceptor, and a fold-back (FB) resulting from self-priming of the full-length product. Locations of the markers are indicated on the left of the panel and product sizes on the right. The schematic on the right indicates the region of primer binding as well as the corresponding regions on the template for the pause sites. C, transfer efficiencies supported by the HIV-1 and EIAV RTs. Transfer efficiency was determined using the formula %TE = TP/(FL + FB + TP) x 100. Transfer efficiencies were determined using donor to acceptor ratios of 1:2 (black bars) and 1:8 (gray bars) for HIV-1 and EIAV RTs in the presence and absence of nucleocapsid proteins at 200% coating. Results represent the average of at least three independent experiments.

 

EIAV RT Has Low Strand Transfer Activity in Hairpin Containing Substrates—Despite similar pausing profiles, a lower transfer efficiency was observed for EIAV RT as compared with HIV-1 RT. The transfer efficiency is a measure of how many transfer products are produced in relation to the total full-length products. It follows the formula TE = TP/(TP + FL + FB) x 100%. Here the transfer efficiency (TE) is related to the amount of transfer product (TP), full-length extension products on the donor template (FL), and the product resulting from foldback and additional extension of the donor extension product (FB). At a 1:2 donor to acceptor ratio (Fig. 2C) HIV-1 RT and EIAV RT supported a TE of 5 and 3%, respectively. The initial "docking" step in the proposed two-step transfer involves an interaction between the acceptor and the nascent cDNA. We therefore reasoned that higher acceptor concentrations would increase the chances for such an intermolecular event and therefore enhance TE. When the concentration of acceptor was quadrupled the TE increased 4-fold to 21% for HIV-1 RT but only doubled to 6% for EIAV RT. The lower TE for EIAV RT and its lesser responsiveness to increases in the acceptor concentration suggested that it was less efficient in the docking step that is dependent on RNase H activity. This lead us to examine and compare the events leading up docking for both RTs.

Differences in RNase H Cleavages by the RTs—To examine RNase H cleavages during synthesis, extension reactions were carried out using unlabeled primer and 5' end-labeled donor. Both RTs made numerous cuts in two regions on the donor RNA as follows: between positions 80 and 100 (Fig. 3, A and B), corresponding to RTs stalled at the base of the stem, and in the descending limb of the hairpin, corresponding to cleavages at the end of the template, which produces fragments smaller than 20 nucleotides in length. The characteristics of those cuts, however, were different for the two RTs.



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  		FIG. 3.
RNase H cleavage profiles of HIV-1 and EIAV RT during synthesis. Reaction conditions for HIV-1 (A) and EIAV RT (B) were the same as those used for extension reactions in the absence (lanes 1–6) or presence of HIV-1 NC (lanes 7–12). The radiolabel (shown as a star) was on the 5' end of the donor RNA instead of the primer, and no acceptor was present. The schematic on the right indicates the region of primer binding as well as the corresponding regions on the template for the cleavage sites. Enough NC for 200% coating of the templates was added to the reaction and incubated for 5 min prior to the addition of RT. The reactions were sampled at the times indicated above each lane, and products were separated through an 8% polyacrylamide gel run under denaturing conditions. Sizes of the cleavage products are indicated. Ctrl lane, reactions done in absence of RT.

 

Whereas HIV-1 RT is paused at the first base of the stem of the hairpin, it creates closely spaced cuts at positions +101, 98, 94, 91, and 83, with the most intense cut at +94. These correspond to cuts in the donor 23, 20, 16, 13, and 5 nucleotides downstream of the +78 pause site. These cleavages could be the result of either single cuts on different RNA molecules or the result of several cuts on a single molecule. If the former were true, then over time one would observe a net accumulation of all cleavage products, and only the full-length RNA band would be reduced. However, products at +101, 98, and 94 appear within 1 min of the reaction, but then their intensities decrease, while other bands become more intense. We interpret this as signifying that these early products are further cleaved into smaller products that appear at later time points. Apparently in our reactions a significant portion of the RNA molecules are cut in multiple locations. We also note that the persistence of some uncut RNA in reactions without NC most likely reflects incomplete priming of the RNA, rather than a low level of cutting.

The cleavage pattern suggests that at least some of the RNAs are fragmented into pieces either small enough to dissociate into solution or to be readily displaced by a docking acceptor RNA molecule. The RNase H cleavage profile of EIAV RT produced a major fragment 98 nucleotides in length and minor cuts at 101 and 94. The resulting segments of RNA may be too large to dissociate, inhibiting docking by an acceptor molecule leading to poor transfer efficiency.

Throughout the template, the cleavage pattern shows that HIV-1 RT cuts the RNA with much more diversity of location, e.g. at positions +63, 50, 27, and 17, than EIAV RT. This provides the opportunity for nearby cuts on the same template, which could allow gap formation that would promote primer-donor interaction. The positive correlation between diverse positions of cleavage and high transfer efficiency suggests that closely spaced cuts are an important prerequisite for efficient docking. The inability of the EIAV RT to make diverse-position cuts suggests the basis for its low TE relative to the HIV RT.

Transfers Carried Out by EIAV RT Take Place by an Inefficient Two-step Mechanism—In a previous study we showed that deleting the docking region of an acceptor leads to a very large reduction in transfers supported by HIV-1 RT (54). Using such an acceptor (ssAcceptor) leads to a 10-fold reduction in transfer efficiency for HIV-1 RT (Fig. 4). Unlike the acceptor that includes the docking region, increasing the acceptor concentration 4-fold did not increase the TE with the shortened acceptor. Similar to HIV-1 RT, EIAV RT also displayed a reduction in transfers in the absence of a docking region. At the lower acceptor ratio the transfer efficiency was reduced from 3% with the original acceptor to 1% with ssAcceptor (Fig. 4). For both RTs, the drop in transfer efficiency with ssAcceptor is greater at the higher acceptor concentration (Fig. 4). This suggests that transfers promoted by EIAV RT were also initiated at the base of the hairpin, although very inefficiently due to poor docking. The reduction in TE observed with the ssAcceptor also implies that the base of the hairpin is the primary docking site for transfers promoted by both RTs. Otherwise the transfer efficiency would have been maintained despite the deletion.



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  		FIG. 4.
Effect of deleting the base of hairpin on transfer efficiency. The ssAcceptor (54), lacking the region 3' to the base of the hairpin, was used instead of AI-2 in standard transfer assays. A schematic of the donor-acceptor substrate pair in indicated above the panel. Transfer efficiencies and NC coating levels were calculated as in Fig. 2. Transfer efficiencies were determined using donor to acceptor ratios of 1:2 (black bars) and 1:8 (gray bars) for HIV-1 and EIAV RTs in the presence and absence of nucleocapsid protein at 200% coating. Results represent the average of at least three independent experiments.

 

Distribution of Transfer Products—The second step in strand transfer, or "lock" step, involves the translocation of the 3' end of the cDNA from the donor to the acceptor RNA. This produces the genetic shift. The position of locking was obtained by analyzing cloned transfer products to determine which markers from the acceptor they incorporated. The data in Fig. 5, A and B, were generated by dividing the number of colonies showing a transfer between two markers by the total number of colonies sequenced. Fig. 5A compares the distribution of crossovers at the lower acceptor concentration for both HIV-1 and EIAV RT. The distributions show some similarities. Both RTs transferred mostly within the hairpin, downstream of the base of the hairpin. They also both had a peak of transfer between markers 4 and 5, with 52 and 40% for HIV-1 and EIAV RT, respectively. There were few transfers on the ascending limb of the hairpin between markers 2–4 or after the last nucleotide had been copied from 5' to 8. Differences arose in the second-most preferred site of transfer; for HIV-1 RT it was the base of the hairpin between markers 1 and 2 (25%) and the 5' half of the loop, between markers 5 and 6 (12.5%), whereas for EIAV RT it was between markers 5 and 8 where segments 5–6, 6–7, and 7–8 supported 19, 21, and 14% of total transfers.



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  		FIG. 5.
Distribution of primer terminus transfers for HIV-1 and EIAV RTs. Transfer reactions using HIV-1 and EIAV RT were carried out using a donor to acceptor ratio of 1:2 (A) or 1:8 (B). Products were gel-purified, amplified, and cloned, and individual colonies were sequenced. To obtain the distribution, the number of clones containing transfers within each individual segment were divided by the total number of clones sequenced and multiplied by 100. Two independent experiments were carried out for each set. The graph shows the average values for each set and the range of values for each experiment within that set. For reactions performed using HIV-1 RT (black bars), a total of 55 clones were sequenced; for reactions using EIAV RT (gray bars), 60 clones were sequenced. A schematic of the donor-acceptor substrate pair is indicated above the panel.

 

Increasing the acceptor concentration 4-fold had an interesting effect on the transfer distributions. Fig. 5B shows that the general pattern of transfers for HIV-1 RT maintained a bimodal nature. However the number of transfers at the base was significantly increased (43%), to the point that they become the most likely inter-marker site of primer terminus transfer. The higher acceptor concentration also triggered a major redistribution of transfers for EIAV RT. The increase from 2 to 6% in the transfer efficiency translated mostly into an increase from 19 to 39% in the fraction of transfers between markers 5 and 6 and from 21 to 33% of those between 6 and 7, whereas the number of transfers between 4 and 5 were reduced from 40 to 21%. The overall data showed that at a lower acceptor concentration the distributions of transfers as well as the transfer efficiencies were similar for both enzymes. However, increasing the concentration of acceptor affected the amount and location of transfers differently for HIV-1 and EIAV RTs.

Nucleocapsid Protein Increases Transfer Efficiency—Because HIV-1 NC has been shown to promote transfers, we tested the effects of both HIV-1 and EIAV NC on the template switching reactions carried out by their respective RTs. We observed that at the low acceptor concentration, HIV-1 NC increased transfers promoted by HIV-1 RT from 5 to 22% (Fig. 2C). Interestingly, at the higher acceptor concentration, NC did not have much of a significant effect on the TE. This suggests that the increase in transfers mediated by HIV-1 NC on HIV-1 RT can be saturated at high acceptor concentrations.

Surprisingly, EIAV NC did not significantly stimulate transfers with EIAV RT at the low or high acceptor concentrations (Fig. 2C). An additional surprise was that HIV-1 NC had a robust effect on transfers by EIAV RT. A 4.5-fold increase was registered at low and high acceptor ratios. This suggested that at least some of the enhancements produced by retroviral NCs are not dependent on specific interactions between autologous RTs and their nucleocapsid proteins. Also, further raising the concentration of acceptor 4-fold increased transfers for EIAV RT in the presence of HIV-1 NC (Fig. 2C). This suggests that HIV-1 NC alleviates but does not eliminate an inefficient intermediate step in the EIAV RT-promoted transfer reaction. Because of the much greater stimulatory effects of HIV-1 NC on both reactions, we carried out additional NC assays for both RTs using HIV-1 NC.

HIV-1 NC Effects on Synthesis and RNase H Activity—In an attempt to define the manner in which NC promotes transfers, we examined both extensions and RNase H cleavages in the presence of HIV-1 NC (Figs. 2 and 3). Fig. 2, A and B, shows extension reactions in the presence of NC. The protein did not significantly alter the extension profile or the pausing profile. However, quantification of band intensities indicated an increase (2–3-fold) of those products making it to full-length (FL) versus those paused at the hairpin base (+78 and +79) for both RTs. This showed that NC enhances synthesis through the major pause site.

The effect of NC on the RNase H profile was more striking (Fig. 3, A and B). For HIV-1 RT, NC creates a new set of cuts at positions +86, 24, and 20, whereas the intensities of bands at position +101 are increased (Fig. 3A). The product at position 94 does not accumulate at the early time points. The overall effect is an increase in the number of cuts between positions +83 and 101, the region adjacent to the base of the hairpin. One could deduce then that this would certainly increase the exposure and availability of the "docking" site on the cDNA primer. Also, the intensity of products 17 nucleotides or smaller in length is significantly increased, consistent with an overall increase in RNase H cleavage.

A similar effect is also observed for EIAV RT (Fig. 3B). In the presence of NC new cleavage sites were observed in the region adjacent to the base of the hairpin, in particular cuts appearing at positions +86 and 83. Intensities of cleavages at +101, 94, and those corresponding to the smaller products were also increased. Thus NC increases cleavages in the expected docking site of EIAV RT as well. This result suggests a mechanism by which NC could substantially increase transfers by EIAV RT.

HIV-1 NC Improves the Docking Step of EIAV RT—The effect of NC on RNase H cleavages suggested that at least part of its enhancement of transfers by EIAV RT could derive from more efficient cuts followed by more docking. To test this hypothesis we measured strand transfers with the shortened ssAcceptor in the presence of NC. In the presence of NC between an 8- and 10-fold increase in transfers was observed with the short acceptor (Fig. 4), regardless of acceptor concentration. This increase in transfers could not have arisen from enhanced docking at the base of the hairpin, because that region of the substrate was deleted. Even if more cleavages were made throughout the donor RNA, the highly annealed structure of the acceptor should have suppressed docking. The robust increase in transfers seen with NC could have resulted from a combination of an increase in cleavages throughout the donor, possibly creating new invasion sites, and a reduction in the stability of the acceptor structure allowing previously inaccessible regions to dock with the cDNA.

We were able to distinguish docking sites by using blocking oligonucleotides. These are short DNA molecules complementary only to a specific region of the cDNA and not to sequences of any other strands in the assay (54, 56). For the original acceptor a blocking oligonucleotide complementary to the cDNA at the base of the hairpin was used (54). Adding this oligonucleotide to the HIV-1 RT reactions reduced transfers by 60%, but it did not have a measurable effect on transfers by EIAV RT (Fig. 6). We attributed this result to the poor hairpinbase docking carried out by EIAV RT. A paradoxical effect was observed when oligonucleotide interference was carried out in the presence of NC. HIV-1 RT transfers were less efficiently blocked in the presence of NC (Fig. 6), a result previously observed (54). However, interference was now observed for the EIAV RT, and as much as a third of all transfers could be blocked. In the case of HIV we determined that NC destabilized the blocking oligonucleotide-cDNA complex, thus reducing its efficiency (data not shown). However, the result with EIAV RT suggested that an NC-induced improvement in invasions at the base of the hairpin was more effective than the destabilization of any blocking oligonucleotide-cDNA complexes made.



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  		FIG. 6.
Effect of using blocking oligonucleotide on transfer efficiency. Transfer assays were carried out with HIV-1 RT and EIAV RT in the absence (filled bars) and presence of a DNA oligomer (gray bars) (see "Experimental Procedures" for details). Assays were performed using DI donor and AI-2 acceptor at a 1:5 ratio, in the absence and presence of NC at 200% coating level. Description and details are same as in Fig. 2. Schematic of the RNA templates showing action of the blocking oligomer during transfer is indicated above the panel.

 

HIV-1 NC Significantly Affects the Distribution of Transfers by EIAV RT—We had previously reported that the presence of NC protein did not significantly change the distribution of transfers by HIV-1 RT at high salt concentration (80 mM KCl) (54). We re-checked the effects of NC on the distributions at the currently used 50 mM KCl. A bi-modal distribution of transfers was still observed for HIV-1 RT (Fig. 7); however, 40% fewer transfers took place at the base of the hairpin and 20% fewer between markers 4 and 5. An increase in transfers occurred between markers 5 and 7 (14.3–26%). Despite these slight differences, the larger features of the distribution were preserved. A substantial alteration in the distribution of EIAV transfer products occurred in the presence of NC. Without NC, most transfers took place between markers 4 and 5, whereas in its presence they occurred mostly beyond marker 5 (Fig. 7). This constituted a shift in transfers toward the 5' end of the template.



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  		FIG. 7.
Effect of NC on distribution of primer terminus transfers for HIV-1 and EIAV RT. Transfer reactions were carried out using HIV-1 and EIAV RTs at a 1:2 donor to acceptor ratio with 200% NC coating. Transfer products were gel-purified, amplified, and cloned, and individual colonies were sequenced. Two independent experiments were carried out for each set. The distributions were calculated as in Fig. 5 legend. For reactions performed using HIV-1 RT (black bars) a total of 60 clones were sequenced; for reactions using EIAV RT (gray bars) a total of 57 clones were sequenced.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We recently proposed that strand transfer in retroviruses occurs through a two-step mechanism (54). An initial docking step starts the process on the donor RNA template, whereas a final "locking" step completes it on the acceptor template. We observed these two steps separately through the use of highly structured RNA templates. In the present work we address the manner in which enzyme-dependent factors, such as pausing and RNase H activity of the RT, as well as template structure and NC protein affect each of the two steps and therefore the overall transfer process.

Pausing during synthesis has been proposed to improve transfer, because the RT can concentrate RNase H cuts while stalled (36–38, 54, 56). We observed an inhibition of transfer with both the ssAcceptor, which lacks sequences at the base of the hairpin (docking site), and a blocking oligonucleotide, which interferes with the docking site. This suggests that docking occurred predominantly at the base of the hairpin, a preferred RNase H cleavage region on the donor template. We propose that docking, which is the first step in the transfer, occurs at locations where the RNase H activity of the RT clears away a segment of the donor RNA so that the cDNA can interact with structurally accessible sections of the acceptor.

Comparisons of the transfers supported by the EIAV and HIV-1 RTs reveal that efficient docking is required for template switching and that this step is strongly influenced by the enzymatic properties of the RTs. We found the pausing profiles of the EIAV and HIV-1 RTs to be very similar. In particular, they both paused strongly once synthesis reached the base of the hairpin. However, HIV-1 RT was much better at promoting strand transfers than EIAV RT. We believe that this difference derives from the nature of the cleavages executed by the RTs while stalled at this major pause site. HIV-1 RT catalyzed a number of closely spaced cuts, which efficiently exposed the cDNA for docking. EIAV RT made a single prominent cut 18–20 nucleotides away from the pause site. This limited cleavage is unlikely to create an efficient docking site for invasion, thereby lowering its efficiency of transfers.

Increasing the acceptor concentration leads to an overall increase in template switching by HIV-1 RT, most likely by increasing the frequency of docking interactions (Fig. 9C). High acceptor concentration did not substantially increase the transfer efficiency of EIAV, presumably because it could not improve the initially poor docking efficiency.



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  		FIG. 9.
Dock and lock model of strand transfer from hairpin structures. Schematic depicts mechanisms in the overall transfer process. Once synthesis on the donor (thick black lines) reaches the base of the hairpin (A), it pauses and creates a set of adjacent cuts (B) that allows the acceptor (thick gray lines) to dock (base pair) with the nascent cDNA (thin black line) at the exposed site (C). This could lead either to continued synthesis on the donor while the acceptor is docked (D), or to immediate transfer of the cDNA to the acceptor (E). In the former case, the invasion can lead to branch migration (F) which eventually locks the cDNA onto the acceptor (H) and transfer is completed. In the latter case, locking occurs in the vicinity of the initial pause site, completing the transfer and synthesis continues on the acceptor (G).

 

Studies have shown that HIV-1 RT makes a primary RNase H cut ~18 nucleotides behind the primer terminus on the RNA strand, followed by secondary cuts 8–9 nucleotides behind the primer terminus (85, 86). To make a secondary cut, HIV-1 RT has to translocate forward to a new position after making the primary cut (86). Once paused at the base of the hairpin, EIAV RT made a cut ~18 nucleotides away, but unlike HIV-1 RT it is not able to make cuts closer to the hairpin. In addition, we observed that EIAV RT had low strand displacement activity when paused at the base of the hairpin. Possibly, structural features of the EIAV RT that restrict strand displacement synthesis also limit the movement necessary to make secondary RNase H cleavages. As described above, this would restrict docking and lower the transfer efficiency. An analogous mechanism has been described in the case of an RT inhibitor which specifically restricts strand transfer by inhibiting RNase H secondary cuts (87).

HIV-1 NC is an RNA chaperone protein that promotes annealing of complementary strands and also influences the folding and stability of nucleic acid structures (64, 88–92). Moreover, it has been shown previously to enhance overall RNase H activity and secondary RNase H cuts by HIV-1 RT (86). HIV-1 NC increased transfers by both RTs. We observed that NC widened the distribution of cuts at the proposed invasion site for both HIV-1 RT and EIAV RT. For EIAV RT, this very likely promoted docking, as evidenced by increased inhibition by the corresponding blocking oligonucleotide, and resulted in more efficient transfers. Both with and without NC, high transfer efficiency correlated with the production of a distribution of cuts. Furthermore, the stimulatory effects of HIV-1 NC on HIV-1 RT transfer efficiency is partly attributable to its chaperone properties, because HIV-1 RT is capable of efficiently cleaving the donor RNA and facilitating docking in its absence. Interestingly, only in the presence of HIV-1 NC were both RTs efficient in promoting transfers with the ssAcceptor. We believe that this results from the creation of new docking sites through increased cutting of the donor RNA and to the relief of structural constraints on the acceptor RNA. This would make previously unavailable regions accessible for base pairing with the cDNA and hence for invasion.

Once docked, what triggers the primer terminus transfer or lock step? We had observed previously that although a fraction of the primers locked close to their docking site (Fig. 9, C and E), for the majority of primers, RT continued synthesis past the pause site on the donor (54). Whereas the site of docking remained unchanged, this synthesis allowed for the locking to occur further downstream (Fig. 9, C and D) (54). The location of genetic shift occurs at the point where the primer terminus is transferred. To compare the locking distributions, the transfer efficiency for each of the RTs at all conditions tested was multiplied by the fraction of transfers in each template segment and re-plotted as shown in Fig. 8.



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  		FIG. 8.
Distribution of primer terminus transfers normalized to the transfer efficiency. Distribution of primer terminus transfers for HIV-1 and EIAV RTs at 1:2 and 1:8 donor to acceptor ratio in the absence of NC (Fig. 5, A and B) and at 1:2 donor:acceptor ratio in the presence of NC (Fig. 7) were normalized for the transfer efficiency measured under that specific condition (Fig. 2C) for each RT. This was done by multiplying the fraction of transfers taking place in each segment for a given set of assay conditions by the transfer efficiency measured for each RT under those conditions. Conditions are as follows: donor to acceptor ratio of 1–2 (black bars), donor to acceptor ratio of 1–8 (gray bars), donor to acceptor ratio of 1–2, and 200% NC coating (white bars).

 

We noted a correlation between the intensity and location of pause sites beyond the base of the hairpin and the distribution of locking. For example, EIAV RT paused more strongly between markers 6 and 7 (pause +16) than HIV-1 RT, and at low acceptor concentration more transfers mapped in this region for EIAV RT than for HIV-1 RT (Fig. 8). Possibly, pause sites observed later in synthesis slow down the polymerizing RT, allowing the invasion initiated at the dock site to catch up to the primer terminus through branch migration, completing the transfer event (Fig. 9, F and H). Another possibility is that the latter pause sites create a second interaction site between the primer terminus and acceptor, in addition to the docking. We have already shown that transfers by HIV-1 RT with ssAcceptor, although very inefficient, result in a similar distribution as transfers from the original length template (54). This suggests that some sequences may initiate locking even before branch migration from the docking site catches up to the primer terminus. DeStefano and colleagues (67) have shown that the process of branch migration is necessary to complete transfers after docking by HIV-1 RT but that it is not efficient in the absence of NC. This second interaction is likely to promote branch migration, which ultimately spans the distance between docking and locking sites.

Better docking at high acceptor concentrations shifted more of the primer terminus transfers closer to the docking site (Fig. 8), probably by forming the docking intermediate before the RT could move very far forward on the donor, favoring step E over step D in Fig. 9. The effect of NC on the location of primer terminus transfer may derive from its chaperone properties. NC has been recently shown to enhance fraying (partial unannealing) at the ends of nucleic acids duplexes (93–96). A reduction of the stability of the structure of the acceptor and increased fraying at the ends of paused primers should make locking events more likely to occur within previously inaccessible regions of the hairpin. Also, we observed that for HIV-1 RT, NC permitted less transfers at the base of the hairpin than are observed at high acceptor concentration (Fig. 8). NC increased primer extension past the base of the hairpin, thus increasing the separation between the site of docking and that of locking (Fig. 9, C and D). Clearly NC and higher acceptor concentration both promote transfer but by at least partially different mechanisms.

Overall, our results suggest that efficient strand transfer requires promotion of both the dock and lock steps. Docking is most efficient when the RT pauses in a way that makes a series of adjacent RNase H cleavages. Invasion at the site is promoted at high acceptor concentration and by the presence of NC. The locking step is most proficient in regions of weak pausing that lack stable structure and is promoted by the chaperone properties of NC.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM49573 and T32-CA09363 (to M. N. H.) and also in part by NIH/NCI Contract N01-CO-12400 with SAIC-Frederick, Inc. 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

§ Trainee in the Medical Scientist Training Program funded by National Institutes of Health Grant T32-GM07356 and is also supported by National Institutes of Health Predoctoral Fellowship 5 F31 GM20911-02. Back

§§ To whom correspondence should be addressed: Dept. of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-2764; Fax: 585-271-2683; E-mail: robert_bambara{at}urmc.rochester.edu.

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase; NC, nucleocapsid; FL, full length; EIAV, equine infectious anemia virus. Back


   ACKNOWLEDGMENTS
 
We thank Roslyn Chang for help in purifying HIV-1 RT. We also thank Yan Chen for helpful discussions.



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