(Received for publication, October 12, 1995)
From the
RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) RNase H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3` DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5` RNA ends. We constructed substrates to test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3` DNA and 5` RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5` end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5` end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5` RNA/3` DNA end. When a DNA primer recessed on an RNA template had a 3` unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5` RNA/3` DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3` end and is directed by the 5` RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3` DNA or 5` RNA end, where it is positioned for catalysis by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5` RNA end structure are discussed.
Human immunodeficiency virus reverse transcriptase (RT) ()catalyzes many essential steps required for viral
replication. RT has been shown to possess DNA-dependent and
RNA-dependent DNA polymerase activities, RNase H activity, strand
transfer and strand displacement activities, all of which are essential
to complete the process of conversion of single stranded viral RNA
genome to double-stranded proviral DNA (reviewed by Goff(1990)).
Because the RT is vital to the life cycle of the virus, it has been the
target of chemotherapy. Biochemical properties of the RT are being
examined in many laboratories in an effort to obtain information needed
to design more effective antiviral drugs.
The native enzyme is a heterodimer of 66- and 51-kDa subunits (diMarzo Veronese et al., 1986; Lightfoote et al., 1986). The amino-terminal region of each subunit harbors a polymerase domain, and the carboxyl terminus of only the larger subunit harbors the RNase H domain (Johnson et al., 1986). The absolute requirement of RT RNase H for retroviral replication has been established by mutational analysis (Repaske et al., 1989; Tanese and Goff, 1988). RNase H activity has been shown to catalyze the necessary destruction of the plus strand RNA and the tRNA primer, and the generation and removal of the polypurine tract plus strand primer (for a review, see Champoux(1993) and Telenitsky and Goff(1993)). RNase H activity has also been demonstrated to be required for the strand transfer reactions needed in the replication cycle (Cirino et al., 1995; DeStefano et al., 1992; DeStefano et al., 1994b; Ghosh et al., 1995; Peliska and Benkovic, 1992).
Several studies have shed light on the mechanism by which the RT uses its RNase H activity for the removal of the plus strand RNA during and after minus strand DNA synthesis (DeStefano et al., 1991a, 1993, 1994a; Schatz et al., 1990; Gopalakrishnan et al., 1992; Fu and Taylor, 1992; Furfine and Reardon, 1991; Huber et al., 1989; Krug and Berger, 1989; Wöhrl and Moelling, 1990). We and others have employed substrates that resemble intermediates of minus strand DNA synthesis. Results show that the RT makes initial cleavages of template RNA about 14-18 nucleotides upstream of the 3` hydroxyl of a DNA primer (DeStefano et al., 1991b; Gopalakrishnan et al., 1992; Furfine and Reardon, 1991; Ghosh et al., 1995;, Schatz et al., 1990; Zhan et al., 1994). These are followed by additional cleavages in the direction of the primer terminus. Crystallographic studies show that the polymerase and RNase H active sites of the RT are separated by a distance of about 20 nucleotides (Jacobo-Molina et al., 1993; Kohlstaedt et al., 1992), suggesting that interaction of the polymerase active site with the primer template determines the positioning of the RNase H active site.
RNase H activity that accompanies minus strand DNA synthesis directed from the growing 3` hydroxyl of the DNA primer was termed ``polymerase-dependent'' (Furfine and Reardon, 1991; Peliska and Benkovic, 1992). A ``polymerase-independent'' mode of RNase H activity has been implicated for cleavages not mediated by the 3` OH of the DNA primer (Furfine and Reardon, 1991; Peliska and Benkovic, 1992). Biochemical analyses indicate that polymerase-dependent RNase H activity does not completely degrade the plus strand RNA during minus strand DNA synthesis, but produces RNA oligomers, many of which are long enough to remain bound to the newly synthesized DNA (DeStefano et al., 1991b; DeStefano et al., 1994a; Dudding et al., 1991; Kati et al., 1992). These RNA segments must then be removed by RTs that act after passage of the growing minus strand DNA primer. Examining the action of RTs on such substrates, i.e. RNA oligomers annealed to longer DNAs, we found that the positions of initial cleavage are about 18 nucleotides from the 5` end of RNA (DeStefano et al., 1993). Again, this distance suggests that the separation between the catalytic sites on the RT is determining the positioning of the RNase H. We proposed that the 5` end of the RNA fixed the position of the polymerase active site as indicated in Fig. 1, model A (DeStefano et al., 1993). This differs from positioning which is apparently caused by the high affinity of the polymerase active site for the 3` end of a DNA primer (Fig. 1, model B). However, there is no obvious reason why the polymerase active site would be expected to have an affinity for the 5` end of the RNA.
Figure 1: Model for the mechanism of RT positioning for RNase H cleavage. Three possible modes of RT positioning are represented. The letters P and H refer to polymerase and RNase H active sites, respectively.
In this paper we investigate the possibility that the structure of the RNA-DNA hybrid region of the substrate, rather than the termini of the RNA or DNA strands, is the major determinant of RT positioning. We propose that the RT could first position at the 3` end of the DNA, and then slide to the nearest region of hybrid (Fig. 1, model C). This would give the impression that the 5` end of the RNA determines positioning, when it actually does not. Results presented here distinguish the influence of the RNA end structure versus the hybrid region structure on positioning of the RT.
Figure 2: Substrates used in this study. Substrates A and B were formed by annealing a 5`-labeled 41-mer RNA transcript to each of two 47 nucleotide DNA templates 1 and 2, respectively. The nucleotide sequences of DNA and RNA are given under ``Experimental Procedures.'' The unannealed region at the 5` end of the 41-mer RNA in substrate B is 10 nucleotides long. Substrates C and D were obtained by annealing DNA primers 2 and 3, respectively, to a 5`-labeled 142-mer RNA template 3. The 20-mer DNA primer 2 fully annealed to the RNA. The 26-mer DNA primer 3 differed from DNA primer 2 by the presence of additional 6 nucleotides, which formed a tail at the 3` end upon annealing to the RNA template 3. Substrates E, F, G, and H were generated by annealing a 189-mer RNA transcript (RNA primer 4) to DNA template 4, DNA template 5, DNA template 6, and DNA template 7, respectively. The 189-mer RNA fully annealed to DNA template 4, but had a 9- and 27-nucleotide unannealed region at the 5` end on DNA templates 5 and 6, respectively. In substrate H a 9-nucleotide annealed region at the 5` end was followed by a 40-nucleotide loop. In this and subsequent figures, the RNA portion is indicated by thin lines and the DNA portion is indicated by thick lines.
The plasmid pCP1 lacks a 9-nucleotide sequence 5`-GGGCGAATT present in the plasmid pBS+ downstream from the T7 promoter.
The plasmid pCP2 lacks 5`-GGGCGAATTCGAGCTCGGTACCCGGGG present in the plasmid pBS+ downstream from the T7 promoter.
DNA primer 2 is 5`-GTGTGGAATTGTGAGCGGAT; DNA primer 3 is 5`-GTGTGGAATTGTGAGCGGATGGTCCG. DNA template 1 is 5`-ATGCTCTAGAGGATCCCCGGGTACCGAGCTCCATGGTCATAGCTGTT; DNA template 2 is 5`-ATGCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCGCCCGCTGTT.
Figure 3: HIV-1 RT RNase H-mediated cleavage on substrate A versus B. A schematic representation of substrates A (right panel) and B (left panel) is shown at the top. The asterisk at the 5` end of RNA indicates the radiolabel. Filled and open arrows represent points of initial and secondary cleavages, respectively. The numbers above the arrows represent the size of the labeled products. An X on the arrows represents the absence of the cleavage. The left panel comprising lanes 1-6 shows reaction products from substrate B, and the right panel comprising lanes 7-12 shows the reaction products from substrate A. The presence or absence of E. coli RNase H is indicated by + or -. The concentration of RT is indicated above each lane. The numbers on the left indicate the product length of RNA determined by an RNA ladder prepared as under ``Experimental Procedures.''
Substrate A, when subjected to HIV-RT RNase H-mediated cleavages, underwent both primary and secondary cleavages at the previously observed positions of 18 and 10 nucleotides, respectively, measured from the 5` terminus of RNA. Since with substrate A both the primary and the secondary cleavages were complete in the 15-min reaction time, the predominant products observable were 9-10 nucleotides long (Fig. 3, right panel, lanes 9-12). However, with substrate B the only products that appeared were cleaved 18 nucleotides from the 5` end of RNA (Fig. 3, left panel, lanes 3-6). There were no other observable products even after long autoradiographic exposures. Cleavage directed from the point of hybridization should have yielded products 28 and 20 nucleotides in length, but these were totally absent. Quantitation of primer 1 using PhosphorImager(TM) analysis showed that the amount of cleavage at any RT concentration was about the same in substrates A versus B (Fig. 3, compare the disappearance of the 41-mer in lanes 3-6 with lanes 9-12, respectively). Therefore, differences observed in product distribution did not result from fundamental differences in the sensitivity of the two substrates to cleavage. Contrary to our anticipation that the point of hybridization determines or influences the positional cleavage by RT, the products obtained from gel electrophoresis analysis indicate that the 5` terminus of the RNA is a primary determinant. Influence of the 5` end predominates even if it is not annealed!
Cleavage of substrate B also consisted of only primary products (Fig. 3, compare amounts of 18-nucleotide products). This was probably because, following the initial cleavage at about 18 nucleotides from the 5` end of the RNA on substrate B, the cleaved product had such a short region of complementarity with the template that it dissociated before secondary cleavage could occur.
E. coli RNase H was employed to demonstrate the presence of RNA/DNA hybrid in substrates A and B (Fig. 3, lanes 1 and 7). E. coli RNase H cleavage does not follow the cleavage pattern of HIV-1 RT. Additionally, the presence of a product of 16 nucleotides in length following E. coli RNase H treatment of substrate B demonstrates that the tail region of substrate B does not undergo cleavage, and therefore is unannealed (Fig. 3, left panel, lane 1).
Figure 4: Time course of RNase H cleavage on substrate A versus B. A schematic representation of substrates A (right panel) and B (left panel) is shown at the top. The time course RNase H reactions were performed as described under ``Experimental Procedures.'' Sampling times are indicated above each lane. All other labels are as described in the legend to Fig. 3.
Cleavage of substrates C and D over a range of RT concentrations is shown in Fig. 5. After 15 min, both substrates C and D sustained some cuts even at the lowest RT concentration tested, and both appeared similarly sensitive to cleavage as the concentration was raised. With both substrates, however, the predominant products observed were the secondary cleavage products at 94 nucleotides. There were also some minor residual primary cleavage products visible with substrate C (Fig. 5, left panel, lanes 1-5). Importantly, the appearance of cleavage products of same sizes with substrates C and D suggests that the enzyme measures the distance from the point of hybridization, indicating that helical structure does influence cleavage position on hybrids with recessed DNA oligomers.
Figure 5: HIV-1 RT RNase H-mediated cleavage on substrate C versus D. A schematic representation of substrates C (left panel) and D (right panel) is shown at the top. The concentration of RT is indicated above each lane. The left panel comprising lanes 1-6 and the right panel comprising lanes 7-12 represent products from substrates C and D, respectively. All other labels are as described in the legend to Fig. 3.
Figure 6: Time course of RNase H cleavage on substrate C versus D. A schematic representation of substrates C (left panel) and D (right panel) is shown at the top. Sampling times are indicated above each lane. The time course RNase H reactions were performed as described under ``Experimental Procedures.'' All other labels are as described in the legend to Fig. 3.
Figure 7: HIV-1 RT RNase H-mediated cleavage on substrate E versus F. A schematic representation of substrates E (left panel) and F (right panel) is shown at the top. The left panel comprising lanes 1-6 and the right panel comprising lanes 7-12 represent products from substrates E and F, respectively. The concentration of RT is indicated above each lane. Control reactions performed with E. coli RNase H are shown in lanes 6 and 12. Products were analyzed on a 12% urea gel. All other labels are as described in the legend to Fig. 3.
Figure 8: HIV-1 RT RNase H-mediated cleavages on substrate E versus G. A schematic representation of substrates E (left panel) and G (right panel) is shown at the top. The left panel comprising lanes 1-7 and the right panel comprising lanes 8-14 represent products from substrates E and G, respectively. The concentration of RT is indicated above each lane. Control reactions performed with E. coli RNase H are shown in lanes 7 and 14. Products were separated on a 12% urea gel. All other labels are as described in the legend to Fig. 3.
Figure 9: HIV-1 RT RNase H-mediated cleavages on substrate E versus H. The autoradiogram on the right is from an analysis of reaction products on a 12% urea gel, and on the left is an analysis of the same products on an 8% gel. A schematic representation of substrates E (right panel) and H (left panel) is shown at the top. The left panel comprising lanes 1-5 and the right panel comprising lanes 6-10 represent products from substrates H and E, respectively. All reactions except lanes 4 and 9 were carried out for 15 min. Lanes 4 and 9 are reaction products after 1-h incubation of substrates H and E, respectively. Concentration of RT is indicated above each lane. Control reactions performed with E. coli RNase H are shown in lanes 5 and 10. All lanes on the left autoradiogram contain identical reaction samples to corresponding lanes in the autoradiogram on the right. All other labels are as described in the legend to Fig. 3.
An additional surprise was a class of cleavage products 69 and 61 nucleotides in length (Fig. 9, left panel, lanes 1, 2, and 4). These appear to derive from positioning of the polymerase active site at the 3` side of the loop, and then measurement of the usual 18 nucleotide distance to the RNase H site. This indicates that the beginning of the RNA/DNA hybrid structure was the determinant of the position of cleavage.
These observations present a complicated picture. The results show that cleavage directed from the point of hybridization is possible but can only be facilitated by the presence of a hybridized 5` RNA terminus. They emphasize the essential role of the 5` RNA end, but also a potentially important role for helical structure.
We have examined the role of RNA/DNA hybrid structure versus the location of the ends of the RNA and DNA strands in the determination of HIV-RT-mediated RNase H cleavage specificity. In previous work, RT positioning either at the 3` end of a DNA primer on an RNA template, or the 5` end of an RNA primer on a DNA template was found to be important for the RNA cleavage specificity (DeStefano et al., 1991a, 1994a; Gopalakrishnan et al., 1992; Ghosh et al., 1995; Schatz et al., 1990; Zhan et al., 1994). Since RT has to extend the DNA primer during minus strand DNA synthesis, it is easy to envision RT binding to the 3` end of the DNA strand, and thereby mediating RNA cleavage of the template RNA at a distance determined by the spatial separation of active sites. It is more difficult to imagine why the 5` RNA end influences the RT positioning on substrates with recessed RNA primers on DNA templates.
As a unified explanation for these observations, we proposed an alternative model whereby the structure of the hybrid influences or determines RT RNase H cleavages as proposed in Fig. 1, model C. In this model the RT binds the RNA/DNA hybrid region as close as possible to the 3` end of the DNA strand and the 5` end of the RNA strand. This, of course, is the same end of the duplex in either case. However, if the DNA is recessed on the RNA, the RT would contact the DNA 3` end, whereas if the RNA is recessed on the DNA the RT would contact the RNA 5` end. The model also suggests initial binding to the 3` DNA end as a means for the RT to load onto the substrate.
We originally anticipated that the helical structure of the substrate would have a dominant role in the positioning of the RT when it was binding over the 5` end of a recessed RNA. To our surprise, the 5` RNA end has a predominant influence on positioning the RT. This was revealed by comparison of substrates having an RNA primer annealed completely to the template DNA, with substrates having an unannealed 5` RNA (Fig. 3). For unannealed regions up to 10 nucleotides long, cleavage continued to be measured from the 5` RNA end. There was no apparent influence of the hybrid region. When the unannealed region was 27 nucleotides long, cleavage was inhibited (Fig. 8). This suggested that the RT could ``hold down'' a short unannealed region to make the measurement, but was frustrated by a longer region.
By careful primer-template design, however, we could still observe a positioning effect of the helical region. The substrate in question had an RNA primer with an internal unannealed loop. The DNA template was circular to eliminate possible influence of the 3` end of the DNA template. In this case, the patterns of cleavages indicated that some RTs positioned at the RNA 5` ends, but others bound the hybrid adjacent to the loop. This latter positioning suggests that the RT could sense the helix orientation, and tried to position as close as possible to the 5` RNA and 3` DNA ends. However, it could not pass the unannealed region and stopped just next to it. The results of this experiment imply that the RT might bind the hybrid at any point and move in the direction of the 5` RNA and 3` DNA ends. However, we do not have direct experimental evidence for a movement process. Also, the means by which the RT downstream of the loop can sense the annealed 5` RNA end is a mystery.
An additional surprising observation was that short unannealed 3` end regions of a DNA primer on an RNA template relinquished their influence on RT positioning. In this case, the helical region dominated the binding location of the RT. In fact, use of circular DNA templates indicates that the presence of a DNA end on the substrate is not a requirement for RT binding or positioning for RNase H activity. This result demonstrates that our mode C model is overly complicated. The RT does not need to first bind a DNA 3` end and then move to the hybrid region. It can bind and position itself through the use of the helix, and the 5` RNA end.
When an RNA primer with a long 5` tail was annealed to a circular DNA template, no cleavage occurred. This shows that in the absence of a DNA 3` end, an RNA end, either annealed or with a sufficiently short unannealed region, is necessary for cleavage. Although on our template with a looped primer (substrate H) we observed cleavage indicative of positioning by helix structure, cleavage was possible only in the presence of an annealed 5` RNA end. This shows a role of hybrid structure in the determination of cleavage specificity, but clearly a critical determinant is the 5` annealed RNA end.
Overall, we have found that the positions of HIV-RT RNase H cleavage of RNA/DNA hybrid structures present during viral replication are determined by the location of strand ends, and by the helical structure of the hybrid. Cleavage of RNA on a substrate with a recessed DNA primer on an RNA template is directed by the binding of the polymerase active site to the DNA 3` end. This is the normal binding configuration of the RT during minus strand viral synthesis. Results presented here show that in the absence of a fully annealed DNA 3` end the RT still positions on the hybrid as close as possible to the DNA 3` end. This suggests that the hybrid structure influences the RT to preferentially bind, and possibly move to, the correct position for minus strand synthesis and accompanying RNA template cleavage.
The RT is also expected to cleave short RNAs left annealed to the newly synthesized minus strand. A surprising finding was that the 5` ends of short RNAs annealed to DNA are a major determinant of the positioning of the RT for such cleavage. We also detected an influence of the helical structure to position the RT as close as possible to the 5` ends of these RNA primers.
Possibly, in both cases, the role of the helix binding specificity is to direct a movement of bound RT to the appropriate strand end for catalysis. The reason for the high specificity of the RT to measure cleavages from 5` RNA end remains elusive. It may simply have evolved to be the means by which short RNA primers are recognized for binding and cleavage.