©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nevirapine Alters the Cleavage Specificity of Ribonuclease H of Human Immunodeficiency Virus 1 Reverse Transcriptase (*)

(Received for publication, August 25, 1994; and in revised form, December 18, 1994)

Chockalingam Palaniappan (1) Philip J. Fay (1) (2) Robert A. Bambara (1) (3)(§)

From the  (1)Departments of Biochemistry and (2)Medicine, and the (3)Cancer Center, University of Rochester, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3` terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA primer terminus allowing cleavage at more sites. The change in specificity occurred whether the primer terminus was at the end or internal on the template. Experiments with RNA primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of primers that underwent synthesis and the processive elongation of those primers. The significance of these results with respect to viral replication and recombination is discussed.


INTRODUCTION

The human immunodeficiency virus type 1 (HIV-1) (^1)is the major etiologic agent of acquired immune deficiency syndrome. The reverse transcriptase of HIV-1 has been demonstrated to be important for viral replication (Goff, 1990; Fauci, 1988). The enzyme has been shown to possess three catalytic activities that are involved in the synthesis of double-stranded proviral DNA from the single-stranded RNA genome: RNA- and DNA-directed DNA synthesis and RNase H. The native enzyme is a heterodimer composed of a 66- and a 51-kDa polypeptide. The larger subunit has been shown to possess a DNA polymerase domain and an RNase H domain. The smaller subunit lacks the RNase H domain and is a maturation product of proteolysis of the 66-kDa subunit. The native enzyme utilizes its RNA-dependent DNA polymerase activity for the formation of minus strand DNA synthesis. The RNase H digestion occurs during and after minus strong stop DNA synthesis (DeStefano et al., 1991; Gopalakrishnan et al., 1992; Furfine and Reardon, 1991; Fu and Taylor, 1992; Schatz et al., 1990; Wöhrl and Moelling, 1990). The DNA-dependent DNA polymerase activity of HIV-1 RT catalyzes the synthesis of plus strand DNA. Viral replication requires an orderly degradation of the RNA template during the formation of proviral DNA. Because of the multifunctional nature of HIV-1 RT and its absolute requirement for viral replication, the enzyme has been the subject of kinetic characterization and an attractive target for antiviral strategies. Any compound that inhibits the polymerase activity of the RT or causes an alteration of RNase H activity is likely to interfere with viral replication.

So far, two classes of such inhibitors have been developed, distinguished by different inhibitory mechanisms. The nucleoside analogs 3`-azido-3`-deoxythymidine, 2`,3`-dideoxycytidine and 2`,3`-dideoxyinosine act by chain termination and are known to inhibit competitively with respect to dNTPs. Although currently in use for treatment of patients with AIDS, emergence of resistant viral isolates to these compounds and their cellular toxicity limit the success of continued therapy (for review, see Larder(1993, 1994)). On the other hand, the second class of chemicals, the nonnucleoside inhibitors, act at a site distinct from the polymerase active site (De Clercq, 1993). Kinetic characterization of HIV-1 RT inhibition by several members of this class has been performed. These include compounds such as nevirapine (Merluzzi et al., 1990; Kopp et al., 1991), calanolide (Kashman et al., 1992), derivatives of coumarin (Taylor et al., 1994), derivatives of benzodiazepines (Debyser et al., 1991; Gopalakrishnan and Benkovic, 1994; Pauwels et al., 1990), pyridinone (Carroll et al., 1993; Goldman, et al., 1991; Olsen et al., 1994), catechin derivatives (Nakane and Ono, 1990), and psychotrine (Tan et al., 1991). Nonnucleoside inhibitors have been reported to exert low levels of cytotoxicity. They are selective for inhibition of HIV-1 RT and in general are inactive against HIV-2 RT. Analysis of the three-dimensional structure of HIV-1 RT complexed with nevirapine has revealed binding of the compound to a hydrophobic site adjacent to, but distinct from, the polymerase active site (Kohlstaedt et al., 1992). Moreover, the binding of nevirapine to two tyrosine residues at positions 181 and 188 in the conserved region has been demonstrated (Cohen et al., 1991; De Vresse et al., 1992). Interestingly a number of nonnucleoside inhibitors seem to share this region for their binding. Mutation at residues 181 and 188 has been shown to develop cross-resistance to a number of nonnucleoside inhibitors (for a review, see Larder(1993)).

Detailed studies on the mechanism of inhibition of DNA polymerase activity of RT by nevirapine have been carried out (Kopp et al., 1991; Merluzzi et al., 1990; Shih, et al., 1991; Tramontano and Cheng, 1992; Wu et al., 1991). Their results indicated that the binding of nevirapine to RT is noncompetitive with respect to template-primer and dNTPs. However, studies to date have not addressed the mechanism of inhibition during a single round of RT binding to a heteropolymeric substrate. Furthermore, the action of nevirapine on RNase H activity of RT has not been characterized in detail.

Several previous reports have suggested that when the RT is bound to a primer-template, the polymerase and RNase H active site contacts are separated by a fixed distance (DeStefano et al., 1991; Gopalakrishnan et al., 1992; Furfine and Reardon, 1991; Fu and Taylor, 1992; Schatz et al., 1990; Wöhrl and Moelling, 1990). When the RT was bound to a DNA primer recessed on a RNA template, the RT bound such that the polymerase active site contacted the 3` region of the primer terminus. Under those circumstances, the primary position of RNA cleavage was 14-20 nucleotides upstream of the primer terminus. This is slightly smaller than the 20-nucleotide separation between polymerase and RNase H active sites predicted from the crystal structure of HIV-RT (Jacobo-Molina et al., 1993; Kohlstaedt et al., 1992). RNase H activity that is coupled to binding of the DNA polymerase active site was termed DNA polymerase-dependent RNase H activity (Furfine and Reardon, 1991; Gopalakrishnan et al., 1992). Minor cleavages are also observed after the first that are the result of a processive action of the RNase H carrying out endonucleolytic cuts that appear on either side of the initial cut (DeStefano et al., 1993). An alternate mode of cleavage, polymerase-independent RNase H activity, is less structure-specific and is the prevalent means of degrading RNA-DNA hybrids with RNA segments less than 19-20 bases long (Peliska and Benkovic, 1992). Hence, an alteration of RNase H activity or specificity by any compound is likely to affect the viral replication.

We have extended the previous studies to investigate the effect of nevirapine on the kinetics of RNase H and RNA-dependent DNA polymerase activity of RT. We have employed model primer-templates of defined length and measured the action of the RT during a single round of synthesis and cleavage on the substrate. Results show that nevirapine alters the cleavage specificity of the RNase H and also stimulates cleavage activity. Features of the inhibition of polymerase activity have also been elucidated.


EXPERIMENTAL PROCEDURES

Materials

Recombinant HIV-RT, having native primary structure, was graciously provided to us by Genetics Institute (Cambridge, MA). This enzyme had a specific activity of approximately 21,000 units/mg. One unit of RT is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37 °C using poly(rA)bulletoligo(dT) as template-primer. Aliquots of HIV-RT were stored frozen at -70 °C, and a fresh aliquot was used for each experiment. T4 polynucleotide kinase and T7 RNA polymerase were obtained from U. S. Biochemical Corp. RNase T1, bovine pancreatic DNase (RNase free), dNTPs, and rNTPs were obtained from Boehringer Mannheim. All other chemicals were from Sigma. Radiolabeled ATP and rCTP were from DuPont NEN. Nevirapine (BI-RG-587) was obtained from Boehringer Ingelheim Pharmaceuticals Inc. The preparation of plasmid pBSM13+(Delta) has been previously described (DeStefano et al., 1992).

Methods

Run-off Transcript

Plasmid pBSM13+(Delta) was digested with BstNI, and 142-nucleotide-long run-off transcript was prepared using T7 RNA polymerase according to the procedure described in the Promega Protocols and Applications Guide(1991). The transcript was internally labeled using [alpha-P]CTP. The DNA template was digested at the end of the reaction with RNase-free bovine pancreatic DNase I (1 unit/mg of DNA). The sample was passed through a Sephadex G-50 spin column (Boehringer Mannheim) to remove free nucleotides. The sample was then mixed with an equal volume of 2 times gel loading buffer, heated at 90 °C for 4 min, and then subjected to electrophoresis on an 8% polyacrylamide gel containing 7 M urea. The full-length transcript was located by autoradiography, excised from gel, and then eluted using the crush and soak method (Sambrook et al., 1989).

To prepare 5`-labeled RNA transcript, run-off transcription was performed using unlabeled rNTPs. The unincorporated nucleotides were removed at the end of the reaction using a Sephadex G-50 spin column. The sample was then dephosphorylated with calf intestinal phosphatase to remove the 5`-phosphate. The enzyme was heat-inactivated and removed by phenol extraction. The dephosphorylated RNA was concentrated using ethanol precipitation and then 5`-end-labeled using [-P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase, according to the protocol described by the manufacturer. The free nucleotides were removed by spin chromatography, and the full-length transcript was gel purified as outlined above.

RNA-DNA Hybridization

Annealing of the RNA and DNA was performed in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 80 mM KCl. The components were mixed together, heated to 65 °C for 10 min, and then slow cooled to room temperature over a period of 90 min. Excess primer was removed by spin chromatography.

Quantitation of Nucleic Acids

Both 5`-end-labeled and internally labeled RNA samples as well as single-stranded DNAs were quantitated by a native gel hybridization ``shift-up'' assay procedure as described previously (DeStefano et al., 1993). In this assay, a labeled synthetic DNA oligomer (20-mer) of known concentration was allowed to hybridize with varying dilutions of RNA or DNA samples. After hybridization, the product was subjected to native gel electrophoresis. The concentration of the nucleic acid being quantitated was evaluated from the amount required to shift-up approximately 50% of labeled synthetic oligomer.

Preparation of Single-stranded Circular DNA

The single-stranded pBSM13+(Delta) was obtained by following the procedure from the Promega Applications guide(1991).

RNase H Assays

Final reaction mixture (25 µl) 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 HIV-1 RT (2 units). The enzyme was preincubated with or without nevirapine at 4 °C for 3 min. All other components except MgCl(2) were added and incubated for 5 min at 37 °C. The reaction was initiated by the addition of MgCl(2) in a volume of 3.75 µl. Reactions were carried out for 15 min at 37 °C, unless specified otherwise, and then terminated by the addition of 25 µl of 2 times gel electrophoresis buffer (90% formamide (v/v), 10 mM EDTA (pH 8.0), and 0.1% (w/v) each of xylene cyanol and bromphenol blue). Products were visualized by autoradiography following separation on urea-polyacrylamide gels.

RNase H Assays in Reactions Challenged with a Heparin Trap

The reaction conditions were the same as for the standard RNase H assays except that 8 µg of heparin trap was included along with MgCl(2) at the start of the reaction. This modification limits the RNase H activity to preformed complexes of RT and the substrate (DeStefano et al., 1991). Any dissociating RT molecule is statistically much more likely to bind to the excess trap polymer present in the reaction mixture. This effectively prevents RTs from returning to the substrate to which they were originally bound. Assays challenged with the heparin trap allowed us to measure products formed during a single binding event of the RT to the substrate. A control reaction always was included to ensure the presence of adequate trap. In this reaction, enzyme was allowed to preincubate for 5 min with trap and substrate with the rest of the components of reaction prior to the initiation by the addition of MgCl(2). This was used to establish the minimum concentration of trap necessary to eliminate synthesis in the control reaction. This level of trap was then used in the experimental reaction, since it can sequester essentially all dissociated RTs.

Assays performed in the absence of heparin trap are referred to as standard RNase H reactions in the text. Assays carried out in the presence of the heparin trap are referred to as challenged RNase H reactions.

RNA-dependent DNA Polymerase Assay

The final reaction mixture (25 µl) contained 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 1.0 mM EDTA, 80 mM KCl, 2 nM substrate, 50 µM each of dNTPs, 6 mM MgCl(2), and HIV-1 RT (2 units). The enzyme was preincubated with or without the nevirapine at 4 °C for 3 min. All other components except MgCl(2) and dNTPs were then added, with further incubation for 5 min at 37 °C. The reaction was initiated by the addition of a mixture of dNTPs and MgCl(2) in a 3.75-µl volume. Reactions were carried out for 15 min at 37 °C and terminated by the addition of 25 µl of 2 times gel electrophoresis buffer, as defined above. Products were separated by denaturing gel electrophoresis and analyzed.

Reactions with trapping polymer were performed by including heparin (8 µg) at the start of the reaction. An appropriate trap control reaction wherein the trap was included in the preincubation mixture was always carried out. As with the RNase H assays, when heparin was included in the reaction, the reaction was referred to as a challenged polymerase assay.

Preparation of RNA G-ladder and Base Hydrolysis Ladder

The RNA G-ladder and base hydrolysis ladder were prepared by employing protocols supplied with the Pharmacia RNA sequencing kit.

Substrate Sequences

The nucleotide sequence of the BstNI-digested pBSM13+(Delta) run-off transcript is 5`-GGGCGAAUUAGCUUUUGUUUAGUGAGGGUUAAUUCCGAGCUUGGCGUAAUCAUGGUCAUAGCUGUUUCCUGUGUUUCCUGUGUGAAAUUGUUGUUGUUAUCCGCUCACAAUUCCACAC-3`. The nucleotide sequences of the synthetic primers are as follows: primer 1, 5`-GTGTGGAATTGTGAGCGGAT-3`; primer 2, 5`-CTCGTATGTTGTGTGGAATTGTGAGCGGAT-3`; primer 3, 5`-GAACAAAAGCTAATTCGCCC-3`.


RESULTS

Effect of Nevirapine on the RNA-dependent DNA Polymerase Activity of HIV-1 RT

The substrate to assay DNA synthesis consisted of a heteropolymeric RNA template primed with a 5`-labeled 20-nucleotide-long DNA oligomer (template-primer A) such that the product of full-length synthesis was 108 nucleotides long (Fig. 1). The effect of nevirapine on the length of products made by a single round of synthesis by the RT was examined. This measurement required use of a challenged polymerization assay as described under ``Methods.'' In this assay, the RT is prebound to the primer-template. Synthesis is then initiated in the presence of a trapping polymer, which sequesters the RT as soon as it dissociates. Fig. 2shows results of an experiment testing the effect of increasing concentrations of the inhibitor on DNA synthesis. There is a dose-dependent effect of the inhibitor on the amount of synthesis. Titration of the inhibitor at lower concentrations does not significantly alter the profile of extension products. At higher concentrations, the average length of extension is clearly shortened. This suggests that the primary mode of action of the inhibitor at the lower concentrations is to prevent the RT from either binding the primer-template or initiating synthesis. At higher concentrations, the inhibitor also interferes with the actual process of primer elongation, decreasing the processivity of RTs that initiate synthesis. Interestingly, even at the highest inhibitor concentration, RTs that initiate synthesis proceed about 15 nucleotides before termination or dissociation.


Figure 1: Substrates used in this study. Plasmid pBSM13+(Delta) was digested with BstNI and then run-off transcripts of 142-nucleotide-long RNA were generated using T7 RNA polymerase, as described under ``Methods.'' Internally labeled or 5` labeled transcripts were hybridized with 20- or 30-nucleotide-long complementary DNAs as shown. Template-primers A, B, and C were formed by annealing with primers 1, 2, and 3, respectively. The nucleotide sequence of the template and primers are given under ``Methods.'' Template-primer D was generated by hybridizing the 142-nucleotide-long RNA transcript with circular single-stranded pBSM13+(Delta) DNA.




Figure 2: Effect of nevirapine on the RNA-dependent DNA polymerase activity of HIV-1 RT. Template-primer A was employed as the substrate. The primer was P-5`-end labeled 20-mer DNA. HIV-1 RT was preincubated with or without nevirapine at various concentrations for 3 min at 4 °C and subsequently preincubated with the substrate (2 nM) for 5 min at 37 °C. Reactions were initiated by the addition of MgCl(2), trapping polymer heparin and dNTPs as indicated in the assay procedure. All reactions were carried out with excess trapping polymer heparin (see ``Methods'') except the reaction that is shown in lane11, in which trap was omitted. Lane10 is a trap control reaction to demonstrate that the presence of excess trap in the preincubation mixture completely sequesters the RT. Products of the reaction were separated on a 10% polyacrylamide, 7 M urea gel and visualized by autoradiography. Lanes1-9 show products of reactions in which the enzyme was preincubated with 800, 400, 200, 100, 50, 25, 12.5, 6.5, and 0 µM nevirapine, respectively. The presence (+) or absence(-) of the trap in the reactions is indicated above each lane.



The above experiments were performed at 80 mM KCl concentration. In order to determine the action of nevirapine on RT polymerization reaction at lower salt concentration, we performed identical reactions at 34 mM KCl (data not shown). There was no noticeable difference in the level of inhibition by nevirapine at either salt concentration, indicating that the observed results are independent of salt concentration in the range we have employed.

Effect of Nevirapine on the RNase H Activity of HIV-1 RT on Template-Primer A

A challenged assay was also employed to evaluate the products made by the RNase H activity of the RT. As described in the Introduction, the primary site of RNA cleavage is expected at a distance of 14-20 nucleotides from the 3` terminus of the primer. Using 5`-end labeled or internally labeled RNA templates, the products formed by the action of RNase H activity were analyzed by gel electrophoresis. The 5` labeled RNA template permitted us to positively identify products spanning the region from the 5`-end to the first cleavage site. The internally labeled substrate enabled us to simultaneously follow the pattern of products resulting from 5`, 3`, and internal regions. The substrate was chosen such that the smallest 5`-end product generated will be bigger than the largest 3`-end-derived products. Much smaller products result from the hybrid region, which we refer to as the internally derived products. By separation on denaturing gels, the origin of each of these labeled products can be easily identified.

In the absence of nevirapine, a 5` labeled product about 102 nucleotides long was observed (Fig. 3A, lane3). However, in the presence of nevirapine, there was an additional cleavage product about 98 nucleotides long (lanes1 and 2). Evidently, nevirapine facilitated movement of the RT in the 3` to 5` direction on the RNA, shifting the primary position of cleavage closer to the 5`-end of the RNA (compare lane3 with lanes1 and 2).


Figure 3: Effect of nevirapine cleavage of template-primer A by the HIV-RT RNase H. PanelA is an autoradiogram of an experiment in which the RNA portion of the substrate is 5`-end labeled with P. In panelB, the RNA is labeled internally. Lanes1-5 are from reactions performed in the presence the trap, and lanes6 and 7 are from reactions without trap. Nevirapine concentrations 100 µM (lane1), 50 µM (lane2), and 0 µM (lane3) were used for trap reactions. Lane4 is a substrate control reaction that lacks the enzyme; lane5 represents a trap control reaction. Lane6 has no trap and no nevirapine and contains only the RT (2 units) and the substrate. Lane7 is same as lane6 except that the RT (2 units) was preincubated with 400 µM nevirapine. G-ladder and B-ladder were prepared using 5` P-labeled template RNA by limited digestion with T1 RNase or base hydrolysis according to Pharmacia RNA sequencing protocol. The numbers in the left designate the length of the RNA (in nucleotides).



A standard reaction was also carried out in the absence of the heparin trap. In this case, the RT could interact multiple times with the primer-template. The difference in products made can be seen by comparing lanes6 and 7. It is clear that exposure of the substrate to repeated interaction with RTs ultimately leads to RNA cleavage products similar to those made by a single RT in the presence of nevirapine.

Products made during cleavage of the internally labeled RNA are shown in Fig. 3B. When nevirapine was added to challenged reactions, distinctly more short internal products were produced. Compare lane3 with lanes1 and 2 in panelB. An increase in these products is also indicative of movement of RT on the substrate. A nevirapine-induced rise in the amount of this class of products is also observed in the standard reaction (compare lanes6 and 7, panel B).

Effect of Nevirapine on the RNase H Activity of HIV-1 RT on Template-Primer B

The results with template-primer A showed that nevirapine facilitates movement of the RT toward the 5`-end of the RNA. This suggested that movement toward the 3`-end of the RNA may also be promoted. However, such movement may be restricted by the short hybrid region. To examine this possibility, a longer primer was employed. We used template-primer B, wherein the primer 2 binds the template such that its 3`-end is in the same position as the 3`-end of primer 1, but its 5`-end extends 10 nucleotides further (Fig. 1). Results of cleavage performed during a single interaction of RT with the 5` labeled substrate are shown in Fig. 4A. The presence of nevirapine results in the expected formation of additional smaller 5`-end cleavage products (compare lane3 with lanes1 and 2). Interestingly, results using internally labeled RNA (Fig. 4B) show the nevirapine-induced formation of additional 3`-derived products (compare lane3 with lanes1 and 2). This indicates that nevirapine also enhances the movement of RT in the 3` direction on the RNA template.


Figure 4: Effect of nevirapine on the cleavage of template-primer B by the HIV-RT RNase H. PanelA is an autoradiogram of an experiment in which the RNA portion of the substrate is 5`-end labeled with P. In panelB, the RNA is labeled internally. Labels are as described in the legend to Fig. 3.



As with template-primer A, nevirapine induced internally derived products can be seen (Fig. 4B, compare lane3 with lanes1 and 2). Again, these are indicative of an increase in the movement of the RT over the hybrid region. Also evident from the autoradiogram is a change in the pattern of products caused by the action of nevirapine in the reaction without trap (Fig. 4B, compare lanes6 and 7).

Overall, these results show that nevirapine alters the cleavage specificity of the RNase H of HIV-RT. The apparent requirement for the primary cleavage site to be a fixed distance from the 3`-end of the DNA primer is relaxed. This results in prominent cleavages both 5` and 3` of the original primary cleavage site.

Effect of Nevirapine on the RNase H Activity of HIV-1 RT on Template-Primer C

The substrates used to this point resemble the structure of the virus during the course of minus strand synthesis. At the completion of synthesis of the strong stop DNA and prior to the first primer strand transfer, the primer is extended to the 5`-end of the RNA template. To examine the effects of nevirapine on RNase H cleavage specificity at the end of the RNA template, template-primer C was employed (Fig. 1). A 20-nucleotide long primer (primer 3), which anneals to the 5`-most nucleotides of the RNA template was used. The products resulting from RNase H cleavage reactions performed in the presence and absence of nevirapine are presented in Fig. 5. When a 5` labeled RNA template was used in the absence of nevirapine, cleavage products 16 and 17 nucleotides long were generated (Fig. 5, lane3). This result shows that the absence of a template extension ahead of the primer does not alter the positioning of the RT. Consequently, points of primary cleavage are the expected distance from the primer terminus. When nevirapine was added, additional products 13 and 14 nucleotides long predominated (Fig. 5, lanes1 and 2). Again nevirapine enhanced the movement of the RT in the 5` direction and resulted in the additional cleavage products (Fig. 5, compare lane3 with lanes1 and 2). As with the previous substrates, smaller cleavage products were observed when the RT was allowed multiple interactions with the primer-template (compare lanes6 and 7). We also observed a slight decrease in the overall efficiency of RNase H cleavage on this substrate (template-primer C) compared with the previous substrates (template-primers A and B).


Figure 5: Effect of nevirapine on the cleavage of template-primer C by the HIV-RT RNase H. The RNA portion of the substrate is 5`-end labeled with P. Labels are described in the legend to Fig. 3.



Time Course of the Effect of Nevirapine on RNase H Activity on Template-Primer B

A time course of RNase H cleavage activity was performed in a challenged assay (Fig. 6). Products generated in the presence and absence of nevirapine are shown in panelsA and B respectively. It is evident that the nevirapine-induced change in cleavage position is essentially complete by the earliest time point of 15 s. A much less efficient secondary cleavage process, as described earlier (DeStefano et al., 1993), also occurs in the absence of nevirapine. Although the cleavage position appears to be the same as in the presence of nevirapine, the process is much slower, with product still continuing to accrue at 960 s.


Figure 6: Time course of the effect of nevirapine on RNase H activity on template-primer B in challenged assays. Reactions were performed as described for the RNase H assay procedure. PanelsA and B show experiments using 5` labeled RNA. PanelsC and D show experiments using internally labeled RNA. Reactions were conducted in the presence (50 µM) (panelsA and C) or the absence of nevirapine (panelsB and D) at varying time periods as indicated above the lanes. Aliquots of 25 µl were drawn at the indicated time periods. Reactions were terminated with loading buffer as described under ``Methods,'' and the products were separated on a urea-polyacrylamide gel.



A similar experiment with internally labeled RNA allowed examination of the formation of 3` and internal RNA products with time (Fig. 6, C and D). Again, shorter 3` products and a distinctly greater amount of internal products were observed in the presence versus the absence of nevirapine. The unique nevirapine-induced products were present at nearly maximum levels, even at the earliest time point of 7.5 s. Similar products were generated in the absence of nevirapine, but over a much longer time course (compare panelsC and D, lanes under different time intervals).

We observed that if the RT were allowed multiple interactions with the primer-template in the absence of nevirapine, the final cleavage products were similar to those observed in trap experiments in the presence of nevirapine (Fig. 4A, compare lanes1 and 6). To determine whether nevirapine has an effect on the generation of cleavage products in nonchallenged reactions, we carried out a time course of cleavage in the absence of trap. Cleavage of template-primer B was measured in the presence and absence of nevirapine (Fig. 7). In the absence of nevirapine, a primary cleavage product about 102 nucleotides long appeared as early as 15 s (panelA). The secondary cleavage product, about 98 nucleotide long, accumulated over a longer period of time, with the concomitant decrease in the primary product. In the presence of nevirapine, the secondary cleavage product is present at nearly maximum level even at the earliest time point (panelB). In the absence of nevirapine, it takes about 4 min to accrue the amount of secondary product as is present at 15 s in the presence of nevirapine. This observation clearly indicates that nevirapine influences the RNase H function of the RT in unchallenged reactions. Identical results were obtained using internally labeled RNA, which also showed much more rapid appearance of secondary 3` products and internal products in the presence versus absence of nevirapine (data not shown).


Figure 7: Time course of the effect of nevirapine on RNase H activity on template-primer B in nonchallenged assays. Reactions were performed under standard assay conditions in the absence of heparin trap. PanelsA and B show experiments using 5` labeled RNA. Reactions were conducted in the absence of (A) or the presence of (B) nevirapine (100 µM) at varying time periods as indicated above the lanes. Aliquots of 25 µl were drawn at the indicated time periods. Reactions were terminated with loading buffer as described under ``Methods,'' and the products were separated on a urea-polyacrylamide gel.



Effect of Nevirapine on the RNase H Activity of HIV-1 RT on Template-Primer D

The nevirapine-induced relaxation of cleavage specificity of the RNase H appears to result in a net increase in cleavages in each reaction. This should increase the specific activity of the RNase H. We also wanted to evaluate whether nevirapine genuinely stimulates the RNase H to perform a greater number of cleavages per time, independent of the change in specificity. To address this issue, we examined the cleavage of a long segment of RNA annealed to a circular DNA template (Fig. 1). This substrate has no DNA 3` terminus to fix the position of the RT and the RNase H cleavage site. The ends of the RNA could also influence cleavage, as discussed below. However, we attempted to minimize such effects by using a long RNA, which should have a large number of potential cleavage sites per end. Cleavage of this substrate was examined in challenged assays. In the absence of nevirapine, we observed essentially no cleavage products (Fig. 8, lane5). However, a series of products about 18 nucleotides long and smaller (compare lane5 with lanes1-4) resulted in the presence of nevirapine.


Figure 8: Effect of nevirapine on cleavage of template-primer D by the HIV-RT RNase H. Autoradiogram of an experiment in which the RNA portion of the substrate is 5`-end labeled with P. Lanes1-7 are from reactions performed in the presence the trap, and lanes8-12 are from reactions lacking trap. The nevirapine concentrations 400 µM (lane1), 200 µM (lane2), 100 µM (lane3), 50 mM (lane4), and 0 µM (lane5) were used in reactions containing trap. Lane6 shows a substrate control reaction which lacks the enzyme, lane7 shows products of a trap control reaction. HIV-1 RT (2 units) was used in reactions in lanes1-7, except in lane6 where the enzyme was omitted. HIV-1 RT 8 units (lane8), 2 units (lane9), 0.2 units (lane10), 0.02 units (lane11), 2 units (lane12) was used for reactions without trap. Lane12, the reaction was performed in the presence of 400 µM nevirapine.



Cleavage products made in reactions allowing multiple interactions of the RT with the substrate are shown in Fig. 8, lanes8-12. At sufficiently high concentration of RT, in the absence of nevirapine, substantial cleavage was observed. At lower concentration of the RT, the presence of nevirapine resulted in a large increase in cleavage activity.

Cleavage activity in standard assays using template-primer D with internally labeled RNA was quantitated by analysis of generation of acid soluble nucleotide and oligonucleotide products. Results demonstrated a 5-6-fold stimulation of acid soluble material in the presence versus absence of nevirapine (data not shown).


DISCUSSION

Nevirapine (BI-RG-587) is a potent inhibitor of reverse transcriptase activity and replication of HIV-1 (Merluzzi et al., 1990). Previous studies have indicated that this compound binds noncompetitively with respect to both dNTPs and primer-template at conserved residues tyrosine 181 and 188 of the p66 subunit of the RT in a hydrophobic pocket adjacent to polymerase active site (Cohen et al., 1991; Kohlstaedt, et al., 1992; Kopp et al., 1991; Smerdon, et al., 1994; Shih, et al., 1991; Tramontano and Cheng, 1992; Wu et al., 1991). This pocket is defined by two beta-sheets composed of amino acid residues 100-110 and 180-190. Viral resistance to nevirapine has been demonstrated to be predominantly due to a mutation of tyrosine at position 181 to cysteine. This mutation also has been demonstrated to be cross-resistant to a variety of nonnucleoside inhibitors (Richman, et al., 1991). Steady-state kinetic data have shown that the major effect of nevirapine is on V(max) rather than K(m) on the polymerization reaction (Kopp et al., 1991). It has also been previously suggested that the mechanism of inhibition by nevirapine may be due to the interference of movement of the so-called thumb domain of the RT, which might result in the suppression of translocation along the primer-template following nucleotide addition (Smerdon, et al., 1994). Alternatively, nevirapine may alter the orientation of some conserved carboxylate side chains believed to be important in polymerization reaction (Smerdon, et al., 1994).

Our results provide additional details of the effect of nevirapine on the polymerization reaction. Binding of the compound to the enzyme clearly diminishes the capacity of the enzyme to initiate synthesis at a primer template. At concentrations of nevirapine greater than 25 µM, the processivity of DNA synthesis is decreased. This means that nevirapine influences RTs that are in the process of primer elongation to dissociate prematurely. Since inhibition is noncompetitive with respect to primer-template, nevirapine molecules should freely equilibrate with the RTbulletprimer-template complex. This suggests an explanation for the observed effect on processivity. An RT without a bound nevirapine molecule could initiate synthesis and begin adding nucleotides. Sometime during the course of nucleotide addition, a nevirapine molecule could bind. Either immediately or earlier than its normal time, the RT would dissociate.

Previous studies have not addressed the action of nevirapine on the RNase H activity of HIV-1 RT on heteropolymeric substrates. However, using model substrates, Gopalakrishnan and Benkovic(1994) have demonstrated an acceleration of polymerase-independent RNase H activity by a thiobenzimidazolone derivative, a different nonnucleoside inhibitor.

We have observed that nevirapine changes the cleavage specificity of the HIV-1 RT. When the RT is bound to a DNA primer on an RNA template, the RNase H normally makes a primary cleavage 14-20 nucleotides from the 3` terminus of the DNA (DeStefano et al., 1991; Fu and Taylor, 1992; Furfine and Reardon, 1991; Gopalakrishnan et al., 1992; Wöhrl and Moelling, 1990). When the action of a single RT is measured in a challenged reaction, some secondary cleavages several nucleotides closer to the 5`-end of the RNA slowly accrue (DeStefano et al., 1993). In the presence of nevirapine, the RT rapidly moves both 3` and 5` of the primary point of cleavage and makes additional cleavages. In less than 15 s, the quantity of these secondary products approaches that of the primary product.

One possible explanation for this result is that the binding of the polymerase active site to the 3`-end of the primer terminus fixes the position of the primary cleavage by the RNase H, preventing the RNase H from cleaving at nearby sites. The binding of a nevirapine molecule may weaken the interaction of the primer terminus with the polymerase active site, allowing the polymerase to slide short distances both forward and backward on the primer-template. Based on available biochemical and structural data, two possible mechanisms by which nevirapine and other nonnucleoside inhibitors exert their action have been proposed (for a review Tantillo et al., 1994). One possibility is that nevirapine alters the so-called ``primer-grip'' region (Jacobo-Molina et al., 1993). Primer-grip is a region within the palm and thumb subdomains of the p66 subunit of RT and is implicated in the positioning of the template-primer relative to the polymerase active site. Nevirapine binds to a site adjacent to the primer-grip in the palm region of p66, presumably interfering with the geometry of structural elements responsible for active polymerization. An alternate possibility is that nevirapine interferes with the translocation of the thumb subdomain (Tantillo et al., 1994). The thumb of p66 appears to make extensive contact with the template and plays an active role in polymerization. The movement of the thumb is thought to be facilitated by a ``hinge'' region. It has been proposed that binding of nevirapine interferes with the hinge elements resulting in altered movement of the thumb (Tantillo et al., 1994). Therefore, in our experiments, alteration of thumb movement by nevirapine may allow frequent RNase H cleavages.

Another possibility is that the binding of nevirapine partially dislodges the RT, allowing it to rebind in a mode in which the positioning of the RNase H is independent of the primer-terminus. This seems less likely, since cleavage at a distance from the primary site occurs in the presence of heparin, which immediately inactivates the RT upon its dissociation from the primer-template. Furthermore, the major nevirapine-induced cleavages are 1-6 nucleotides on either side of the primary cleavage site. This suggests that the RT has restricted freedom of movement forward and backward. This observation is more consistent with a limited sliding process rather than a new terminus-independent mode of binding.

Extension of the primer to the 5`-end of the RNA is a model of the strong stop viral replication intermediate. In challenged reactions, the primary RNase H product is again formed by a cleavage made at a fixed distance from the DNA 3` terminus. The presence of nevirapine allows a relaxation of the allowable positions of cleavage. Induced cleavages appear 1-6 nucleotides 5` and 3` to the primary cleavage site. This result suggests that there is little or no sequence dependence on the alteration in cleavage specificity. There was the same forward and backward movement of the RT cleavage sites at two widely separated sites on the RNA, having different sequences in the region of cleavage and the DNA primer terminus. Furthermore, these results show that the presence of a template extension beyond the primer terminus has no significant influence on the positions of cleavage in either the absence or presence of nevirapine. However, we observed some reduction in the efficiency of cleavage in the absence of a template extension.

The products made in the presence of nevirapine, can also be made in its absence if the RT is allowed to repeatedly bind to the same primer-template. However, it is evident from the time course experiment conducted in the absence of trap that nevirapine results in much more rapid accumulation of secondary RNase H cleavage products. This observation suggests that the presence of nevirapine could alter the balance of competing processes that occur during viral replication. For instance, if nevirapine increases cleavage of the RNA template during synthesis, primer strand transfer leading to recombination could be promoted. Misincorporation has also been known to accompany strand transfer (Peliska and Benkovic, 1994). It may then be possible that such events enhanced by the compound could lead to the emergence of resistant strains.

Our results also suggest that nevirapine causes an intrinsic stimulation of the RNase H, independent of stimulation caused by the broadening of cleavage specificity. To measure RNase H activity with a minimum influence of cleavage specificity, we employed a substrate consisting of a long RNA primer on a circular DNA template. The only termini were the 3`- and 5`-ends of the primer. Our previous results showed that the 3`-ends of RNA primers do not influence the position of RNase H cleavage, but the RT appears to interact with the 5`-end such that some of the cleavages are directed by this site (DeStefano et al., 1993). However, this influence is not as dominant as that of a DNA primer terminus, allowing many cleavages that are greater than 20 nucleotides distant from the 5`-end. Even with this substrate, we have observed an approximately 5-fold stimulation of RNase H activity by nevirapine. This is likely to be a direct stimulation. Similar to our observation, Gopalakrishnan and Benkovic(1994) have reported an acceleration of RNase H activity and an inhibition of polymerase activity of HIV-1 RT by a thiobenzimidazolone derivative. This feature may be common to other nonnucleoside inhibitors as well.

Overall, our results indicate that nevirapine has a complex effect on the HIV-1 RT. It inactivates the polymerase active site and reduces processivity of RTs in the process of primer elongation. It alters the cleavage specificity of the RNase H, presumably by allowing the RT to slide over forward and backward on the primer terminus. It also appears to directly stimulate the activity of the RNase H, independent of its effects on binding specificity.


FOOTNOTES

*
This work was supported by National Insitutes of Health Grant GM 49573 and in part by core grant CA 11198 to the University of Rochester Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, P. O. Box 607, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-2764; Fax: 716-271-2683.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; dNTP, deoxynucleoside triphosphate; RT, reverse transcriptase.


ACKNOWLEDGEMENTS

We thank Drs. Peter M. Grob, Johanna Griffin, Cheng-Kon Shih, and James Crute of Boehringer Ingelheim Pharmaceuticals, Inc. for invaluable suggestions and assistance with this work. We also thank Drs. John McCoy and Jasbir Seehra of Genetics Institute for the gift of HIV-1 RT.


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