The Sequential Mechanism of HIV Reverse Transcriptase RNase H*

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978–11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.

Several reports have examined the spatial arrangement of the polymerase and RNase H active sites of HIV-1 RT (3, 6, 7, 30 -34). Based on crystallographic, biochemical, and cross-linking data, RT positions the polymerase active site to the 3Јhydroxyl of the replicating DNA primer and places the RNase H active site18 nt upstream on the RNA (30,(35)(36)(37)(38). This mode of binding allows RT to catalyze DNA synthesis ahead of the RNase H site, which creates the RNA/DNA substrate for degradation. The cleavage of this RNA by the synthesizing RT is referred to as the polymerase-dependent mode of RNase H cleavage. This mode of cleavage by HIV-1 RT degrades the majority of the RNA genome.
Several studies have examined the coordination of polymerization and RNase H activities (30,(35)(36)(37)(38)(39)(40). Kati et al. (40) compared the rate of dNTP addition to that of RNase H degradation. This study showed that the nucleotide addition rate was 7-10 times faster than the RNase H degradation rate. In addition, DeStefano et al. (14,35,38) showed that the polymerizing RT did not remove all of the RNA during minus strand DNA synthesis. Both studies demonstrated that the polymerase and RNase H activities are not strictly coupled and that the RT leaves behind RNA fragments. Because a virus contains 50 -100 copies of RT and two copies of the RNA genome, many RTs are available to bind and degrade these residual RNAs. We and others have shown that RT will position independent of the 3Ј terminus of the DNA primer to degrade these fragments (12,18,(41)(42)(43)(44)(45)(46). This is referred to as the polymerase-independent mode of cleavage. We have shown RT to align the polymerase active site near the 5Ј-end of an RNA fragment recessed on a DNA template. This places the RNase H active site 18 nt downstream from this end, to the site where RT makes a primary cut. This mode of binding is referred to as 5Ј-directed binding.
Previously, we examined the process by which the RT identifies and binds to the 5Ј-end of the RNA (43). We considered that RT might bind the 3Ј-end of the DNA template and then scan back until it finds an RNA fragment. However, RT bound to the 5Ј-end of an RNA segment annealed to a circular DNA template. This result showed that the 3Ј-end of the DNA template does not participate in the 5Ј-end directed binding. We also determined the binding specificity of RT for the 5Ј-end, by use of an RNA with a 5Ј unannealed tail of 10 nucleotides. The RT forced the tail into a configuration that simulated annealing and still cleaved 18 nt from the 5Ј-end. These results show that RT binds directly to the 5Ј-ends of an RNA with high specificity.
Several studies have questioned whether degradation or displacement is the most common way that RT clears a template of residual RNA downstream of a growing primer (11,47). We examined polymerization on a DNA template with an annealed downstream RNA oligomer (47). The RT was found to have a higher affinity for the RNA fragment versus the DNA primer. In enzyme excess, RT cleaved away the RNA before it interfered with primer elongation. This result suggests that RNA fragments are effectively degraded before plus strand synthesis. Also Kelleher and Champoux (11) showed that the wild type murine leukemia virus RT supports a greater amount of synthesis on a DNA template with an annealed downstream RNA than the corresponding RNase H minus RT. This indicates the importance of RNase H-directed cleavage of RNA to clear the template for synthesis. Overall, both studies further support that RT preferentially removes residual RNAs by the polymerase-independent mode of cleavage.
Recently, we clarified the overall cleavage mechanism for the removal of RNA fragments (48). We examined the specificity and the rate of each cut to reveal the order of cleavage events (see Fig. 1B). First RT positions to the 5Ј-end of the RNA to make the primary cut. Next the enzyme can slide forward toward the 5Ј-end to make the 8-nt secondary product or backward to the 3Ј-end of the RNA, creating a 5-nt product. This latter cut occurs by alignment of the carboxyl region of the RT with the 3Ј-end of the annealed RNA. The RNase H active site is then placed 5 nt in from the 3Ј-end to make the cut (see Fig.  1B). Lastly, RT aligns to newly generated 5Ј-ends from the primary and secondary cuts to create the next 18-nt primary products. Because this is an ordered mechanism, RT may have to complete an earlier cleavage before a later cleavage can take place. In this way a particular cleavage would depend on the ones occurring before it. Another possibility is that the order of cuts is based on their relative rates, i.e. that each cut is an independent event. In this study we inhibit each cut and examine how this affects the next cuts of the ordered mechanism.

EXPERIMENTAL PROCEDURES
Materials-The DNA template was purchased from Integrated DNA Technologies, Inc. The chimeric 41-nt RNA with the 2Ј-O-methyl group at positions 12-22 and the 18-nt 2Ј-O-methyl RNA were purchased from Dharmacon Research, Inc. The 18-nt RNA was purchased from Oligos Etc. The 41-nt RNA oligomer was prepared from the Ambion T7-ME-GAshortscript kit. All RNAs were quantified by a Ribogreen assay supplied by Molecular Probes. Purified HIV reverse transcriptase (40,000 units/mg) was generously provided by Genetics Institute (Cambridge, MA). AccI, Klenow fragment, and T4 kinase were purchased from Roche Molecular Biochemicals. T4 DNA ligase was from New England Biolabs. All reactions were performed using conditions specified by the manufacturers.
Transcription in Vitro-The pBSM13ϩ plasmid was linearized by AccI and added to a transcription reaction in vitro (Ambion T7-ME-GAshortscript kit) creating the 41-nt RNA transcripts.
5Ј-End Labeling of the RNA-The 41-nt, chimeric 41-nt with the 2Ј-O-methyl groups, the 18-nt, and the 18-nt 2Ј-O-methyl RNAs were labeled at the 5Ј-end using 6000 Ci (222 TBq)/mmol [␥-32 P]ATP and T4 kinase. Excess radionucleotides were removed by Tris RNase-free P30 Micro Bio-Spin columns from Bio-Rad. The RNAs were polyacrylamide gel electrophoresis purified and eluted overnight with 500 l of a buffer containing 0.1% SDS, 1 mM EDTA, and 0.5 M ammonium acetate. All RNAs were ethanol precipitated and resuspended in 10 mM Tris-HCl, 1 mM EDTA buffer (pH 8.0).
3Ј-End Labeling of the RNA-The 41-nt and chimeric 41-nt RNAs were 3Ј-end labeled by the addition of one [␣-32 P]dATP using the Klenow fragment polymerase. Both RNAs were purified as described above.
Site-specific Internal Labeling-The 41-nt and chimeric 41-nt RNAs were ligated to either the 5Ј-end labeled 18-nt or 18-nt 2Ј-O-methyl RNAs by T4 DNA ligase. The RNAs were annealed to a DNA template such that the 18-nt RNA was adjacent to the 3Ј-end hydroxyl of the 41-nt RNA. The reactions were incubated at 15°C for 15 h and purified as described above.
Hybridization-The 41-nt RNAs were both annealed to a 77-nt DNA template with a ratio of 1 RNA to 3 DNAs. The 41-nt, chimeric 41-nt, 18-nt, and 2Ј-O-methyl 18-nt RNAs were annealed to an 84-nt DNA template with a ratio of 1:5:2 of 41-nt RNA to 18-nt RNA to DNA template. The annealing reaction was performed in 50 mM Tris-HCl (pH 8.0), 80 mM KCl, and 1 mM dithiothreitol. Components were mixed, heated to 95°C, and slowly cooled to room temperature.
RNase H Assays-Reactions were performed the same as described by Palaniappan et al. (43). Briefly, final reactions contained 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 1.0 mM EDTA, 34 mM KCl, 6 mM MgCl 2 , 2 nM substrate, and 4 nM HIV-1 RT. The reaction mixture without the 6 mM MgCl 2 was incubated for 2 min at 37°C. The reaction was started by the addition of the MgCl 2 and terminated with a 2ϫ termination dye consisting of 10 mM EDTA (pH 8.0), 90% formamide (v/v), and 0.1% each of xylene cyanole and bromphenol blue. Samples were subjected to 10% denaturing polyacrylamide gel electrophoresis and analyzed by a Molecular Dynamics PhosphorImager using Imagequant (version 1.2). An RNA ladder created by base hydrolysis was employed to determine the lengths of RNA products.

RESULTS
The removal of RNA fragments created during minus strand synthesis is essential for minus strand transfer and plus strand synthesis. Free RTs bind and cleave these fragments in a specific ordered mechanism (Fig. 1B). Cleavages occur in following order: primary, then secondary or 5-nt, and then second primary. In this study we examined the nature of the sequential mechanism. We determined whether each cut is dependent or independent of the previous cut. We inhibited each cut position to reveal the significance of that cut for the overall mechanism.
Determining the Importance of the Primary Cut on the Formation of the Secondary Cut-In a previous paper, we showed by quench flow analysis that the secondary cut occurs after the primary cut. Because the primary cut is first, we considered that the execution of this cut might be essential to allow the secondary cut, by releasing the enzyme from the primary position so that it can slide or rebind to the secondary site. To test this hypothesis, we created a 5Ј-end labeled chimeric 41-nt RNA that contained 2Ј-O-methyl groups within and around the primary cut site (Fig. 1A, Substrate 2). Cleavage of this substrate was compared with a 5Ј-end labeled 41-nt RNA with the same sequence but without chemical modification (Fig. 1A, Substrate 1). Each 41-nt RNA was annealed to the 77-nt DNA template. Fig. 2 shows a time course comparing the formation of the secondary cut products for both substrates. Fig. 2A shows the experiment with the 41-nt RNA. Two products appear over time: the 18-nt product, which is from the primary cut, and the 8-nt product, which is from the secondary cut. The primary product accumulates more rapidly in the initial time points and then decreases as the secondary cut is made. With the chemically modified 41-nt RNA substrate, the primary cut is not allowed (Fig. 2B). Remarkably, this complete inhibition has no effect on the apparent rate of the secondary cut. This result shows that the secondary cut occurs independently of the primary cut. The primary cut must occur first because its rate is intrinsically faster and not because this cut is required to release RT for the secondary cut. This result does not exclude the possibility that RT might require binding to the primary cut position to slide forward toward the 5Ј-end of the RNA to make the secondary cut.
Determining the Importance of the Primary Cut on the Formation of the 5-nt Product-Previously, we showed that RT generates a 5-nt product, apparently measured from the 3Ј-end of 41-and 50-nt RNAs. On the 41-nt substrate, this cut might also have been produced by RT aligning to the new 5Ј-end created by the first 18-nt primary cut. In that case it would functionally be the next primary cut. If this 5-nt product results from a next primary cut, then inhibiting the first primary cut would inhibit formation of this product. The same substrates were used as in Fig. 2 except that the RNAs were 3Ј-end labeled. When the 41-nt RNA was examined, two products appeared over time, 23 and 5 nt in length, corresponding to the primary and next primary or 5-nt cuts (Fig. 3A). The primary cut product rapidly accumulated in the early time points and then decreased as the 5-nt product increased. Fig. 3B shows a time course of cleavage of the 3Ј-end labeled 41-nt RNA that contained 2Ј-O-methyl groups within and around the primary cut site. Two products were generated over time, 33 and 5 nt in length, corresponding to the secondary and 5-nt cuts. The 23-nt primary product was completely absent. This result shows that the 5-nt product did not require the primary cut. It strongly suggests that the this cut is not a second primary cut, because it does not require the 5Ј-end generated by the primary cut. Most likely this cleavage has been generated by alignment of the RT to the 3Ј-end of the RNA (Fig. 1B). This result is consistent with earlier quench flow data, which showed that the 5-nt cuts occurred at the same rate for the 41-and 50-nt substrates, whereas the second primary cuts on the 50-nt RNA substrate did not occur during the time course of the quench flow reaction.
Determining the Importance of the Secondary Cut on the Formation of the 5-nt Cut and Primary Cut-To examine whether the secondary cut has an influence on the primary or the 5-nt cuts, we inhibited this cut by use of a substrate in which the 3Ј-end of the DNA template does not extend past the 5Ј-end of the RNA to create a blunted ended substrate (Fig. 1A, Substrate 3). This substrate configuration apparently does not allow the RT to slide further toward the RNA 5Ј-end than the original primary cut binding site. Fig. 4A shows the results obtained with the 5Ј-end labeled 41-nt RNA annealed to the 50-nt DNA template. The secondary cut was very effectively inhibited during the first 4 min of the reaction. A 15-nt product does accumulate over time, which presumably corresponds to the farthest position RT can move to before falling off the blunt end of the template. The 18-nt primary product appeared normally, clearly not dependent on the secondary cut. This is expected because the primary cut generally precedes the sec-ondary cut. When this same substrate was 3Ј-end labeled, the primary and 5-nt cuts both could be seen to occur independently of the secondary cut (Fig. 4B). To confirm that the 5-nt product appears independently of both the primary and secondary cuts, we used a 3Ј-end labeled 2Ј-O-methyl chimeric 41-nt RNA annealed to the 50-nt DNA substrate to create a blunt end (Fig. 1A, Substrate 4). This substrate does not sustain either primary or secondary cuts, as is evident from the lack of 33-and 23-nt products (Fig. 4C). However, the 5-nt product still occurred over time with this substrate, independent of the other cuts.
Determining the Importance of the Primary and Secondary Cuts for the Formation of the Second Primary Cut-By definition, second primary cuts require the newly generated 5Ј-ends of the first primary or the secondary cuts. Inhibition of these earlier required cuts should prevent the second primary cuts. To examine this hypothesis, we used an RNA substrate sufficiently long such that second primary products would be easily identified by their distinctive sizes. We created a 59-nt RNA substrate that was designed to inhibit the primary cut, the secondary cut, or both (Fig. 1A, . We internally labeled the RNA at position 42 from the 5Ј-end. The location of the label is approximately 5 residues 3Ј of the cleavage expected to make the second primary products, allowing us to follow the second primary cut over time. To create this internally labeled substrate, we ligated a 5Ј-end labeled 18-nt RNA to the 3Ј-hydroxyl of the either the 41-nt or 2Ј-O-methyl 41-nt RNA. These two internally labeled RNAs were annealed to either an 85-nt or a 65-nt DNA template. The latter DNA template creates a flush end with the RNA, which inhibits secondary cuts. Fig. 5B shows the image of the time course using the internally labeled RNA without a modification. The most predominant product within the first 30 s of the reaction is 40 nt in length and corresponds to the first primary cut (Fig.   FIG. 1. A, the sequence of eleven substrates used in this study. The top strand is the sequence of the RNA for each substrate and is shown in the 5Ј-3Ј direction, left to right. The sequence that is underlined and in bold type represents the 2Ј-O-methyl modified residues. The bottom strand is the DNA template and is shown in the 3Ј-5Ј direction, left to right. B, the stepwise mechanism of the RNase H cleavages by HIV-1 RT on a recessed RNA fragment. Patterned lines represent DNA, whereas the solid lines represent RNA. The rectangle represents RT with the indents corresponding to the polymerase (P) and the RNase H (H) active sites. The numbered arrows indicate the length of cleavage products. The schematic shows a linear order of cleavage events: primary, then secondary and 5-nt cut, and then primary cut. 5A). Products around 35 nt in length possibly correspond to the 5-nt cut of the 40-nt products. Minor products around 24 -30 nt with predominant products of 26 and 29 nt in length possibly correspond to a second primary cut aligning from the 5Ј-end created by the secondary cut. Products ranging from 19 to 23 nt with a predominant product at 20 nt may correspond to the second primary cut aligning from the 5Ј-end created by the first primary cut. The 18-nt product may result from RT cleaving a 5-nt cut from the 3Ј-end of the 20 -23-nt second primary product. The 11-13-nt products may occur by the 5-nt cut cleaving the 3Ј-end of the 18-nt products. Also 18-nt products are cleaved by the secondary cut creating 6 -9-nt products. Clearly this is a very complex pattern.
To examine whether the second primary cut is dependent on the first primary cut, we inhibited this first cut by creating a 59-nt RNA with 2Ј-O-methyl groups within and around the primary cut site (Fig. 1A, Substrate 6). We expected that formation of the products ranging from 20 to 23 nt in length would be affected by the inhibition of the primary cut if these products were truly the result second primary cuts. Fig. 5C shows the results of the time course with this internally labeled chimeric 59-nt RNA. Because the 40-nt primary product is completely inhibited in this experiment, a product of 52 nt appears as a result of secondary cleavage. This product was not observed in the previous 59 nt RNA reactions because the primary cut cleaved the RNA substrate into a shorter product before the secondary cut appeared (Fig. 5B). Interestingly, the 20 -23-nt products still occurred even though the first primary cut was inhibited. This result shows that these products are independent of RT alignment with the newly generated 5Ј-end of the primary cut. Furthermore this demonstrates that these prod-ucts are not second primary cuts but may result from RT binding and cleaving internally on this RNA/DNA hybrid. To further examine this concept, we inhibited the secondary cuts by using substrate 7, to determine whether this would affect the 24 -30-nt second primary products (Fig. 5D). These products would be affected by the secondary cut if RT aligns to the new 5Ј-end created by this cut to make the second primary product. We used the internally labeled 59-nt RNA annealed to the 65-nt DNA templates to create a blunt ended substrate. We found that the secondary cut was not necessary for the 24 -30-nt products, demonstrating that these products are made independently of the secondary cut and result from RT binding and cleaving internally on the RNA/DNA hybrid. That is, the positions of these cuts are not determined by an RT-directed measurement from a terminal nucleotide. Thus, these cleavage events are termed the internal cuts.
Lastly we inhibited both the primary and secondary cuts. We used the internally labeled 59-nt RNA with the 2-O-methyl group with and around the primary site annealed to the 65-nt DNA template to create a blunt end to inhibit these two cleavage events (Fig. 1A, Substrate 8). We found that the minor products of 24 -25 nt and those 30 nt in length were inhibited (Fig. 5E). The 26-and 29-nt products still occurred but less accumulated over time, whereas more of the 22-23-nt products accumulated over time. We do not believe that the inhibition of 24 -25-and 30-nt products or the lower accumulation of products 26-and 29-nt-long were dependent on the secondary or primary cuts. Most likely the inhibition of these products is due to a different mechanism. Because this assay was performed with enzyme excess, potentially an RT would remain bound to the 5Ј-end of the RNA, since the primary and secondary cut were not made, whereas another RT binds internally. The enzyme at the 5-end of the RNA would sterically hinder the other bound RT from moving to or binding at positions to make the 24 -30-nt products. Because minor products of 26 and 29 nt in length did occur, this represents the portion of RNA not bound at the 5Ј-end by RT. The 22-23-nt products accumulated efficiently presumably because the RT bound to the 5Ј-end did not sterically interfere with these further away cleavage sites. A shows the experiment with substrate 1. The 18-and 8-nt products correspond to the primary and secondary cuts. B shows the experiment with substrate 2, which inhibits the primary cut. The 18-nt primary cut is completely inhibited, and the 8-nt secondary product still occurs. Also minor products ranging from 25 to 37 nt in length appear over time.

FIG. 3. Examining the effect that inhibition of the primary cut has on the formation of the 5-nt cut. A and B
show the time course with the 3Ј-end labeled 41-nt and modified 41-nt RNA substrates, respectively. The substrates are represented above each panel, and the same symbols were used as in Fig. 2. A shows the experiment using 3Ј-end labeled substrate 1. Products 22-23 and 5-7 nt long occur and correspond to the primary and 5-nt cuts. B shows the experiment using the 3Ј-end labeled substrate 2, which inhibits the primary cut. The 22-23-nt products are completely inhibited, and the 5-7-nt products still occur. The 31-34-nt products correspond to the secondary cut.
Determining the Importance of the 5-nt Cut Alone or in Conjunction with the Primary or Primary and Secondary Cuts on the Internal Cuts-RT might require the 5-nt cut from the 3Ј-end to release the enzyme to scan upstream and bind internally on the RNA. We determined the effect of the 5-nt cut on the formation of the internal products by using an internally labeled 59-nt RNA with the 3Ј-most 18-nt modified with 2Ј-Omethyl groups to inhibit it (Fig. 1A, Substrate 9). A time course was performed with this substrate to examine the internal cut pattern (Fig. 6A). A product 40 nt in length appeared representing the primary cut. If the 5-nt cut were made in this experiment, products 35 and18 nt in length would be observed. Products 20 -30 nt in length were not affected by the inhibition of the 5-nt cut. Over time the majority of the RNA was cleaved into 20-and 23-nt products (Fig. 6A). The 18-nt product did not occur because this cut is the result of a 5-nt from cut the 3Ј-end of the 20 -23-nt products. The 23-nt product accumulated to a greater extent in the absence of the 5-nt cuts. Also the products 6 -13 nt in length were inhibited because they were within the 2-O-methyl region. Additionally, we tested whether the primary and 5-nt cuts affect the formation of the internal cuts. Possibly, RT binds to either end and makes a cut that releases the enzyme to move internally. Inhibiting both cuts from either end of the RNA might have prevented internal binding. To examine this premise, we used an internally labeled 59-nt RNA with 2Ј-O-methyl groups within and around the primary site and the 5-nt site (Fig. 1A, Substrate 10). A time course experiment using this substrate showed two sets of products occurring over time (Fig. 6B). A 52-nt product resulted from a secondary cut of the substrate, and 19 -29-nt products arose from internal cuts. The 40-and 18-nt products of the primary and 5-nt cuts were inhibited. Also the 6 -13-nt secondary cuts of the 18-nt product were inhibited. The inhibition of these cuts had no effect on the formation of the internal products, showing that these cuts are independent events. Last we inhibited secondary, primary, and 5-nt cuts to examine the effect on the internal cuts. We used the same RNA in the previous experiment except this modified RNA was annealed to the 65-nt DNA template (Fig. 1A, Substrate 11). Inhibition of every cut had no effect on the formation of the internal cuts, again showing that these cuts do not depend on earlier cuts. Less of the 29-nt product accumulated over time, suggesting that an RT was bound to the 5Ј-end of the RNA causing steric hindrance of an internally bound RT. Also more of the 20 -23-nt products accu-mulated over time, suggesting that these sites are not affected by an RT bound to the 5Ј-end of the RNA.
The Overall Model for the Polymerase-independent Mode of RNase H Cleavage by HIV-1 RT-In our previous paper, we showed that the polymerase-independent mode of cleavage progresses in a stepwise order of events. Based on our determination of the rates, the primary cut is first, the secondary and 5-nt cuts are next, and the second primary cut is last. In this study, we further defined the mechanism of these progressive cuts. We found that each cut is created independently of the previous cut. This result shows that the order of events of this stepwise mechanism occurs by the rate of the cut only. We also showed that the second primary cuts are not necessary because RT does not require a 5Ј-end to bind and cleave internally on the RNA/DNA template. We initially believed RT cleaved internally only by aligning the polymerase active site to the 5Ј-end of the RNA created by previous cuts to create the next primary cut. Because inhibiting the primary, secondary cuts, and 5-nt cuts had no effect on this cut, we show that RT can bind internally at random sites on the RNA fragments. Overall this result shows that the mechanism for the polymerase-independent mode is the primary cut first, the secondary and 5-nt cut next, and the internal or next primary cuts last (Fig. 7). DISCUSSION Previously, we examined the mechanism for the polymeraseindependent mode of HIV-1 RT RNase H cleavage by determining the rates and specificity of each cut. We found that these cuts occurred in a stepwise order. The primary cut was fastest, followed by secondary and 5-nt cuts, and then second primary and internal cuts. This finding suggested an ordered mechanism, in which later cleavages require the execution of earlier cleavages. Alternatively, the RT cuts RNAs in a temporal order based on rates of each individual cut. In the latter case each cut would be an independent event. In this study we inhibited cuts at each specific site to determine the effect of earlier cuts on later cuts. We found that the inhibition of a particular cleavage had no effect on subsequent cleavage events, proving that each cut is independent. Importantly, this result reveals that execution of an early cut does not release the RT from its binding site, allowing it to position for the next cut.
Our results also demonstrated that second primary cuts are not necessarily a major source of cleavage after the primary FIG. 4. Examining the effect that inhibition of the secondary cut has on the formation of the primary and 5-nt cuts. The same nomenclature was used as in Fig. 2. A shows the time course using the 5Ј-end labeled substrate 3, which inhibits the secondary cuts. The 18-nt segment is the product of the primary cut. Formation of the 8nt secondary product is inhibited. B displays the experiment using 3Ј-end labeled substrate 3. The 22-23-nt products derive from the primary cut. The 5-7-nt segments are from the 5-nt cuts. C shows the experiment with the 3Ј-end labeled substrate 4, which inhibits the primary and secondary cuts. The 5-7-nt segments are from the 5-nt cuts. The primary and secondary products are not made. and secondary cuts. The evidence for this conclusion is that generation of new 5Ј-ends by the primary or secondary cuts is not required to align the enzyme for internal cleavages. Hence we believe that cleavages downstream of the primary cut in RNA/DNA hybrid result largely from internal binding of RT and not from a binding position measured from a strand terminus. Of course, we cannot exclude the occurrence of second primary cuts as a component of the cuts in this region. Overall, this study shows that a stepwise mechanism is based solely on the rate of formation of each cut.
Because the same catalytic site on the enzyme is used to produce each cleavage, the order of cuts must depend on the binding affinity of RT for each site cleaved in the stepwise process. Because the primary cut is first, RT would have the highest affinity for this site. Our previous studies provide evidence for this interpretation. Palaniappan et al. (43) showed that RT persisted in making the primary cut even if a 10nucleotide region at the RNA 5Ј-end was unannealed, making the substrate more difficult to bind. This result emphasizes the strong specificity that the RT displays for binding in the position for the primary cut. RT apparently has less affinity for the sites of the secondary and 5-nt cleavages, causing these cuts to have similar but slower rates. We hypothesize that the relative affinities determine the movement of the RT during the cleavage process. The enzyme is anticipated to first bind the primary site and then slide in either direction on the RNA to position for the secondary or 5-nt cuts. Alternatively, RT may position to these sites directly from solution. In fact we previously examined the processivity of the cleavage mechanism (35). Results indicated that some of the RT that cleaves at the secondary site moves from the primary site, whereas the rest arrives from solution. In this study we found that inhibiting the primary cut had no affect on the formation of the secondary or 5-nt cuts. This shows that the actual primary cleavage event is not essential for the next steps in RNA removal but remains consistent with a mechanism in which RT first binds to the primary site then moves to the next cut site.
RT appears to have the lowest binding affinity for the inter- The bold vertical arrow above the substrate points to the cleavage site and labeled above it is the type of cut. The horizontal double arrows with the numbers above them represent the length of each RNA product that remains after the indicated cut is made. The light gray rectangle with indents is the RT with the polymerase active site located on the DNA template and the RNase H active site located on the RNA strand. A bold arrow indicating the cut site is located within the RNase H indent. This figure shows the cuts that occur in these experiments with the 59-nt RNA substrates and the products expected from each cut. The primary cut would create a 40-nt product. If the second primary cuts occur by aligning to the first primary and secondary cuts, then these cleavages would create the 20 -30-nt products. The 5-nt cut of the second primary products would create the 18-nt product. This segment is cut into 6 -13-nt products by secondary and 5-nt cuts. B shows the time course experiment with internally labeled substrate 5. The corresponding products of each cut are described in A. C displays the experiment using internally labeled substrate 6, which inhibits the primary cut. The band at position 52 is created by the secondary cut. The primary cut was inhibited because the 40-nt band does not appear. The remaining products are described above. The 20 -23-nt products are not affected by the inhibition of the primary cut. Because these products do not require a 5Ј-end generated by the primary cut, they must occur because RT binds internally on the RNA/DNA hybrid. D is the time course experiment using internally labeled substrate 7. The secondary cut is inhibited. All products occurred as seen in B. The secondary cut had no affect on the formation of the 24 -30-nt products. These cuts must not depend on RT aligning to the 5Ј-end generated by the secondary cut. RT must bind internally on the RNA/DNA hybrid to create these cleavages. E shows the experiment using substrate 8, which inhibits the primary and secondary cuts. Products at positions 52 and 40 nt did not occur. Internal cuts ranging from 20 to 29 nt still occurred regardless of the inhibition of primary and secondary cuts. Also the products from the 5-nt and secondary cuts occurred. nal cut sites, because these cuts are the slowest cleavages to appear in this stepwise mechanism. Apparently, alignment of the RT with a strand terminus produces higher affinity binding for the primary, secondary, and 5-nt cuts. Numerous bands are created by internal cuts, suggesting that the RT is distributing among a number of low affinity positions on the RNA/DNA hybrid.
Specific amino acid residues are presumably responsible for the different binding affinities of RT to each site on the RNA/ DNA substrates (26 -28, 44, 49, 50). In support of this idea, mutations of specific residues of RT can alter the binding affinity for a particular cut. Several studies have examined properties of mutant RTs with altered residues within the primer and template binding domains. We showed that mutations P226A and F227A of the primer grip domain lower the binding affinity of RT for the primary cut site (44). Also, mutations Y232A (28) and Y181C enhance the binding of RT to the secondary cut site. 2 Mutations in the thumb domain can prevent RT from recognizing the PPT RNA primer and can cause RT to position and cleave within this fragment (26). These results indicate that the RT has specific amino acids for binding and positioning correctly to each site. For the wild type enzyme, the specific residues that interact with the primary site apparently make stronger contacts with the substrate versus the residues that contact the secondary cut site.
Specific sequences in the substrate are likely to influence the exact position, efficiency, and distribution of cuts (39,41). The primary cut has a dominant positioning preference based on distance from the RNA 5Ј-end. As such, sequence differences have a minor effect on this cut, changing the peak distribution of products over a range from 15 to 19 nt in length. At this and other sites the distribution of cuts presumably depends on which binding position has lowest energy and how much the RT wobbles around this site. For the secondary cuts the majority of products are 7-10 nt in length, and for the 5-nt cut, products range from 4 to 7 nt in length. Again sequence has a minor role and can cause one site within that size range to be predominantly cleaved. In the case of the 41-nt RNA, the 18-nt product is predominant for the primary cut, the 8-nt product is predominant for the secondary cut, and the 5-nt product is predominant for the 5-nt cut. For internal cuts, adjacent RTs bound to terminal sequences may influence positioning of an internally bound RT. In that case different sequences would only have a minor effect on product distribution. Alternatively, because there is no strong positioning influence from the RNA terminus, the sequence might have a significant effect on the distribution of internal cuts.
Certain polymerase-independent cuts are likely to be very important for viral replication (6,7,30,51,52). In vitro studies examining minus strand transfer and RNase H activity at the 5Ј-end of the HIV genome have shown the accumulation of 15-18-nt products (6,30,52). The secondary cut on these fragments is slow, showing that RT must requires a 3Ј overhang on FIG. 6. Examining the effect that inhibition of the 5-nt cut alone or in conjunction with the primary cut alone or with the primary and secondary cuts has on the formation of the internal cuts. A displays an experiment using substrate 9, which inhibits the 5-nt cut and the other cuts within the 18-nt product. The 40-nt product is created by the primary cut and the 20 -29-nt products result from internal cuts. The 6 -18-nt products do not occur because the 5-nt cut is inhibited. B shows the time course experiment using internally labeled substrate 10, which inhibits the primary and 5-nt cuts. Products 20 -29 nt in length correspond to the internal cuts. The 40-and 6 -18-nt products from the primary cut, other cuts within the 18-nt product, and the 5-nt cuts are inhibited. C shows the experiment using internally labeled substrate 11, which inhibits the primary, secondary, and 5-nt cuts. Products of 20 -29 nt occur and correspond to the internal cuts.
FIG. 7. The stepwise mechanism of the polymerase-independent mode of RNase H activity by HIV-1 RT. The same nomenclature was used as in Fig. 1. The schematic shows the order of cleavage events: primary cuts, then secondary and 5-nt cuts, and then next primary or internal cuts. the DNA template for efficient positioning for this cut. Until the 8-nt secondary cut is made, the 3Ј-end of the DNA is not free to complete minus strand transfer. In addition a newly described compound, PD126338 appears to specifically inhibit the secondary and 5-nt cuts preventing minus strand transfer in vitro (51). This demonstrates the importance of the polymerase-independent secondary and 5-nt cleavages to viability of the virus.
Overall, our results have defined the basic elements of the 5Ј RNA directed RT RNase H cleavage mechanism. The order of cleavages starting with primary, followed by secondary and 5-nt, and lastly by internal and possibly second primary, shows the process to have a stepwise mechanism. However, each step does not depend on completion of the previous step. Instead, the order of steps is apparently determined by relative RT affinity for the cleavage sites. Why should the virus have evolved an unlinked stepwise mechanism? With such a mechanism the RT is not obligated to follow a strict order of cuts. Cutting out of order may be important for the formation of the PPT RNA primers, needed for priming plus strand DNA synthesis. During the synthesis of the minus strand of DNA, RT degrades the RNA genome and leaves behind fragments. The PPT sequence may be imbedded at any position within a fragment. The PPT sequence would then inhibit cuts at certain distances from the 5Ј-or 3Ј-ends of the RNA. However, because each cut is an independent event, the inhibition of any cut would not prevent the execution of other cuts needed for the correct processing of the PPT. This would be true for any sequence within the RNA genome that may restrict cleavage. Ultimately, this result shows that short segments of RNA are efficiently cut to products that readily dissociate from the DNA template, as is necessary before the DNA template can be efficiently copied to make double-stranded DNA during viral replication.