Mutations in Human Immunodeficiency Virus Type 1 RNase H Primer Grip Enhance 3′-Azido-3′-Deoxythymidine Resistance

Abstract

We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3′-azido-3′-deoxythymidine (AZT) resistance up to 536 times over wild-type (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is responsible for converting the single-stranded RNA viral genome into double-stranded DNA (3, 27). To accomplish this, HIV-1 RT contains two enzymatic properties: a DNA polymerase, which can incorporate nucleotides using RNA or DNA as a template, and an RNase H, which degrades the RNA template after it has been copied into DNA (9, 26). The process of reverse transcription requires that RNase H make both specific and nonspecific cleavages. Nonspecific cleavages are required to degrade the viral RNA genome, and specific cleavages are required to remove the tRNA primer, cleave the polypurine tract (PPT) to make it available for use as a plus-strand primer, and finally degrade and remove the PPT primer.

For reverse transcription to be completed accurately, the nucleic acid hybrid must be properly aligned at both the polymerase and RNase H active sites (2, 13, 22). Crystal structure studies of HIV-1 RT show the presence of several amino acids that contact the DNA primer strand and the RNA template strand near the RNase H active site (6, 7, 11, 22). These amino acids (G359, A360, H361, K390, K395, E396, T473, Q475, K476, Y501, and I505) are collectively referred to as the RNase H primer grip and lie either in the p51 or p66 subunit of RT. In vivo and in vitro studies of the RNase H primer grip indicate that these amino acid residues are important for the proper binding and positioning of the nucleic acid to RT. Several point mutations in the HIV-1 RNase H primer grip have been shown to decrease the efficiency of DNA synthesis initiation, reduce RNase H activity, alter RNase H cleavage specificity, decrease the ability of RT to excise the PPT primer, and/or reduce strand transfer efficiency (1, 15-17, 21). Furthermore, the Y586F mutation (equivalent to Y501F in HIV-1) in the RNase H primer grip for murine leukemia virus RT has been shown to be important for the proper positioning of the template-primer at the polymerase active site and fidelity of DNA synthesis (29). Overall, these results indicate that amino acids in the RNase H primer grip can affect the positioning of the nucleic acid at both the polymerase and RNase H active sites and significantly influence the process of DNA polymerization and RNA degradation.

Antiviral therapy results in the selection of drug resistance mutations that allow HIV-1 to replicate in the presence of antiviral drugs (5, 14, 23). The acquisition of thymidine analog resistance mutations (TAMs) allows the virus to increase the efficiency of nucleotide excision and continue reverse transcription, thus escaping the effects of nucleoside RT inhibitors (NRTIs) (10). Recently, we proposed another mechanism for NRTI resistance in which mutations that reduce RNase H activity increase NRTI resistance by increasing the time available for RT to undergo nucleotide excision (18, 19). We examined two RT mutants with reduced RNase H activity, D549N and H539N, and showed that these mutants exhibited increased 3′-azido-3′-deoxythymidine (AZT) resistance 12 and 185 times, respectively. Additionally, we reported that eight amino acid substitutions (E312Q, G335C/D, N348I, A360I/V, V365I, and A376S) in the connection domain of RT that were present in viral sequences isolated from treatment-experienced patients, but not treatment-naïve patients, also increased AZT resistance and decreased template switching, suggesting that the AZT resistance-associated mutations in the connection domain also reduced RNase H activity (18). Interestingly, the A360 residue is a part of the RNase H primer grip, and the A360I/V substitutions were associated with enhanced AZT resistance. These observations suggested that the AZT resistance-associated mutations in the connection domain could influence NRTI resistance and RT template switching by influencing the RNase H primer grip structure.

Since mutations in the RNase H primer grip were previously shown to decrease RNase H activity (1, 15-17, 21), and mutations in the RNase H primer grip (A360I/V) from the connection domains of NRTI-treated patients were shown to enhance AZT resistance and decrease template switching (18), we sought to determine whether other mutations in the RNase H primer grip would also increase NRTI resistance. We observed substantial increases in AZT resistance for RNase H primer grip mutants in the context of a polymerase domain containing TAMs. These results further underscore the importance of the connection and RNase H domains in conferring AZT resistance during antiviral therapy.

MATERIALS AND METHODS

Plasmids, cloning, and mutagenesis.

pHCMV-G expresses the G glycoprotein of vesicular stomatitis virus (VSV-G) (28). Construction of vector pHL[WT] was previously described (18). Briefly, pHL[WT] expresses the firefly luciferase reporter gene and all of the HIV-1 proteins except Nef and Env; the vector also contains a natural MscI site at the beginning of polymerase (at amino acids 25 to 26), a unique Eco47III site (at amino acids 288 to 289), a unique SpeI site (at amino acids 423 to 424), and a unique ClaI site (flanking integrase amino acids 4 to 5). These restriction enzyme sites were used to subclone polymerase (MscI to Eco47III), connection (Eco47III to SpeI), and/or RNase H (SpeI to ClaI site) domain combinations into pHL[WT]. Site-directed mutagenesis was carried out using the QuikChange XL site-directed mutagenesis kit (Stratagene), and the presence or absence of each mutation was verified by DNA sequencing. The details of the cloning steps are available upon request. pHL[TAM] contains mutations D67N, K70R, T215Y, and K219Q introduced into the pHL[WT] polymerase domain, and pHL[K65R] was constructed by introducing K65R into the pHL[WT] polymerase domain. RNase H primer grip mutations (G359A, A360K, H361A, K390A, K395A, E396A, T473M, Q475A, K476A, Y501A, or I505A) were added to pHL[WT], pHL[TAM], or pHL[K65R].

Antiviral drugs.

NRTI inhibitors AZT, 2′,3′-didehydro-3′-dideoxythymidine (d4T), and 2′,3′-dideoxyinosine (ddI) were obtained from Sigma-Aldrich. 2′,3′-Dideoxy-3′-thiacytidine (3TC) was obtained from Moravek Biochemicals. Non-NRTI (NNRTI) inhibitor (4S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one (efavirenz [EFV]) and NRTI inhibitors (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (abacavir [ABC]) and 9-[(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine (tenofovir [TDF]) were obtained from the NIH AIDS Research and Reference Reagent Program.

Cells, transfection, and virus production.

Human 293T cells (American Type Culture Collection) and a 293T-based cell line, GN-HIV-GFFP, were maintained at 5% CO₂ and 37°C in Dulbecco's modified Eagle's medium (CellGro) supplemented with 10% fetal calf serum (HyClone), penicillin (50 U/ml; Gibco), and streptomycin (50 μg/ml; Gibco). Hygromycin (Calbiochem) selection was performed at a final concentration of 270 μg/ml. To produce pHL[WT]-based virus containing primer grip mutations, 293T cells were plated at 5 × 10⁶ cells per 100-mm-diameter dish and transfected by calcium phosphate precipitation in the presence of pHCMV-G. Forty-eight hours later, virus was harvested, filtered through a Millex GS 0.45-μm-pore-size filter (Nalgene), concentrated 20-fold by centrifugation at 25,000 rpm for 90 min (Surespin; Sorvall), and stored at −80°C.

Single-replication-cycle drug susceptibility assay.

Drug susceptibility assays were performed as previously described (19). Briefly, fresh media containing serial dilutions of drug or no drug were added to 293T target cells plated the previous day at 4,000 cells/well in a 96-well plate. Four hours later, normalized virus was added to each well, and 48 h postinfection, luciferase activity was measured using a 96-well luminometer (LUMIstar Galaxy; BMG LABTECH). Data were plotted as the percent inhibition of luciferase activity versus the log₁₀ drug concentration, and the percent inhibition was calculated as follows: [1 − (luciferase activity in the presence of drug/luciferase activity in the absence of drug)] × 100. The drug concentration at which virus replication was inhibited by 50% (IC₅₀) was calculated using inhibition curves defined by the following four-parameter sigmoidal function: y = y₀ + a/[1 + (x/x₀)^b] (SIGMAPLOT 8.0 software).

Assay of replicative capacity.

Utilizing a single-cycle replication assay, virus containing the vector of interest was harvested from 293T cells, normalized by using p24 capsid (CA) values (HIV-1 p24 ELISA kit; Perkin-Elmer), and used to infect target 293T cells plated at 4,000 cells/well of a 96-well plate. Normalized luciferase light units measured 48 h postinfection were used to determine the replicative capacity.

RT assay.

RT assays were performed using the Quant-T-RT assay system (Amersham) according to the manufacturer's instructions. Normalized p24 CA values were assayed for RT activity for each mutant, and the resulting activities were then normalized to the WT or the TAM control. RT activity was determined using the DNA/RNA hybrid provided in the kit: 16-mer DNA primer [poly(T)] annealed to a ∼300-mer polyadenylated RNA.

RT template-switching frequency.

RT template-switching frequency was determined as previously described by using a direct repeat deletion assay (20). Briefly, target 293T cells were infected with virus produced from the GN-HIV-GFFP cell line cotransfected with VSV-G and the corresponding pHL[TAM]-based helper construct containing the RNase H primer grip mutation. Four hours later, the culture media of infected plates were replaced with fresh media and placed under conditions of hygromycin selection 24 h later. Every 3 days for 10 to 14 days, the culture medium was replaced with fresh medium containing hygromycin. Approximately 500 to 2,000 hygromycin-resistant colonies were pooled per infection, expanded, and analyzed by flow cytometry to determine the percentage of green fluorescent protein (GFP)-expressing cells (FACScan; Becton Dickinson).

Statistical analysis.

Statistically significant differences were determined using a two-sample t test (SIGMAPLOT 8.0). Linear regression analysis was performed using SIGMAPLOT 8.0 software, and the significance of the negative correlation was determined first by calculating the t statistic, t = r√[(n − 2)/1 − r²)], where n is the number of data points and r is the correlation coefficient. The P value was determined using a standard t distribution table (8).

RESULTS

Mutations in the RNase H primer grip region increase AZT resistance.

The HIV-1 RNase H primer grip, shown in Fig. 1, is composed of amino acid residues G359, A360, H361, T473, Q475, K476, Y501, and I505 from the p66 subunit of HIV-1 RT and amino acids K390, E395, and E396 from the p51 subunit (22). These amino acids form hydrogen bonds and other interactions with the primer strand and help to position the template-primer complex at the RNase H and polymerase active sites. Mutations in the RNase H primer grip were introduced into an HIV-1 vector containing either a wild-type (WT) polymerase domain (pHL[WT]; WT control) or a polymerase domain containing TAMs (D67N, K70R, T215Y, and K219Q) (pHL[TAM]; TAM control), and single-cycle drug susceptibility assays were performed to determine their effects on drug resistance. The AZT concentration needed to reduce luciferase activity to 50% (IC₅₀) was determined to be 0.05 ± 0.003 μM for the WT control (onefold) and on average 0.50 ± 0.016 μM for the TAM control (9 to 10 times over WT levels) (Fig. 2A and B).

FIG. 1.

Positions of the 11 HIV-1 RNase H primer grip amino acids in complex with the DNA primer strand. The HIV-1 p51 subunit is tan, while the thumb, connection, and RNase H domains of the p66 subunit are purple, blue, and green, respectively. The DNA primer strand is red, and the RNA template strand is white. Each RNase H primer grip amino acid is correspondingly labeled. RNase H primer grip amino acids K390, K395, and E396 are from the p51 subunit of RT. The RNase H primer grip amino acids G359, A360, H361, I505, K476, T473, Q475, and Y501 are from the p66 subunit of RT. Q475 contacts both the RNA template and the DNA primer, while K390 contacts only the RNA template. The remaining RNase H primer grip amino acids contact only the DNA primer strand. The figure was generated using RasMol using the HIV-1 RT crystal structure data reported previously by Sarafianos et al. (22).

FIG. 2.

AZT drug resistance associated with HIV-1 RNase H primer grip mutants in the context of a WT or TAM-containing polymerase domain. (A) Mutations in the RNase H primer grip were tested in the context of a WT polymerase domain, and AZT IC₅₀s were determined. Changes (n-fold) in IC₅₀s versus the WT control vector are indicated above each bar. Error bars represent standard errors from two to five experiments per mutant. Statistically significant differences (*) in IC₅₀ values were calculated (P < 0.05 by t test) versus the WT control (dotted reference line). (B) Mutations in the RNase H primer grip were tested in the context of a polymerase domain (T) containing TAMs (D67N, K70R, T215Y, and K219Q), and AZT IC₅₀s were determined. Changes (n-fold) in IC₅₀s versus the WT control vector are indicated above each bar. Error bars represent standard errors from 2 to 11 experiments per mutant. Statistically significant differences (*) in IC₅₀ values were calculated (P < 0.05 by t test) versus the TAM control (dotted reference line).

Single-alanine mutations G359A, H361A, K390A, K395A, E396A, Q475A, K476A, Y501A, and I505A were introduced individually into either pHL[WT] or pHL[TAM] for each RNase H primer grip residue. Two exceptions were the A360K and T473M substitutions; a charged lysine replaced the hydrophobic alanine at position 360 to create a drastic amino acid change, and a methionine was substituted for threonine because it was previously observed that the T473A substitution reduced viral titers to undetectable levels (16).

The effects of the RNase H primer grip amino acid substitutions on AZT resistance were tested in the context of a WT polymerase domain (Fig. 2A). With the exception of the H361A and Q475A mutations, the AZT susceptibilities observed with the RNase H primer grip substitutions were not significantly different (P > 0.05 versus WT control); only the Q475A substitution altered AZT sensitivity by more than two times. In comparison, the TAM control increased AZT resistance by 10 times over the WT control.

Next, we tested the effects of the amino acid substitutions in the RNase H primer grip region on AZT resistance in the context of a polymerase domain containing TAMs (Fig. 2B). All RNase H primer grip mutations increased AZT resistance 7 to 243 times above WT (one time). For 10 of the 11 RNase H primer grip mutants, this increase in AZT resistance was significantly higher than the nine-times increase observed in parallel experiments for the TAM control (P < 0.05 versus TAM control). H361A resulted in an increase of seven times in AZT resistance, which was not significantly different from the TAM control (nine times).

The effect of RNase H primer grip mutations on replicative capacity.

The effects of the RNase H primer grip amino acid substitutions on the viral replicative capacity were determined by comparing the firefly luciferase expression levels after infection of target cells with equivalent amounts of virus, as determined by the quantities of p24 CA in the virus preparations (Fig. 3A). Overall, the RNase H primer grip amino acid substitutions had greatly variable effects on the viral replicative capacity. In the background of a WT polymerase domain (Fig. 3A), all RNase H primer grip mutants, except G359A and K476A, significantly decreased the replicative capacity of the virus compared to the WT control (P < 0.05). K476A slightly increased the replicative capacity. H361A, T473M, Q475A, Y501A, and I505A substitutions substantially reduced the viral replicative capacity to less than 10% of the capacity observed for the WT control, while G359A, A360K, K390A, K395A, and E396A substitutions decreased the viral replicative capacity less than two to three times compared to that of the WT. Since the TAM control significantly reduced the viral replicative capacity by approximately 32% (P < 0.05 versus WT control), we also determined whether the primer grip mutations rescued viral replicative capacity in the context of TAMs (Fig. 3B). The effects of G359A, A360K, K390A, K395A, and Q475A substitutions on the viral replicative capacity in the context of a polymerase domain containing TAMs were statistically similar to those observed for the RNase H primer grip mutants in the context of a WT polymerase domain. H361A, E396A, K476A, and Y501A had less severe reductions in the viral replicative capacity in the context of TAMs relative to their effects in the context of a WT polymerase domain (P < 0.05). On the other hand, T473M and I505A had more severe reductions in viral replicative capacity in the context of TAMs relative to their effects in the context of the WT polymerase domains (P < 0.05).

FIG. 3.

Effects of HIV-1 RNase H primer grip mutants on replicative capacity and RT activity. (A) Mutations in the RNase H primer grip were tested in the context of a WT polymerase domain. Target 293T cells were infected with normalized p24 CA concentrations for each RNase H primer grip mutant, and changes (n-fold) in replicative capacity (black bars) and RT activity (gray bars) were normalized to the WT control. Error bars represent the standard errors from two to four independent experiments. All RNase H primer grip mutants except G359A (WT) were statistically different (P < 0.05 by t test) in replicative capacity versus the WT control (dotted line). All primer grip mutants except G359A (WT) and Y501A (WT) were statistically significant in their RT activities (P < 0.05 by t test) versus the WT control. (B) Mutations in the RNase H primer grip were tested in the context of a TAM-containing polymerase domain (T). Target 293T cells were infected with normalized p24 CA concentrations for each RNase H primer grip mutant, and changes (n-fold) in replicative capacity (black bars) and RT activity (gray bars) were normalized to the WT control. Mutants H361A (T), K390A (T), K395 (T), T473M (T), Q475A (T), K476A (T), Y501A (T), and I505A (T) were statistically significant in their replicative capacities (P < 0.05 by t test) versus the TAM control. Mutants H361A (T), T473M (T), and Y501A (T) were statistically different in their RT activities (P < 0.05 by t test) compared to the TAM control. Error bars represent the standard errors from two to four independent experiments. (C) Regression analysis. Increases (n-fold) in AZT resistance were plotted versus replicative capacity for RNase H primer grip mutants containing a TAM polymerase domain.

Further analysis was completed to determine if the changes in replicative capacities were due to differences in RT activity or some other defect in the viral life cycle. Normalized p24 CA values for each RNase H primer grip mutant in the context of a WT or TAM-containing polymerase domain were assayed for RT activity (Fig. 3). For RNase H primer grip mutants in the context of a WT polymerase domain, G359A, A360K, H361A, K390A, K395A, T473M, and K476A all had RT activity levels similar to their replicative capacities (Fig. 3A). E396A, Q475A, Y501A, and I505A mutants had RT activities higher than their replicative capacities. For RNase H primer grip mutants in the context of a TAM-containing polymerase domain, G359A, A360K, K390A, K395A, E396A, K476A, and Y501A all had RT activity levels similar to their replicative capacities (Fig. 3B). T473M, Q475A, and I505A mutants had RT activities higher than their replicative capacities. Mutant H361A had an RT activity level that was lower than its replicative capacity. Overall, these data suggest that the observed replicative capacities were influenced by the RT activity levels. Interestingly, some mutants (for example, T473M, Q475A, and I505A in the context of TAMs) had higher levels of RT activity than expected compared to their replicative capacity. Since the RT assay measures polymerase activity only on a poly(A) template, it is possible that these mutants have further defects that affect specific steps in viral DNA synthesis. For example, the mutations may reduce the efficiency of minus-strand synthesis initiation or polypurine tract cleavages, which are not detectable in our RT assay.

To determine if the increased AZT resistance was correlated with the replicative capacity of the RNase H primer grip mutants in the presence of TAMs, linear regression analysis was performed (Fig. 3C). An R² value of 0.39 was obtained, suggesting that the increases in AZT resistance observed with mutations in the RNase H primer grip failed to show a correlation with a reduction in viral replicative capacity (P > 0.1 by t test). Although there appeared to be a relationship between a decrease in replicative capacity and an increase in AZT resistance, the regression analysis did not reach a level of significance. Further regression analyses also failed to show a correlation for RNase H primer grip mutants in the presence of TAMs between RT activity and replicative capacity or AZT resistance (R² = 0.013 and 0.0054, respectively).

Mutations in the RNase H primer grip decrease RT template switching in the context of a polymerase domain containing TAMs.

We previously proposed that reducing RNase H activity increases AZT resistance by increasing the time available for the excision of a chain-terminating nucleotide from a blocked primer (18, 19). Furthermore, it has been shown that reducing RNase H activity also decreases RT template switching, while reducing the rate of DNA polymerization increases RT template switching (4, 12, 20, 25). In an effort to understand the mechanism by which mutations in the RNase H primer grip enhance AZT resistance, we determined their effects on RT template switching by using an established direct repeat deletion assay (20) (Fig. 4A). Briefly, the assay used a cell line (GN-HIV-GFFP) that contains a provirus derived from an HIV-1 vector that expresses a hygromycin resistance gene from an internal ribosomal entry site. The vector also contains two overlapping fragments derived from the GFP gene, GF and FP, which cannot express a functional GFP protein. The “F” portion of the overlapping fragments constitutes a homologous directly repeated sequence; a homologous template switch by RT during reverse transcription functionally regenerates the GFP gene. Virus harvested from the GN-HIV-GFFP cell line cotransfected with an HIV-1 Gag-Pol expression vector and a VSV-G envelope vector was used to infect 293T target cells, which were subsequently selected for the expression of hygromycin. The resulting colonies were pooled and analyzed by fluorescence-activated cell sorter (FACS) analysis to determine the frequency of cells that express a functional GFP. The proportion of GFP-positive cells provides a measure of the frequency with which a functional GFP is reconstituted through an RT template switch during a single cycle of retroviral replication.

FIG. 4.

Effect of HIV-1 RNase H primer grip mutants in the context of a TAM polymerase domain on the RT template-switching frequency. (A) Single-cycle direct repeat deletion assay to determine the percentage of template switching in vivo. Proviruses containing a direct repeat of the GFP gene were mobilized by cotransfecting the cell line GN-HIV-GFFP with VSV-G envelope expression plasmid and the RNase H primer grip mutant vector. Virus from the transfected cells was used to infect 293T target cells, and these cells were placed under conditions of hygromycin selection. Hygromycin-resistant (hygro^R) colonies were then analyzed by FACS to determine the frequency of cells that underwent a homologous template switch during reverse transcription and reconstituted a functional GFP gene. IRES, internal ribosomal entry site; hygro, hygromycin gene; Ψ, RNA packaging sequence; arrows, directly repeated sequences in GFP. (B) The RT template-switching frequency for RNase H primer grip mutants in the context of a TAM (T) polymerase domain. Each bar represents the RT template-switching frequency (percent GFP reconstitution) as it was measured in the single-cycle direct repeat deletion assay (mean of two independent experiments ± standard errors). Statistically significant differences (*) in RT template switching were measured by t test (P < 0.05 versus TAM control) (dotted reference line). (C) Regression analysis. Increases (n-fold) in AZT resistance were plotted versus the RT template-switching frequency for RNase H primer grip mutants containing a TAM polymerase domain.

Previous data showed that TAMs increase the frequency of template switching above WT (18). As shown in Fig. 4B, the frequency of template switching for the TAM control was increased 2.2 times above WT levels. As expected, the D549N mutation, which has significantly reduced RNase H activity, exhibited a decreased RT template-switching frequency to 0.5 times relative to the WT control (one time) (P < 0.05) and 0.2 times relative to the TAM control (2.2 times) (P < 0.05). In comparison to the TAM control, all of the RNase H primer grip mutations in the presence of TAMs significantly reduced the RT template-switching frequency, suggesting that these mutations reduced the RNase H activity. For most of the RNase H primer grip mutations (A360K, H361A, E396A, T473M, Q475A, Y501A, and I505A), the template-switching frequency was substantially reduced (25 to 40%), whereas for other mutations (G359A, K390A, K395A, and K476A), only a minor but statistically significant reduction in RT template switching was observed.

To determine whether the increase in AZT resistance observed with RNase H primer grip mutations was correlated with their effect on reducing RT template-switching frequency, linear regression analysis was performed (Fig. 4C). An R² value of 0.39 was observed, consistent with a lack of correlation between the reduction in RT template-switching frequency and the increase in AZT resistance (P > 0.1 by t test). The process of reverse transcription, which includes polymerization, RNase H degradation of the template, nucleotide excision, and strand transfers, is highly complex, and therefore, a simple linear regression analysis may not be expected to show a correlation between AZT resistance and RT template-switching frequency.

Mutations in the RNase H primer grip do not increase resistance to other NRTIs or NNRTIs.

We sought to determine whether mutations in the RNase H primer grip enhance resistance to other NRTIs that are currently used to treat HIV-1 infection by analyzing the effect of the RNase H primer grip mutations in the context of a polymerase domain containing TAMs on susceptibility to d4T, ABC, 3TC, ddI, and TDF (Table 1). With few exceptions, most RNase H primer grip mutations had little or no effect on susceptibility to the NRTIs tested. The A360K and K476A mutations slightly increased resistance to ABC 3.4 and 3.3 times, respectively, relative to the WT and about 2 times relative to the TAM control (1.7 times); the Q475A mutation increased resistance to ddI 2.5 times relative to the WT and TAM control; in addition, the Q475A and Y501A mutations increased resistance to TDF 2.3 and 2 times relative to the WT and the TAM control, respectively.

TABLE 1.

NRTI drug IC₅₀s for primer grip mutants in the context of a TAM-containing polymerase domain

Mutationa	d4T		ABC		3TC		ddI		TDF
	Mean IC50 (μM) ± SEb	Fold increasec	Mean IC50 (μM) ± SE	Fold increase	Mean IC50 (μM) ± SE	Fold increase	Mean IC50 (μM) ± SE	Fold increase	Mean IC50 (μM) ± SE	Fold increase
WT	1.5 ± 0.23	1.0d	5.1 ± 0.40	1.0d	6.50 ± 1.50	1.0	19.2 ± 2.5	1.0	2.4 ± 0.22	1.0
TAM	2.6 ± 0.26	1.7	8.8 ± 1.00	1.7	18.8 ± 4.17	2.9	17.0 ± 2.7	1.1	2.4 ± 0.16	1.0
G359A (T)	2.6 ± 0.24	1.7	11.4 ± 0.25	2.2	15.1 ± 3.39	2.3	19.8 ± 0.4	1.1	3.0 ± 0.16	1.2d
A360K (T)	2.6 ± 0.56	1.7	17.4 ± 1.28	3.4d	30.5 ± 7.57	4.7	20.3 ± 0.9	1.1	2.8 ± 0.13	1.1
H361A (T)	2.2 ± 0.24	1.5	9.8 ± 1.33	1.9	13.3 ± 1.56	2.0	14.7 ± 1.1	0.9	1.5 ± 0.10	0.6d
K390A (T)	1.9 ± 0.77	1.3	11.8 ± 0.63	2.3	21.0 ± 6.01	3.2	12.2 ± 1.3	0.8	3.1 ± 0.05	1.3d
K395A (T)	1.9 ± 0.33	1.3	11.2 ± 0.99	2.2	10.3 ± 6.22	1.6	14.6 ± 0.7	0.8	2.2 ± 0.20	0.9
E396A (T)	1.4 ± 0.45	0.9	13.1 ± 3.01	2.6	12.0 ± 0.92	0.6	13.9 ± 7.0	0.8	2.7 ± 0.21	1.1
T473M (T)	2.4 ± 0.99	1.6	9.5 ± 0.60	1.9	20.0 ± 3.46	1.8	25.7 ± 5.8	1.8	2.5 ± 0.46	1.0
Q475A (T)	2.8 ± 0.15	1.9	12.3 ± 1.20	2.4	26.8 ± 12.09	4.1	35.8 ± 3.3	2.5d	5.4 ± 0.42	2.3d
K476A (T)	3.7 ± 1.01	2.5	16.8 ± 1.03	3.3d	30.0 ± 12.80	4.6	14.4 ± 3.9	0.9	3.9 ± 0.70	1.6d
Y501A (T)	2.1 ± 0.58	1.4	9.3 ± 0.80	1.8	23.5 ± 4.24	3.6	13.2 ± 0.3	0.9	4.9 ± 0.32	2.0d
I505A (T)	4.0 ± 1.84	2.7	7.6 ± 1.10	1.5	ND		14.1 ± 6.2	1.0	2.5 ± 0.32	1.0

^aT, TAM containing.

^bData are the averages of two to five experiments.

^cIncrease (n-fold) in resistance over WT.

^dIncreases (n-fold) were significant compared to TAMs (P < 0.05 by t test).

We also determined the effects of RNase H primer grip mutations on susceptibility to NNRTIs in the context of a polymerase domain containing TAMs (Table 2). Again, with few exceptions, the mutations in the RNase H primer grip had little or no effect on susceptibility to the NNRTIs EFV and NVP. Mutations H361A and T473M increased sensitivity to EFV about five times relative to the WT and about two times relative to the TAM control. Several mutations (G359A, A360K, H361A, K390A, K395A, E396A, T473M, Q475A, and I505A) increased susceptibility to NVP about two times relative to the WT (1.0 times) and TAM (0.8 times) controls.

TABLE 2.

NNRTI drug IC₅₀s for primer grip mutants in the context of a TAM-containing polymerase domain

Mutationa	EFV		NVP
	Mean IC50 (μM) ± SEb	Fold increasec	Mean IC50 (μM) ± SE	Fold increase
WT	1.32 ± 0.12	1.0d	0.08 ± 0.012	1.0
TAMs	0.51 ± 0.03	0.4	0.06 ± 0.003	0.8
G359A (T)	0.47 ± 0.14	0.4	0.04 ± 0.002	0.5d
A360K (T)	0.38 ± 0.05	0.3	0.03 ± 0.006	0.4d
H361A (T)	0.25 ± 0.02	0.2d	0.02 ± 0.005	0.3d
K390A (T)	0.41 ± 0.05	0.3	0.03 ± 0.002	0.4d
K395A (T)	0.41 ± 0.06	0.3	0.02 ± 0.002	0.3d
E396A (T)	0.41 ± 0.01	0.3d	0.03 ± 0.005	0.4d
T473M (T)	0.20 ± 0.07	0.2d	0.03 ± 0.003	0.4d
Q475A (T)	0.50 ± 0.02	0.4	0.04e	0.6
K476A (T)	0.53 ± 0.11	0.4	0.04 ± 0.012	0.5
Y501A (T)	0.42 ± 0.05	0.3	0.08 ± 0.012	1.1
I505A (T)	0.40 ± 0.05	0.3	0.03 ± 0.004	0.3d

^aT, TAM containing.

^bData are the averages of two to three experiments.

^cIncrease (n-fold) in resistance over WT.

^dIncreases (n-fold) were significant compared to TAMs (P < 0.05 by t test).

^eThe experiment was done only once.

Mutations in the RNase H primer grip do not increase NRTI resistance in the context of a polymerase domain containing a K65R mutation.

Resistance to the NRTIs d4T, ABC, and TDF is associated with the presence of a K65R mutation in the polymerase domain (14). We sought to determine whether mutations in the RNase H primer grip increased resistance to NRTIs in the absence of TAMs but in the presence of other NRTI resistance-associated mutations in the polymerase domain. We therefore determined the effects of mutations in the RNase H primer grip on susceptibility to d4T, ABC, and TDF in the presence of the K65R mutation (Table 3). In general, most mutations in the RNase H primer grip had little or no effect on susceptibility to the NRTIs d4T, ABC, and TDF in the context of a K65R-containing polymerase domain. Only the Q475A and Y501A mutations increased resistance to d4T approximately twofold (2.6- and 2.2-fold) relative to the WT RT and less than twofold relative to the K65R control (1.4 times).

TABLE 3.

NRTI drug IC₅₀s for primer grip mutants in the context of a 65R-containing polymerase domain

Mutation	d4T		ABC		TDF
	Mean IC50 (μM) ± SEa	Fold increaseb	Mean IC50 (μM) ± SE	Fold increase	Mean IC50 (μM) ± SE	Fold increase
WT	5.9 ± 1.3	1.0	4.5 ± 0.00	1.0c	2.2 ± 0.1	1.0c
65R	8.5 ± 1.5	1.4	14.9 ± 0.8	3.3	5.4 ± 0.4	2.5
G359A (65R)	8.5 ± 1.4	1.4	2.9 ± 4.2	2.9	4.2 ± 2.0	1.9
H361A (65R)	8.3 ± 0.5	1.4	2.8 ± 0.3	2.8	5.0 ± 2.2	2.3
K395A (65R)	15.0 ± 4.7	2.5	2.8 ± 2.6	2.8	4.4 ± 0.7	2.0
E396A (65R)	8.4 ± 1.2	1.4	2.6 ± 1.4	2.6	ND
Q475A (65R)	15.5 ± 0.3	2.6c	2.9 ± 1.6	2.9	ND
Y501A (65R)	13.2 ± 0.9	2.2c	2.8 ± 3.4	2.8	4.6 ± 0.8	2.1
I505A (65R)	13.5 ± 0.1	2.3	2.6 ± 2.6	2.6	4.2 ± 1.7	1.9

^aData are the averages of two to three experiments.

^bIncrease (n-fold) in resistance over WT.

^cIncreases (n-fold) were significant compared to 65R (P < 0.05 by t test).

DISCUSSION

The results of these studies show for the first time that mutations in the HIV-1 RNase H primer grip significantly enhance AZT resistance when the polymerase domain contains TAMs and further underscore the importance of the connection domain in conferring AZT resistance. We previously observed that several substitutions in the HIV-1 connection domain were selected in response to antiviral therapy and enhanced AZT resistance (18). Indeed, two of the substitutions identified in treatment-experienced patients directly altered one of the amino acids in the RNase H primer grip (A360I/V), resulting in an increase in AZT resistance. Our results further support the view that some of the connection domain substitutions selected in response to therapy enhance AZT resistance by directly or indirectly influencing the structure and function of the RNase H primer grip.

Several amino acid substitutions in the RNase H primer grip are observed in clinical isolates, as reported in the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu). Because most commercial genotypic analyses do not include results beyond amino acid 313 of RT, sequence data for the connection and RNase H domains are very limited. Less than 350 patient sequences reach the end of the RNase H domain for subtype B HIV-1. Nevertheless, all RNase H primer grip residues except residues 473, 501, and 505 exhibited polymorphisms in treatment-naïve as well as treated patients. For example, the G359S mutation was observed in 6% of treatment-naïve patients and 12% of treatment-experienced patients. The K476Q mutation was observed in 0.8% of the treated patients but was not observed in treatment-naïve patients. At this time, the potential contribution of any RNase H primer grip amino acid polymorphisms to antiviral drug resistance is unknown. Interestingly, approximately 55% of all of the sequences that had complete connection domain sequences (986 total) contained one or more TAMs; a higher proportion of the sequences containing the G359 mutation (72% of 115 total) and the A360 mutation (73% of 180 total) contained one or more TAMs, suggesting that these replacements are positively associated with TAMs (P < 0.001 by two-proportions test). Therefore, it is possible that these connection domain mutations are selected in response to drug therapy.

It is noteworthy that with the exception of the Q475A mutation, the RNase H primer grip mutations did not significantly increase AZT resistance in the context of a WT polymerase domain. Similarly, the patient-derived connection domain mutations that we recently reported only modestly increased AZT resistance four to six times in the context of a WT polymerase domain (18). We previously observed that the RNase H mutation D549N increased AZT resistance 13 times in the context of WT polymerase and synergistically increased AZT resistance 1,250 times in the context of a TAM-containing polymerase domain (19). The D549N mutation causes the most severe reduction in RT template-switching frequency, suggesting that it drastically reduces RNase H activity. In comparison to the D549N mutation, the RNase H primer grip mutations increased AZT resistance to a lesser extent in the context of a TAM-containing polymerase domain (20 to 243 times versus 1,250 times) and did not reduce RT template-switching frequency as severely as the D549N mutant. These observations suggest that the RNase H primer grip mutations have a less drastic effect on RNase H activity than the D549N mutation. We postulate that since the D549N mutation increases AZT resistance only 13 times in the context of a WT polymerase domain, the effect of most of the RNase H primer grip mutations on AZT resistance in the context of a WT polymerase domain is quantitatively too small (one- to threefold) to be statistically significant. Interestingly, the Q475A mutant had the greatest effect on AZT resistance in the context of a TAM-containing polymerase domain (243 times) and exhibited a statistically significant increase of 3.1 times in the context of a WT polymerase domain.

Since TAMs are known to enhance nucleotide excision, and AZT is excised more efficiently than other NRTIs, the results suggest that RNase H primer grip mutations increase AZT resistance by directly or indirectly increasing the rate of nucleotide excision. One hypothesis is that the RNase H primer grip mutants increase the rate of nucleotide excision by indirectly affecting the RNase H active site. We previously proposed that the balance between polymerase and RNase H activities is an important determinant of nucleotide excision (18, 19) and postulated that mutations reducing RNase H activity would increase the time period available for nucleotide excision. We have also observed that mutations that reduce RNase H activity reduce RT template switching, whereas mutations that slow down the rate of DNA synthesis increase template switching. To determine whether mutations in the RNase H primer grip enhance AZT resistance by reducing RNase H activity, we determined the effects of these mutations on RT template switching. We observed that all of the RNase H primer grip mutations reduced RT template switching in the presence of TAMs, which is consistent with the hypothesis that these mutations likely reduced RNase H activity. These observations are consistent with previously reported studies indicating that RNase H primer grip mutations decrease RNase H activity in vitro and in vivo. Y501E/A/H mutations were shown to have decreased RNase H activity in vitro (1), and mutations T473A, N474A, Q475A, N474A and Q475A, and Y501A were shown to have decreased RNase H activity, as indicated by a decrease in −8 cleavage product accumulation (21). As expected for mutations that alter RNase H activity, mutants H361A, E396A, Y501A, and Q475A showed two- to threefold increases in aberrant 2-long-terminal-repeat circles (15-17), and mutants T473A, N474A, Q475A, N474A plus Q475A, and Y501A also showed a decrease in the specificity of PPT cleavage as well as slower kinetics in PPT removal (17, 21). Furthermore, Smith et al. previously showed that the addition of connection domain sequences to the RNase H domain increased in vitro RNase H activity (24). Taken together, our observations and those previous studies support the hypothesis that RNase H primer grip mutations enhance AZT resistance by reducing RNase H activity and shifting the balance between the rates of DNA synthesis and RNA degradation.

An equally plausible hypothesis that is consistent with these observations is that the RNase H primer grip mutations increase nucleotide excision by directly influencing the structure of the polymerase active site and/or the rate of DNA polymerization. Our results failed to show a correlation between the increase in AZT resistance and the reduction in RT template switching, suggesting that other effects of the RNase H primer grip mutations that do not involve a reduction in RNase H activity may contribute to the increase in AZT resistance. The RNase H primer grip mutations have been shown to influence the overall efficiency of DNA synthesis (15-17, 21) and fidelity of DNA synthesis (29) by influencing the positioning of the primer-template at the polymerase active site. A reduction in the overall rate of DNA synthesis, as was previously shown with mutants Y501A, Q474A, Q475A, and N474A plus Q475A (15-17), might cause RT to reside for a longer period of time with the terminus of the blocked primer residing at the active site before translocation, thus allowing more efficient pyrophosphorolysis and nucleotide excision. It must also be pointed out that the RNase H primer grip mutations may influence both the polymerase and RNase H activities by influencing the positioning of the primer-template at both active sites, since these two effects need not be mutually exclusive.

The RNase H primer grip mutations may also influence both polymerase and RNase H activities by changing the affinity of the RT for the template-primer complex. It is likely that the RNase H primer grip mutations reduce, rather than enhance, the affinity of the RT for the template-primer complex. Indeed, Rausch et al. previously analyzed the interactions between the template-primer complex and T473A, N474A, and Y501A mutants and observed modest decreases in template affinity (21). The RNase H primer grip mutations that reduce template affinity may also alter the structure of the polymerase and RNase H active sites. Thus, a reduction in template affinity could influence nucleotide excision and RT template switching by influencing the overall rates of DNA polymerization and RNA degradation.

The results of these studies substantially increase the number of amino acid replacements in the connection and RNase H domains that increase AZT resistance in the presence of TAMs. These results also have important clinical implications in that HIV-1 variants containing TAMs can considerably increase resistance to NRTIs, specifically AZT, if the virus acquires further mutations in the RNase H primer grip. The data presented here support the hypothesis that mutations in the connection and RNase H domains can increase AZT resistance by altering the RNase H primer grip region, which in turn disrupt interactions between RT and the template-primer complex and/or shifts in the balance between polymerase and RNase H activities.