(Received for publication, August 25, 1994; and in revised form, December 18, 1994)
From the
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.
The human immunodeficiency virus type 1 (HIV-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.
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.
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.
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.
Figure 1:
Substrates used in this study. Plasmid
pBSM13+() 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+(
) 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
, 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.
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).
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.
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.
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.
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).
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 -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
rather than K
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 RTprimer-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.