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. 2010 Aug 27;285(35):26966-75.
doi: 10.1074/jbc.M110.105775. Epub 2010 Jun 8.

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-Mediated Bias Toward RNase H Cleavage During Plus-Strand Initiation

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Free PMC article

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-Mediated Bias Toward RNase H Cleavage During Plus-Strand Initiation

Mia J Biondi et al. J Biol Chem. .
Free PMC article

Abstract

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.

Figures

FIGURE 1.
FIGURE 1.
HIV-1 RT forms distinct complexes during (−)- and (+)-strand DNA synthesis. A, during (−)-strand DNA synthesis, the polymerase-dependent conformation occurs when the polymerase active site (white cylinder) is in contact with the 3′-end of the DNA primer. The RNase H active site (black arrow) is situated 18 base pairs upstream and can simultaneously cleave the RNA template. Once the enzyme has progressed along the template, DNA synthesis is not possible, and polymerase-independent RNase H activity occurs. B, synthesis of the (+)-strand DNA occurs when RT sits in a polymerase-dependent orientation. This conformation is in equilibrium with the polymerase-independent orientation at which point the RNase H active site is near the 3′-end of the primer and cleaves at the RNA·DNA junction to remove the PPT.
FIGURE 2.
FIGURE 2.
The effect of N348I on RNase H degradation during (−)-strand DNA synthesis. A, polymerase-dependent RNase H cleavage (−18, −19), intermediate products (−14, −15), and polymerase-independent RNase H cleavage (−12) were monitored on the PBS sequence 22dpol·*52r, in the presence of increasing concentrations of the next nucleotide. The corresponding sequence indicates the location of these cuts. B, quantification of WT compared with N348I RNase H cleavage is achieved by comparing all cleavage products with respect to total RNase H activity (the sum of all cuts).
FIGURE 3.
FIGURE 3.
The effect of N348I on polymerization and RNase H degradation during (+)-strand DNA synthesis. A, removal of the PPT primer (indicated as the 17r cleavage product) was examined in the presence of increasing concentrations of nucleotide. The 17r cleavage occurs at the RNA·DNA junction indicated on the sequence below. B, RNase H activity was quantified as a percentage of initial substrate.
FIGURE 4.
FIGURE 4.
RNase H degradation of the PBS substrate is enhanced by NVP and EFV. A, in the presence of increasing concentrations of NVP and EFV, polymerase-dependent (−18, −19) versus -independent cleavage (−12) of the 52r template was monitored for WT and N348I-contaning RT. B, graphical representation of subsequent RNase H cleavage seen with respect to the sum of polymerase-dependent and -independent cuts.
FIGURE 5.
FIGURE 5.
N348I counteracts the effect of NVP on the primer removal reaction. A, RNase H activity of WT and N348I was monitored in the presence of increasing concentrations of NVP and EFV. B, graphical representation of RNase H cleavage products 17r, 16r, and 15r seen in A, presented as a percentage of total RNase H product compared to the initial amount of substrate.
FIGURE 6.
FIGURE 6.
The effects of NNRTIs on the initiation of (+)-strand DNA synthesis. Incorporation of a single nucleotide (17r9d) and RNase H activity (17r, 16r, 15r) were compared between N348I and classical NNRTI resistance mutations K103N and Y181C. This equilibrium was monitored in the presence of increasing concentrations of NVP (A) and EFV (B).
FIGURE 7.
FIGURE 7.
The effect of NNRTIs and N348I on (+)-strand DNA synthesis under multiple-turnover conditions. The PPT-based sequence 17r3d*·57d was used to monitor full-length DNA synthesis and the corresponding cleavage of each product. After the incorporation of 12 nucleotides, RT dissociates, changes orientations, and cleaves at the RNA·DNA junction (12d product). As time increases, the final product accumulates at 60 min in conjunction with the disappearance of the 12d cleavage. Two alternate possibilities also exist. RT can process though the 17r12d pausing site, forming the full-length DNA product, or can cleave the initial substrate 17r3d, resulting in the 3d product. This process was monitored with both WT and N348I-containing RT in the presence of 500 nm NVP (A) and 10 nm EFV (B). To demonstrate that resistance is RNase H-dependent, formation of the final product was quantified with respect to initial substrate and total RNase H cleavage. The graphical representation of RNase H activity in the absence and presence of inhibitor is expressed as previously described. C and D, the oligonucleotide sequence for the chimeric hybrid duplex. The arrows indicate cleavage products 12d, after the incorporation of nine additional nucleotides (italics), which results in 17r12d product (C) and 3d, the cleavage of the initial primer (D). Both cuts are the result of the RNase H cut at the RNA·DNA junction.
FIGURE 8.
FIGURE 8.
RNase H-independent full-length DNA synthesis. A, the RNase H active site mutant enzyme, E478Q, was used with the 3′-end-labeled PPT chimeric primer to monitor full-length synthesis in the absence of RNase H activity. The formation of each synthesis product was monitored over 30 min in the absence and presence of 500 nm NVP. B, formation of full-length products are quantified in the corresponding graph.
FIGURE 9.
FIGURE 9.
Conformational differences between RT complexes that permit polymerase-independent RNase H cleavage during (−)-strand DNA synthesis and (+)-strand initiation. The RT enzyme is schematically shown (white oval) with its polymerase active site (white cylinder) within the polymerase domain (red oval) and its RNase H active site (black arrow). The NNRTI-BP (blue diamond) is occupied by an NNRTI (yellow diamond) in the polymerase domain and is depicted to be in close proximity to the polymerase active site. During (−)-strand DNA synthesis, as subsequent cleavage occurs, RNase H cleavage becomes polymerase-independent, at which point the polymerase domain gradually loses contact with the double-stranded DNA·RNA. We suggest that at this stage, the effect of N348I within the connection domain (green oval) is dominant and that NNRTIs lose their ability to enhance RNase H cleavage. The effects of N348I are much more pronounced during the initiation of (+)-strand synthesis. When RT is in the polymerase-independent orientation, the polymerase domain is still in contact with the double-stranded RNA·DNA, and both NVP and EFV increase the removal of the PPT primer. This effect is counteracted by N348I solely in the presence of NVP because its tendency to dissociate allows the N348I mutation to dominate.

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