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Originally published In Press as doi:10.1074/jbc.M412190200 on January 18, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14443-14452, April 15, 2005
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Acceptor RNA Cleavage Profile Supports an Invasion Mechanism for HIV-1 Minus Strand Transfer*

Yan Chen{ddagger}§, Mini Balakrishnan{ddagger}§||, Bernard P. Roques**, and Robert A. Bambara{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Biochemistry and Biophysics and the {ddagger}{ddagger}Cancer Center, University of Rochester Medical Center, Rochester, New York 14642 and the **Departement 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, 2004 , and in revised form, January 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously proposed that HIV-1 minus strand transfer occurs by an acceptor invasion-initiated multi-step mechanism. During synthesis of minus strong stop DNA, reverse transcriptase (RT) transiently pauses at the base of TAR before continuing synthesis. Pausing promotes RT-RNase H cleavage of the donor RNA, exposing regions of the cDNA. The acceptor RNA then invades at these locations to interact with the minus strong stop DNA. Whereas primer extension continues on the donor RNA, the cDNA-acceptor hybrid expands by branch migration until transfer of the primer terminus is completed. We present results here showing that the interaction of the acceptor RNA and the cDNA can be determined by examining the time-dependent cleavage of the acceptor RNA by RNase H. Our approach utilizes a combination of RT-RNase H and Escherichia coli RNase H to allow assessment of acceptor-cDNA interactions at high sensitivity. Results show an initial interaction of the acceptor RNA with cDNA at the base of TAR. We observe a time-dependent shift in RNase H susceptibility along the length of the acceptor toward the 5' end, suggesting hybrid propagation from the initial invasion point. Control experiments validate that the RNase H cleavage profile represents the formation and expansion of the acceptor-DNA interaction and that the process is promoted by the nucleocapsid. Observations with this new approach lend additional support to the proposed multistep transfer mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During retrovirus reverse transcription, minus strand DNA synthesis initiates near the 5' end of the RNA genome. In HIV-1,1 synthesis to the genome 5' end results in a DNA fragment about 200 nt long called minus strand strong stop DNA (–sssDNA). Direct repeat (R) sequences at either ends of the genomic RNA facilitate transfer of the strong stop DNA from the 5' end to the 3' end of either the same or the copackaged RNA (Ref. 1 and references therein). The 5' and 3' repeat regions thereby serve as the donor and acceptor templates, respectively, in this transfer process. The transfer process is obligatory to the completion of reverse transcription and synthesis of double-stranded viral DNA.

The HIV-1 genomic RNA contains 97-nucleotide-long repeat regions, the 5'-terminal 56 nts of which form the TAR hairpin. Deletion and mutational analysis demonstrate that strong stop transfer in HIV-1 does not require all of the R-region homology (2–7). Studies in murine leukemia virus have suggested that homology-independent factors, such as template structure and sequence, are also involved in facilitating minus strand transfer (8).

The RT-RNase H activity is essential at various steps of reverse transcription, including minus strand transfer (Ref. 9 and references therein). During minus strand synthesis, RT-RNase H cleaves the donor RNA within the RNA-DNA hybrid regions. This enables the –sssDNA to transfer to the acceptor. Viruses defective in RT-RNase H activity are unable to complete reverse transcription and show accumulation of –sssDNA in the cytoplasm (10–13). Addition of RNase H in trans can rescue minus strand transfer and viral replication (14, 15). Pausing of RT during synthesis enhances local RNase H cleavages within the template (16, 17). Template features that promote pausing, such as secondary structures or homopolymer sequences (17, 18), enhance RNA template cleavage by RT.

The viral nucleocapsid (NC) protein is also an important component in viral reverse transcription and strand transfer. NC displays nucleic acid chaperone activities, folding the polymer strands into their most thermodynamically stable conformations (19, 20). NC promotes the annealing of complementary strands (21–24). Such properties of NC very likely promote the displacement of the fragmented donor and annealing between the acceptor and cDNA, thereby promoting the transfer process. Additionally, NC has been shown to enhance RT-RNase H activity promoting rapid degradation of the donor RNA (25–29). NC inhibits the –sssDNA from folding back and self-priming, a process that would result in dead-end intermediates incapable of completing transfer (30–33).

Our recent studies (27, 29) analyzing HIV-1 minus strand transfer in vitro led to the proposal of an acceptor invasion-induced transfer mechanism. Results showed that cleavages within internal regions of the donor RNA are important for transfer. However, cleavages at the 5' terminus of the donor template are not a prerequisite for transfer. Additionally, transfer products accumulated prior to formation of cleavages at the donor 5' terminus, further supporting the idea that transfer occurs via an acceptor invasion-initiated mechanism, not involving cleavages near the donor 5' end. Our invasion model predicts that during minus strand transfer, the homologous region of the acceptor RNA invades the donor-cDNA hybrid at gaps created by RT-RNase H cleavage of the donor RNA. Although RT continues synthesis on the donor RNA, the acceptor-cDNA interaction initiates the transfer event well behind the polymerizing RT. The acceptor-cDNA hybrid then propagates by branch migration, strand-displacing the donor RNA fragments. Eventually, the primer terminus switches from the donor to the acceptor RNA, completing transfer of the cDNA. RT then resumes synthesis using the acceptor RNA as the new template.

In a recent study of HIV-1 minus strand transfer, we identified potential acceptor invasion sites within the donor RNA-cDNA hybrid (29). RT pausing at the base of the TAR hairpin promotes extensive cleavage of the donor RNA about 20 nt behind the pause site (29, 34). We designed a series of short DNA oligonucleotides that could compete with specific regions of the acceptor RNA for interaction with the cDNA and block transfer. Blocking oligomers complementary to the region around the base of the TAR hairpin were the most effective at inhibiting transfer. These combined observations suggest the region around the base of TAR to be an invasion site.

In the current study, we developed a technique to further analyze the acceptor invasion mechanism. Using a combination of RT and E. coli RNase H we analyzed the acceptor RNA cleavage profile during the course of the transfer reaction. Sensitivity to RNase H cleavage was used as an indication of RNA-DNA hybrid formation. Results showed that acceptor RNA was first cleaved 60–90 nt from the U3-3'R junction, agreeing with initiation of invasion at the base of TAR. A shift of the cleavage pattern toward the acceptor 5' end was then observed, suggesting hybrid propagation and primer terminus switch. We also show that productive invasion depends on the viral NC protein, clarifying why NC promotes transfers. Results correlate with the previous observations from the donor cleavage and blocking oligomer assays. All indicate that RNase H cleavages at the base of TAR allow acceptor invasion that initiates the transfer process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Recombinant HIV-1 reverse transcriptase (p66/p51 heterodimer) was purified as described previously (28, 35). HIV-1 NCp7 (72 amino acids) was prepared by solid phase chemical synthesis as described previously (36). DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA), and 2'O-methyl-modified RNA oligomers were from Dharmacon Research Inc. (Lafayette, CO). The oligo(dA) and oligo(dT) were purchased from the W. M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT). The 32P isotope was purchased from PerkinElmer Life Sciences. The T7 MEGA shortscript and Escherichia coli RNase H were purchased from Ambion (Austin, TX), and Pfu Turbo DNA polymerase was from Stratagene (La Jolla, CA). All other enzymes, dNTP mixture, and ddNTP mixture were purchased from Roche Applied Science.

Preparation of Templates—The D199, D130, and A97h RNA templates were prepared by run-off transcription in vitro, as described previously (29). The 2'O-M-(4-20) D199 donor RNA was generated by ligating a 31-nt 5' fragment containing the 2'O-methyl modification, to a 168-nt 3' fragment using DNA ligase and bridging DNA oligomer as described previously (37). The 31-nt 5' fragment was synthesized by Dharmacon, whereas the 3' RNA fragment was generated by run-off transcription. All RNA templates used in the study were purified by denaturing PAGE and resuspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA buffer. RNAs were quantified using the Ribogreen assay (Molecular Probes, Eugene, OR). The cDNA130 was purchased from IDT Inc. (Coralville, IA).

Transfer Assay—Reactions were performed as described previously (29) with slight modifications. The PBS DNA primer or RNA templates were 5' end-labeled using [{gamma}-32P]ATP (6000 (222 TBq) Ci/mmol) and polynucleotide kinase to follow either the primer extension or the acceptor degradation, respectively. Primer was annealed to donor RNA by incubating at 95 °C for 5 min and slow cooling to room temperature. Unless described otherwise, substrates were incubated with an appropriate amount of NC and incubated at 37 °C for 5 min. One NC molecule per 7 nts of RNA or DNA was taken as 100% coating (38, 39). RT was pre-bound to substrate at 37 °C for 2 min before reactions were initiated with MgCl2 and dNTPs. Final reactions contained 2.5 nM primer/template, 7.5 nM acceptor RNA, 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 16 nM HIV-1 RT, 6 mM MgCl2 and 17 µM dNTPs, respectively. Reactions were incubated at 37 °C and were terminated at appropriate time points with 2x termination dye (10 mM EDTA (pH 8.0), 90% formamide (v/v), and 0.1% each of xylene cyanol and bromphenol blue). Products were resolved by denaturing PAGE, and visualized and analyzed using a Storm PhosphorImager (Amersham Biosciences) and ImageQuant software (version 2.1). Sizes of DNA products were determined by using 10-bp DNA ladders, whereas sizes of RNA products were estimated using RNA ladders generated by RNase T1 digest.

Detecting Acceptor Cleavage Using E. coli RNase H—Transfer assays were set up as described above, using 5' end-labeled acceptor RNA. At appropriate time points, reverse transcription was terminated with a mixture of oligo(dA):oligo(dT) and ddNTP at a final concentration of 1 nM oligo(dA):oligo(dT) and 50 µM ddNTP. Following termination, the reaction mixture was incubated at 37 °C for 3 min with 8 units of E. coli RNase H. Reactions were then terminated by adding 1 volume of 2x termination dye. Products were resolved on 9% denaturing polyacrylamide gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Acceptor RNA Cleavage and Transfer Mechanism—Our previous analyses suggested that minus strand strong stop transfer in HIV-1 is accomplished in a multistep process (29). During the course of minus strand synthesis, RT paused at the base of the TAR hairpin. While paused, RT cleaved the donor template, creating gaps within the donor-cDNA hybrid. The acceptor template invaded the donor-cDNA complex at such sites, forming a hybrid. Hybrid formation and subsequent events leading to the terminus transfer, as proposed by our acceptor invasion model, are diagrammed in Fig. 1A. The acceptor-cDNA hybrid was susceptible to RNase H cleavage. We hypothesized that the time-dependent profile of RNase H cleavage of the acceptor template would reveal the stepwise progression of the transfer reaction. This is best envisioned when the acceptor is labeled at its 5' end. The site of initial interaction and the course of the hybrid propagation should be seen.



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  		FIG. 1.
Proposed acceptor invasion-induced transfer mechanism. A, steps involved in the acceptor invasion model of transfer. As cDNA (gray line) synthesis proceeds, RT-RNase H cleaves the donor RNA (patterned line). The homologous acceptor RNA (black line) interacts with the cDNA at gaps created within the donor-cDNA hybrid, initiating the invasion. As acceptor-cDNA interactions expand, the invasion propagates, eventually resulting in primer terminus switch from the initial template on to the acceptor. RT resumes DNA synthesis utilizing the acceptor RNA as template. B, schematic of substrates used in the study. Synthesis was initiated on a 199-nt RNA, corresponding to the 5' end of the HIV-1 genomic RNA, designated as the donor template. A 119-nt RNA corresponding to the 3' end of the genome served as the acceptor. The donor comprises of the 97-nt R and 102-nt U5 regions, whereas the acceptor contains the 97-nt R region and 20 nt from the U3. An 18-nt DNA primer complementary to the PBS sequence served as the primer. Star indicates the position of the 32P label.

 
Acceptor RNA Cleavage during Minus Strand Transfer—The HIV-1 minus strand transfer assay system used in this study (Fig. 1B) has been described previously (29). The D199 donor RNA is a 199-nt segment from the 5' end of the genomic RNA, containing the R, U5, and PBS regions. The A97h acceptor RNA comprises the 97-nt R region and 20 nt of the U3 sequence. Synthesis was primed from an 18-nt DNA primer complementary to the PBS. Assays were performed at 200% NC coating level (1 NC molecule per 3.5 nt). Acceptor degradation was followed during the course of the transfer reaction using a 5' end-labeled acceptor RNA. The experimental approach is outlined in Fig. 2A. Reactions contained excess RT over primer/template, thereby increasing the opportunity for RNase H cleavage.



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  		FIG. 2.
HIV-1 RT-catalyzed acceptor cleavage during minus strand transfer. Description of the substrates is the same as in Fig. 1B. A, schematic of experimental design for analysis of acceptor cleavage by RT-RNase H. Transfer reactions were performed with 5' endlabeled A97h acceptor RNA at 200% NC coating. Reactions were sampled at various time points and acceptor cleavage products resolved by denaturing PAGE. B, representative gel showing products of the acceptor cleavage by RT-RNase H. Schematic of the donor and acceptor RNAs is shown alongside the gel, indicating regions of intense (black arrows) and moderate (gray arrows) cleavage as predicted from the size of the specific cleavage products. Reactions were terminated at 1, 2, 4, 8, 16, 32, and 45 min. Lane C, reactions without Mg2+ and dNTPs. Lane L, 10-bp DNA. ladder. C, time course of transfer reaction using D199 donor and A97h acceptor templates and 5' end-labeled PBS DNA primer. Reactions were terminated at 0.5, 1, 2, 4, 8, 16, 32, and 64 min. Synthesis products corresponding to pausing at TAR hairpin, full-length extension on the donor RNA, transfer, and fold back are indicated.

 
Fig. 2B shows the acceptor cleavage profile during the course of the RT-catalyzed transfer reaction. By about 4 min, bands appeared indicating cleavage at a distance of 80–105 nucleotides from the 5' end of the acceptor. This is the expected region of invasion at the base of TAR, as suggested previously by blocking oligonucleotide and homology length experiments (29). A series of intermediate size cleavage products, 60–20 nts in size, observed through the 4–45-min period suggested a progressive acceptor-cDNA hybrid formation from the base of TAR (initial invasion site) to the 5' end of the acceptor. By 16–45 min, a dense set of cleavage products 5–19 nt in size appeared. The size of these cleavage products and their time of appearance indicate cleavage of the acceptor 5' terminus, following completion of synthesis to the end of this template. The cleavage products observed indicate that the RNase H activity intrinsic to the RT cleaves the acceptor RNA during the course of the transfer reaction. Regions of cleavage are therefore indicative of acceptor hybridization with the cDNA. The profile shows an early appearance of cuts at the base of TAR and a time-dependent progression of cuts toward the 5' end of the acceptor RNA. The observed acceptor cleavage profile is consistent with the proposed multistep transfer mechanism in which acceptor-cDNA interaction initiated at the base of TAR and propagated toward the 3' end of the cDNA.

Analysis of cDNA synthesis under the same conditions (Fig. 2C) showed that by 2 min some primer extensions were paused at the base of TAR, whereas a similar amount had completed full-length extension on the donor template. By 8 min a similar amount of primers were paused at the base of TAR and considerably more were fully extended. Concomitant with RT pausing, extensive cleavages were observed on the donor at the base of TAR, during the 2–8-min reaction time (see Ref. 29 and data not shown). Taken together, these results suggest that for those substrates undergoing transfer, invasion can occur just after the primer has extended past the pause site. The propagating hybrid can then follow behind the RT, which is continuing synthesis on the donor RNA.

Analyzing Acceptor Cleavage during Transfer by Subsequent Treatment with E. coli RNase H—The previous experiment used the intrinsic RNase H activity of RT to make the cleavages that mark interaction sites of the acceptor RNA and the donor-cDNA. This approach relies on the capacity of the RT-RNase H to make endonucleolytic cleavages in the acceptor RNA as it interacts and forms partial hybrids with the cDNA. Studies by DeStefano and co-workers (40) have shown that certain RNA-DNA hybrid structures that mimic the acceptor-invasion intermediates are not efficiently cleaved by HIV-1 RT-RNase H. This would explain the observed inefficient acceptor cleavage at the early time points, although transfer products are already formed. Effective probing of acceptor-cDNA interactions would require use of a nonspecific and robust RNase H activity. We therefore developed a second method to analyze acceptor cleavage during transfer, as out-lined in Fig. 3A. RT-catalyzed transfer reactions were set up as described previously. The reaction was sampled at various time points, and RT activity was terminated by a process that traps the RT molecule. The reaction intermediates were then incubated with E. coli RNase H for 3 min, and the time-dependent acceptor cleavage profile was visualized.



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  		FIG. 3.
Analysis of acceptor-cDNA interaction during minus strand transfer using E. coli RNase H. Description of the substrates is the same as in Fig. 1B. A, schematic of experimental design. Experimental set-up was essentially the same as described in Fig. 2A, except that an E. coli RNase H treatment step was included, following termination of the RT reaction. The 5' end-labeled acceptor RNA (A97h) was included at the start of the reaction (B), as described in Fig. 2, or 5 min after initiating cDNA synthesis on donor RNA D199 (C). Reverse transcription was terminated at different time points with non-EDTA terminators to stop polymerase and RNase H activities. The terminated reaction mix was treated with E. coli RNase H for 3 min to allow cleavage of acceptor-cDNA hybrid regions. The cleavage products were resolved by denaturing PAGE. B and C, representative gels showing acceptor cleavage profiles when acceptor RNA is included at the start of the reaction (B) or 5 min following initiation of cDNA synthesis (C) on donor. Cleavage products result from the combined actions of RT-RNase H and E. coli RNase H. Schematic of the donor and acceptor RNAs is shown alongside the gels, indicating the regions of intense (black arrows) and moderate (gray arrows) cleavage as predicted from the size of the specific cleavage products. Reactions were terminated at 1, 2, 4, 6, 8, 10, 15, 20, 25, and 30 min of addition of acceptor. Lane 30-H, 30-min reaction without E. coli RNase H treatment. Lane C, reactions in the absence of RT and E. coli RNase H. Lane L, 10-bp DNA ladder.

 
Fig. 3B shows the time-dependent pattern of acceptor cleavage using E. coli RNase H. E. coli RNase H was titrated to determine the minimal amount required to amplify acceptor cleavage (data not shown). Compared with cleavages by RT (Fig. 2B), much more of the acceptor molecules sustained cleavages in the E. coli RNase H analysis, producing denser bands of cleavage products and the expected greater sensitivity. Remarkably the band pattern, although darker, showed important similarities to the cleavage profile observed in experiments with RT alone (Fig. 2B). It showed dense bands at the proposed invasion site and a progression of cleavages moving toward the 5' end of the acceptor RNA. In the similarities of the two patterns, the two techniques lend validity to each other in showing the site of initial interaction between the acceptor and cDNA. The pattern of the E. coli RNase H analysis also had additional features not present in the analysis with RT alone. These include dense acceptor cleavages 25–30 nucleotides from the 5' end and the early appearance of the 5–9-nt terminal cleavage products. These will be addressed below.

One feature of the acceptor cleavage profile was that it closely matched the cDNA synthesis profile. This suggested the possibility that the progressive cleavages observed from the 3' to 5' end of the acceptor is dictated by the length of cDNA synthesized at any given time point. If so, the initial acceptor cleavages at the base of TAR would not be from the creation of a site-specific invasion site. To address this issue, we performed the assay by adding the acceptor RNA 5 min after RT reaction with the primer/donor template (Fig. 3C). This ensured that full-length cDNA synthesis was completed in a substantial proportion of the templates before acceptor interactions were initiated. If cleavages at the acceptor 3' end were indeed associated with partial cDNA synthesis, then the delayed addition of the acceptor should cause a decrease in intensity of the acceptor 3' cleavages. On the other hand, if acceptor invasion is facilitated by the creation of a specific invasion site on the donor RNA at the base of TAR, then addition of the acceptor after completion of cDNA synthesis should still generate the same profile of acceptor cleavage as observed in Fig. 3B. As observed in Fig. 3C, acceptor cleavage pattern remained basically unchanged from that in Fig. 3B. The predicted invasion site at the acceptor 3' end was still very evident. The time requirement for cDNA synthesis and creation of invasion site within the donor-cDNA hybrid is very likely the reason for the slight delay in formation of the 3' cleavage products in Fig. 3B as compared with Fig. 3C. Overall, these results suggest that cleavages at the 3' end of the acceptor result from a site-specific invasion that is independent of length of cDNA synthesized.

Control Experiments Show That Trapping Conditions Stop Synthesis and Cleavage by RT—The purpose of the E. coli RNase H procedure was to detect acceptor-cDNA interactions that are progressively formed during the course of a transfer event. To assess this accurately, it is important that termination of RT activity and subsequent E. coli RNase H probing be done under the following condition: 1) minimally disrupt the structures of the RT-generated transfer intermediates, and 2) efficiently terminate RT activity while still allowing for subsequent treatment with E. coli RNase H. We therefore used a mixture of dideoxynucleoside triphosphates to terminate synthesis and oligo(dT):oligo(dA) to trap the RT as it dissociates from the substrate. The effectiveness of this method is demonstrated in Fig. 4. The RT reactions were sampled at various time points and terminated with the ddNTP + oligo(dT):oligo(dA) mixture. One-half of the terminated reaction was immediately mixed with an EDTA:formamide stop solution, a method known to be absolutely effective at terminating catalysis. To verify if any enzymatic activity continued after the addition of the termination mixture, the other half was incubated for 10 min at 37 °C before addition of the EDTA:formamide stop solution. The effectiveness of the ddNTP termination mixture was tested for both synthesis (Fig. 4A) and RNase H activities (Fig. 4B). As observed, the 10-min incubation after addition of the ddNTP termination mixture did not yield any additional synthesis or RNase H cleavage products, confirming that the ddNTP + oligo(dT):oligo(dA) mixture served as an effective terminator for both the polymerase and RNase H activities of HIV-1 RT. Therefore, this verifies that during E. coli RNase H incubation, the substrate is no longer susceptible to the activities of the RT.



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  		FIG. 4.
Termination of reverse transcription with the ddNTP + oligo(dA)-oligo(dT) combination terminator. Schematic of the donor and acceptor RNAs is shown above the gels. A, cDNA synthesis was examined during the course of the transfer reaction, using 5' end-labeled primer DNA. B, RT-RNase H activity was examined during the course of cDNA synthesis using 5' end-labeled D199 RNA template. RT reactions were terminated at 0.25, 0.5, 1, 2, 4, 8, and 16 min with the ddNTP + oligo(dA):oligo(dT) mixture. One-half of the terminated reaction was immediately mixed with EDTA: formamide 2x stop dye (–), whereas the other half was incubated at 37 °C for 10 min, before the addition of the 2x stop dye (+). Lane C, reactions without Mg2+ and dNTPs. Lane L, 10-bp DNA ladder.

 
Retention of the Donor RNA Cleavage Profile during E. coli RNase H Treatment—The E. coli RNase H probing was designed to track the initiation and progression of interactions between the acceptor and cDNA by following acceptor degradation. Correct analysis of this interaction therefore required that E. coli RNase H not alter the cleavage profile of the donor, thereby altering acceptor invasion site. To address this issue, we analyzed donor RNA degradation under identical experimental conditions as described for the acceptor cleavage assays (Fig. 3). In these reactions donor RNA D130 was labeled at its 5' end. Degradation of the donor RNA was followed in the absence (Fig. 5A) and presence of E. coli RNase H treatment (Fig. 5B). During synthesis, the donor RNA sustained a series of adjacent cuts as RT paused at the base of the TAR hairpin. We previously related these cuts to the preferred site of invasion of the acceptor RNA for minus strand transfer. Although the E. coli RNase H degrades the donor RNA more extensively, the adjacent cuts at the base of TAR remained a prominent feature of the cleavage profile. Moreover, despite the increased cleavage, the great majority of the RNA remained as oligomeric intermediates, long enough to remain bound to the cDNA.



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  		FIG. 5.
Analysis of donor cleavage in E. coli RNase H probing assays. Transfer reactions were performed using D130 donor RNA and A97h acceptor RNA, at 200% NC coating level. The donor RNA was 5'-labeled to examine its degradation during the course of the reaction (Rxn). Donor cleavage was examined in the absence (A) and presence (B) of E. coli RNase H treatment. Reactions were terminated at 0.25, 0.5, 1, 2, 4, 8, and 16 min of reaction, using the oligo(dA)-oligo(dT) + ddNTP combination terminator. Terminated reactions were immediately mixed with EDTA termination dye (A) or treated with E. coli RNase H for 3 min at 37 °C before addition of EDTA termination dye (B). The products were resolved on a 9% denaturing polyacrylamide gel. Lane C, reactions without Mg2+ and dNTPs. Lane L, 10-bp DNA ladder.

 
We do observe increased cleavages near the 5' end of the donor RNA. This accumulation of short products and a faster disappearance of starting RNA are the expected consequences of additional but random RNase H cleavage. Generally, the above control experiments indicated that the E. coli RNase H treatment performs the role of amplifying the sensitivity of detection of the acceptor-cDNA interaction, without substantially altering the structure of important intermediates in the transfer reaction.

The Observed Acceptor Cleavage Profile Is Not Caused by E. coli RNase H Sequence-specific Cleavage—We also considered the possibility that the pronounced cleavage at the acceptor 3' end (initial invasion site) reflected a site of preferential RNase H cleavage, by virtue of its sequence. To test this we performed the E. coli RNase H procedure in the absence of donor RNA and RT. Single-stranded cDNA130 containing all of the R region was mixed with 5' end-labeled acceptor RNA and incubated with limiting amounts of E. coli RNase H for 1, 3, 6, or 12 min at 37 °C. Reactions were performed in the absence or presence of 200% NC coating (Fig. 6). In the absence of NC, minimal cleavages were observed even at 12 min, possibly because of poor acceptor-cDNA annealing under the tested conditions. In the presence of NC, the acceptor RNA sustained several internal cuts as early as 1 min. The cleavage profile, however, was very distinct and different from that observed in reactions where acceptor invasion was tested during the course of minus strand synthesis (compare Figs. 6 and 3B). First, the prominent set of cuts associated with acceptor invasion at the base of TAR (80–105 nts) was not very evident in the reactions with cDNA130. Second, the acceptor cleavage profile during synthesis showed characteristics suggestive of a time-dependent propagation of acceptor-cDNA interactions initiating from the acceptor 3' end and moving toward the 5' end. Acceptor cleavage profile with the cDNA130 did not show such a pattern. Overall, these results suggested that the acceptor cleavage profile observed in Fig. 3B is not dictated by sequence preference for RNase H cleavage. The donor cleavage profile, by inference, influenced the positions of acceptor interaction.



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  		FIG. 6.
Probing acceptor RNA cleavage using E. coli RNase H in the absence of synthesis. The 5' end-labeled acceptor RNA A97h was mixed with pre-synthesized cDNA130 and incubated at 37 °C for 1, 3, 6, or 12 min, in the presence of 200% NC coating (+NC) or in the absence of NC (–NC). Following incubation, the substrates were treated with 2 units of E. coli RNase H for 3 min at 37 °C. Reaction products were resolved on a 9% denaturing polyacrylamide gel. A schematic of the cDNA130 and A97h acceptor RNA is shown alongside the gel, indicating regions of intense (black arrows) and moderate (gray arrows) cleavage. Lane C, reactions in the absence of RT and E. coli RNase H. Lane L, 10-bp DNA ladder.

 
Evidence for Transfer Initiated at the DNA Terminus—One distinct feature of the acceptor cleavage pattern produced by the experiment with E. coli RNase H is the appearance of a series of adjacent cleavages around 25–30 nucleotides from the acceptor 5' end (Fig. 3, B and C). The position of these cleavages suggested a strong interaction between the acceptor and the cDNA 3' end. These cleavage products appeared early in the reaction along with the cleavages at the base of TAR. One interpretation for this is that a high proportion of the transfers was initiated by early acceptor interaction with the 3' end region of the fully extended cDNA. However, the 25–30-nt cleavage products did not appear when RT alone was tested (Fig. 2B). Furthermore, the conclusion that there is a substantial amount of direct terminal transfer is inconsistent with the results of earlier experiments (27, 29). In view of this, we further tested whether these cleavages relate to minus strand transfer.

The approach was to chemically modify the 5' end region of the donor RNA so that it could not be cleaved. We generated a 199-nt donor RNA in which positions +4 to +20 were substituted with 2'O-methylated nucleotides. When the modified donor RNA was substituted for the unmodified donor RNA in the E. coli RNase H assay, the bands around 30 nucleotides disappeared (Fig. 7A). We interpreted this to mean that because the donor RNA could not be cleaved near its 5' end, acceptor interaction with the DNA in this region was suppressed. If acceptor interaction with the cDNA 3' terminus were essential for the transfer process, then inhibiting donor 5' end cleavages should cause a drop in transfer efficiency. However, the modified donor RNA actually promoted strand transfer with higher efficiency compared with the unmodified D199 donor (Fig. 7B). The combined results clarify that in data presented in Fig. 3, B and C, the cleavages 25–30 nt from the acceptor 5' end are not related to the natural processes leading to transfer. These cleavages do indicate acceptor-cDNA interactions at the 3' end of the cDNA. However, these interactions were detected only during the E. coli RNase H probing, most likely because E. coli RNase H increases donor 5' end cleavages (Fig. 5B), thereby enabling the cDNA to interact with the acceptor.



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  		FIG. 7.
Effect of inhibiting donor 5'-end cleavages on acceptor-cDNA interaction and transfer. Transfer assays were performed using donor RNA, 2'O-M(4-20)D199, and A97h acceptor RNA at 200% NC coating level. A, analysis of acceptor-cDNA interaction during minus strand transfer using E. coli RNase H. Experimental set-up was identical to that described in Fig. 3B but using 2'O-M(4-20)D199 donor. Reverse transcription was terminated at 1, 2, 4, 8, 15, 20, 25, and 30 min with non-EDTA terminator, followed by treatment with E. coli RNase H for 3 min at 37 °C. Reaction products were resolved by denaturing PAGE. Schematic of the donor and acceptor RNAs is shown alongside the gel, indicating the regions of intense (black arrows) and moderate (gray arrows) cleavage as predicted from the size of the specific cleavage products. Lane C, reaction in the absence of HIV-1 RT. B, transfer assays were compared using the D199 (left panel) and 2'O-M(4-20)D199 (right panel) donor templates in the presence of A97h acceptor and 5' end-labeled PBS DNA primer. Reactions were terminated at 1, 3, 5, 15, 20, and 30 min using EDTA termination dye. Full-length extension, transfer, and fold back products are indicated. Lane L, 10-bp DNA ladder.

 
Efficient Invasion Depends on the Presence of NC—Previous analysis of the stimulatory effects of NC had suggested that NC increases the pause-associated cleavages at the base of TAR. It was therefore of interest to see whether such effects of NC were reflected in the E. coli RNase H acceptor cleavage assay. For this, we performed the E. coli RNase H assays at varying levels of NC (Fig. 8). In the absence of NC, very minimal cleavage of the acceptor was observed (Fig. 8, 0% NC). As increasing amounts of NC were included in the reaction, there was a progressive increase in the intensity of bands indicating invasion at the base of TAR. These results support earlier observations that properties of NC are required for efficient invasion leading to strand transfer.



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  		FIG. 8.
Productive acceptor invasion requires the viral NC protein. Acceptor cleavage with E. coli RNase H was examined during minus strand transfer using various concentrations of NC protein. Assays were performed using the D199 donor and A97h acceptor RNA templates. Reaction (rxn) set-up was the same as described in Fig. 3B. Assays were performed in the absence of NC or at 50, 100, and 200% NC coating levels. Reactions were terminated at 0.5, 1, 2, 4, 8, 16, and 32 min of reaction with non-EDTA terminator, followed by treatment with E. coli RNase H for 3 min at 37 °C. Reaction products were resolved by denaturing PAGE. Lane C, reaction in the absence of RT and E. coli RNase H. Lane L, 10-bp DNA ladder.

 
A Time-dependent Increase in Transfer Efficiency—A time course of the transfer reaction showed that full-length extension product on the donor template appeared by 1 min (Fig. 2C). Cleavage of the donor RNA at the base of TAR, creating the invasion site, also occurred very efficiently and as early as 0.25 min (Fig. 5A). However, significant transfer product did not appear until 8 min (Fig. 2C). Time course analysis shows a substantial increase in transfer efficiency over time (Fig. 9), suggesting that there may be one or more steps or events in the overall reaction that limit the formation of transfer products. The RT reactions (Fig. 2C) do not show acceptor cleavages indicative of invasion until 4–8 min. The E. coli RNase treatment accelerated cleavages, and therefore timing of cleavages in that assay was not necessarily an accurate indication of the time of invasion. Although evidence of invasion was apparent, some step or steps in the transfer mechanism must limit the rate of formation of the final transfer product.



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  		FIG. 9.
Time course of accumulation of transfer products. Transfer efficiency was calculated as: 100 x transfer product/(transfer products + full-length donor extension products). Transfer efficiency at various time points was quantitated from transfer reaction shown in Fig. 2C and plotted as a function of time.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have developed a method to analyze the invasion mechanism for RT template switching during strand transfer. The approach enables us to track acceptor-cDNA interactions during the course of the transfer event by following degradation of the acceptor RNA. Our proposed acceptor invasion model for transfer describes a branch migration step, whereby following initial invasion, the acceptor-cDNA hybrid propagates until it catches up with the primer terminus to complete the transfer event. We had previously obtained evidence for the invasion step, through analysis of donor cleavage profile and blocking oligomer assays (29). In the absence of direct evidence, we could only infer the propagation step. In fact, mechanisms could be envisioned that did not use hybrid propagation for transfer. For example, transfer could be promoted through an ordered two-point interaction, whereby acceptor RNA contacts the cDNA at the initial invasion site and then at primer terminus.

By using acceptor degradation analysis, we proposed to address such mechanistic details of the transfer process. Our approach involved use of a 5' end-labeled acceptor RNA, the degradation of which was followed during the course of the transfer reaction. Reactions were first done with RT to reveal the natural profile of cuts occurring over time. Although RT cleaves the donor RNA very efficiently, the acceptor is not cleaved as much, limiting the sensitivity of the cleavage profile. In a complementary procedure we used E. coli RNase H to accentuate the observed cleavages. The nonspecific and robust activity of E. coli RNase H enabled us to visualize more effectively the acceptor-cDNA interactions. The combined profile of the two sets of data revealed details of the acceptor invasion and propagation. Both provided consistent results, showing that substantial acceptor invasion occurred at the base of TAR, after cDNA synthesis over that region. The interaction then propagates by branch migration to the 3' end of the cDNA.

The relatively low level of cleavage of the acceptor compared with the donor RNA suggests that RNase H cleavages made on the donor during synthesis are major contributors to the overall RNase H activity observed during the reaction. Polymerase-dependent cleavages made during RT pausing generate RNA fragments with 5' ends. Such RNA fragments generated at the base of TAR very likely are then efficiently degraded by the polymerase-independent mode of RT-RNase H, generating gaps in the donor-cDNA hybrid. In the absence of synthesis, the acceptor sustains fewer RT-RNase H cuts. This is consistent with a proposal by Pathak and colleagues (15) that polymerization-dependent rather than polymerization-independent RNase H activity is the major promoting factor in strand transfer. DeStefano and co-workers (40) examined the stability of the intermediates of strand transfer having both donor and acceptor RNAs annealed to a cDNA. They concluded that the conformation of the acceptor-cDNA invasion intermediate is not very susceptible to RNase H cleavage, as RT-RNase H does not bind and cleave hybrids effectively in an endonucleolytic mode. On RNA-DNA hybrid substrates, RT preferentially binds at the DNA 3' end or RNA 5' end (41, 42) producing efficient RNase H cuts. This interpretation is consistent with our observation that both the initial invasion intermediate and the longer hybrid formed during propagation resist extensive cleavage by RT-RNase H. We also performed control assays to rule out the possibility that the acceptor cleavage profile observed is not because of sequence preference of RNase H cleavage. By inference, this suggests that the segments of the original donor RNA left after the RT reaction is sampled are influencing the positions where the acceptor RNA forms hybrids with the cDNA. The acceptor cleavage profile in Fig. 3B is therefore indicative of sites of specific interaction between the acceptor with the cDNA.

Because of the low level of RT-directed cleavage of the acceptor, we chose to augment its activity by using E. coli RNase H. Through use of E. coli RNase H, we attempted to track the progressive interactions between the acceptor RNA and cDNA by following acceptor degradation. However, the use of E. coli RNase H has some shortcomings. During DNA synthesis, the RT places cleavages in the donor RNA that we propose favor invasion at specific locations. The E. coli RNase H can place additional cleavages into the donor RNA, potentially altering the kinetics and mechanism of transfer. However, we present evidence that the level of E. coli RNase H chosen did not substantially alter the pattern of donor RNA digestion. This implies that the acceptor cleavages observed in the experiments with E. coli RNase H are indicative of interactions of acceptor RNA and cDNA during minus strand transfers.

The E. coli RNase H assays showed acceptor cleavages around 30 nt from the 5' end (corresponding to the TAR 5' region), very early in the reaction. The presence of these cuts can be taken to indicate that a significant fraction of transfers occurred by a mechanism involving direct transfer of the primer terminus after completing cDNA synthesis. Direct transfer of the primer terminus would require donor 5' cleavages. However, substitution of 2'O-methylated nucleotides near the 5' end of the donor RNA showed that inhibiting cuts in this region did not suppress transfer. It is significant that these particular cuts only appear in the E. coli RNase H assay and appear to be a unique consequence of E. coli RNase H treatment. Enhanced cleavages at the donor 5' end in the presence of E. coli RNase H (Fig. 5B) also support this explanation. These results point out the value of the RNase H assay as a qualitative method for obtaining information on the sites of invasion and propagation. However, results with the RT alone are more reliable to assess the quantitative timing of events. One unexpected outcome of using the modified donor was an increase in transfer efficiency. We have observed previously that inhibiting site-specific RNase H cuts through 2'O-methyl modification of the RNA increased cleavages in the adjacent regions of the RNA (27, 37). With the 2'O-M-D199, we observed a similar effect, with generally increased RT-RNase H cleavages within the R region (data not shown). In addition, in E. coli RNase H assays using this modified donor, we observed increased acceptor cleavage in the 3' region of TAR (Fig. 7A, products in the size range of 45–60 nts), indicating acceptor-cDNA interactions within this region. These data indicate that the increase in transfers very likely results from an increase in donor cleavage promoting better internal acceptor invasion.

Our results show that the presence of NC at a level sufficient to fully coat the substrate strands is necessary for efficient acceptor-cDNA interaction. In particular the intensity of RNase H cleavage bands marking the primary site of invasion at the base of TAR is strongly dependent on the presence of NC. NC very likely enhances acceptor invasion process in two ways. It increases donor cleavages at the base of TAR (29). Additionally, as suggested by data in Fig. 8, NC promotes acceptor invasion at these gaps. Evidently the series of adjacent RNase H cleavages made at the base of TAR in the absence of NC does not clear an area big enough to allow stable binding of the complementary acceptor strand.

These results also suggest that the interaction of the cDNA and acceptor templates is most effective where the single strands have the least secondary structure. Since the cDNA-donor RNA is thought to be largely double-stranded, with short gaps, the structure of the single-stranded acceptor RNA strand may be most influential in determining the favored sites of interaction. We have done structure prediction of the acceptor using m-fold, and indeed we do see that the region at the base of the TAR is mostly single-stranded. This suggests why the first interactions are likely to occur at the base of TAR.

We found previously that transfer efficiency rises during the course of minus strand transfer assays (27, 29). This delay in the formation of transfer products is much too long to represent the time required to synthesize the extra 20 nucleotides on the acceptor template. The delay suggests that the overall process involves one or more steps or events that limit the formation of the transfer product. The proposed invasion and propagation mechanisms are consistent with the idea that the transfer process can include slow steps that do not occur during DNA synthesis on the donor RNA. For example, following invasion, propagation of the hybrid through the structured TAR hairpin in the acceptor is one possible slow step.

Overall, our results provide additional and direct support for the invasion-propagation terminal transfer mechanism for minus strand transfer. This mechanism was previously suggested by homology overlap and blocking oligonucleotide methods (27, 29). The results also address possible mechanisms for the role of NC in promoting transfer.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM 049573 (to R. A. B). 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

§ Both authors have contributed equally to this work. Back

Present address: Dept. of Molecular Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030. Back

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

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase; PBS, primer binding site; nt, nucleotide; –sssDNA, minus strand strong stop DNA; NC, nucleocapsid; R, repeat. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Jeff DeStefano for critical reading of the manuscript and to Drs. Ricardo Roda, Vandana Purohit, and Mark Hanson for helpful discussions.



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 DISCUSSION
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