Incorporation of Oxidatively Modified 2 '-Deoxynucleotide

One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold...
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654

Chem. Res. Toxicol. 2002, 15, 654-661

Incorporation of Oxidatively Modified 2′-Deoxynucleotide Triphosphates by HIV-1 RT on RNA and DNA Templates Gerald E. Wuenschell, Michael R. Valentine, and John Termini* Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010 Received October 26, 2001

Oxidatively modified deoxynucleotide triphosphates (dNoxoTPs) present in nucleotide precursor pools may contribute to retroviral mutagenesis as a result of incorporation and ambiguous base pairing during reverse transcriptase mediated replication. We have examined the incorporation of 5-hydroxy-2′-deoxycytosine triphosphate (5-HO-dCTP) and 2′-deoxyinosine triphosphate (dITP) by HIV-1 reverse transcriptase (HIV-1 RT) on DNA and RNA templates of the same sequence in order to evaluate their mutagenic potential. Significant variations in insertion frequencies at homologous nucleotide positions were observed for each dNoxoTP, in general favoring the RNA template. A comparison of steady-state kinetics revealed a 10-fold preference for 5-HO-dCTP incorporation opposite G in RNA. Insertion frequencies for dITP were 2- to 20-fold greater on RNA for every base position examined. One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold. Deoxyinosine triphosphate was inserted opposite C with an 8-fold higher frequency compared to dGTP in RNA, while on DNA templates, the incorporation frequencies were equivalent. However, incorporation of dITP opposite other bases was characterized by relatively low frequencies. The RNA template bias observed for dNoxoTP incorporation is discussed in terms of recent efforts to utilize 5-OH-dCTP as an anti-HIV agent.

Introduction The HIV virus is able to evade immune system neutralization and evolve resistance to a wide variety of antiviral therapeutics due to an extraordinary ability to generate genetic variability. The driving force for such rapid evolution is a mutation frequency which is ∼106fold higher than that estimated for eucaryotic genomes. It is generally thought that the error prone nature of HIV-1 reverse transcriptase (HIV-1 RT),1 coupled with high turnover of the virus (1), gives rise to a large number of genetic variants. The base substitutions observed in frequently mutated HIV genes such as env are highly biased toward A to G and G to A transition mutations. Recently, an analysis of base substitution frequencies was performed using data from the HIV Sequence Database site (http://hivweb.lanl.gov) for the env gene and the trans activation response (TAR) element (2). For both sequences, A to G and G to A transition mutations accounted for more than 40% of base substitution mutations. Examination of recently compiled mutations found to confer resistance to HIV-1 RT and protease inhibitors similarly revealed a preponderance of these transitions (3). These striking * To whom correspondence should be addressed. Phone: (626) 3018169. Fax: (626) 301-8271. E-mail: [email protected]. 1 Abbreviations: dNoxoTP, oxidized deoxynucleotide triphosphate; 5-OH-dCTP, 5-hydroxy-2′-deoxycytidine-5′-triphosphate; 5-OH-dUTP, 5-hydroxy-2′-deoxyuridine-5′- triphosphate; dITP, 2′-deoxyinosine-5′triphosphate; HIV-1 RT, human immunodeficiency virus type 1 reverse WC transcriptase; fins, insertion frequency; kns 2 , k2 , second-order rate constants for nonstandard and Watson-Crick base pairs, respectively; KF, Klenow fragment.

hypermutation phenomena cannot readily be explained by mismatch errors of HIV-1 RT on RNA templates since the frequency of A‚C and G‚T mispair formation does not appear to be substantially higher than other mismatches.2 Several proposals have been advanced in the literature to account for A to G and G to A hypermutation phenomena. Deamination of adenine in RNA to yield hypoxanthine (Hx) has been proposed to contribute to A to G transitions, in view of the kinetic and thermodynamic preference for Hx‚C base pair formation (4). This view is supported by steady-state kinetic studies of Hx containing RNA templates which favor Hx‚C pairings by HIV-1 RT over A‚T and G‚C by approximately 10-fold (2). Other potential contributors to retroviral mutagenesis may originate from the host nucleotide precursor pools. Nucleotide precursor pool imbalances (low dCTP/dTTP ratios) within virally infected host cells have been postulated to explain G to A hypermutation in HIV (57). Although the in vitro biasing of dNTP concentrations has the expected effect of increasing the frequency of mismatch-induced mutations by HIV-1 RT, there is as yet no direct biological evidence that HIV infection perturbs dNTP pool imbalances in the required manner. Another potential source of mutational bias is the presence of base-modified nucleic acids. Base modifications which alter the relationship of hydrogen bond donor/ acceptor groups from the normal Watson-Crick arrangements enhance the probability for mutations. Oxidized 2

Valentine et al., submitted for publication.

10.1021/tx010167l CCC: $22.00 © 2002 American Chemical Society Published on Web 04/27/2002

5-HO-dCTP and dITP Incorporation by HIV-RT

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Figure 1. Preparative ion exchange (MonoQ 10/10) HPLC chromatogram of synthetic 5-OH-dCTP (second purification). The inset shows the UV spectrum (H2O, pH 7) of the main peak, which was collected and used in the primer extension assays.

bases, either in the template or in the dNTP precursor pool, likely contribute to mutagenesis by this mechanism. Enhanced nucleic acid damage has been correlated with the incidence of HIV infection (8, 9). Phagocytes activated by viral infection release hydrogen peroxide and cytokines, both of which have been shown to promote oxidative lesions in cellular nucleic acids (10). HIV patients display increased levels of malondialdehyde in plasma (11), indicating enhanced lipid peroxidation. Lowered anti-oxidant defenses in these patients exacerbates the potential for oxidative nucleic acid damage (12, 13). Direct measurement of oxidatively modified nucleic acid bases by mass spectrometry has revealed significantly higher levels of 5-hydroxycytosine and several other oxidatively modified bases in symptomatic HIV patients relative to uninfected and asymptomatic individuals (14). Thus, the incorporation of oxidized dNTPs from the nucleotide precursor pool (dNoxoTPs) may constitute an important route for base substitution mutations in HIV. Recently, it has been demonstrated that 8-oxo-2′-deoxyguanosine present in nucleotide precursor pools may influence base substitution patterns in HIV (15). A single cycle of retroviral replication results in the production of a double-stranded DNA provirus from a single-stranded RNA genome. Incorporation of dNoxoTPs by HIV-1 RT from the nucleotide precursor pool may occur during the initial first strand cDNA synthesis using the genomic (+) RNA template or during second strand cDNA synthesis on the (-) strand DNA template. This “dual template” mechanism of replication is unique to retroviruses and the relative fidelity of HIV-1 RT on RNA or DNA templates will influence the mutational outcome in different ways. We have used steady-state primer extension kinetic analyses (16) to evaluate the incorporation of 5-OH-dCTP, an oxidation product of dCTP, and dITP, resulting from the oxidative deamination of dATP. Insertion frequencies were determined for each dNoxoTP opposite the standard bases in RNA and DNA templates

of identical sequence in order to determine the mutation inducing potential resulting from incorporation during either first or second strand DNA synthesis by HIV-1 RT.

Materials and Methods 2′-Deoxynucleotide Triphosphates. The synthesis of 5-OHdCTP was carried out according to the procedure described by Purmal et al. with some minor modifications (17). Briefly, dCTP was brominated at 4 °C, then hydrolyzed in situ via the addition of collidine in water. The desired product was purified using a preparative Pharmacia MonoQ (10/10) column. A gradient of 0 to 100% NH4OAc (pH 7.5) over 30 min at a flow rate of 1 mL/ min was used to separate the reaction products. 5-HO-dCTP eluted ∼7 min after dCTP. This material was repurified on HPLC prior to use in polymerase reactions, and a typical chromatogram is shown in Figure 1. Lyophilized fractions were redissolved in 10% acetonitrile/water prior to treatment with ammonium modified DOWEX 50WX8-200 ion-exchange resin to remove sodium and potassium ions. Isolated product was then analyzed by mass spectrometry using a Mariner electrospray ionization orthogonal time-of-flight mass spectrometer (ESI-OTOF, Applied Biosystems). Data obtained in the negative ion mode is shown in Figure 2 and was consistent with C9H15N3O14P3- (observed mass, 481.9765; calculated, 481.98). Positive ion mode spectra yielded an observed mass of 483.7983 (calculated, 483.99) for C9H17N3O14P3+ (data not shown). We were unable to detect the presence of more than trace amounts of 5-OH-dUTP in samples of 5-HO-dCTP by mass spectrometry even after storage for several months at -20 °C. Deoxyinosine triphosphate was purchased from Sigma. Traces of mono- and diphosphate material were removed by HPLC purification as described above. Template Oligonucleotides. The RNA and DNA template sequences used for primer extension reactions corresponded to nucleotides 962-996 of the V3 hypervariable domain of the envelope glycoprotein (env) of the WMJ-1 strain of HIV-1 (18). RNA templates were synthesized by Dharmacon (Boulder, CO) using 2′-O-bis(2-acetoxyethoxy)methyl phosphoramidites. The DNA primer and RNA/DNA template sequences used are shown in Figure 3. DNA primers were synthesized by the Synthesis Core Lab, City of Hope Cancer Center. Primers used for the single nucleotide primer extension assays were designed to

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Figure 2. Electrospray ionization mass spectrum of 5-HO-dCTP acquired in the negative ion mode. The main ion at m/z 481.9765 is consistent with C9H15N3O14P3-. See text for details. then plotted using the method of direct linear plots (20) to determine Vmax and KM for each base pairing reaction. The frequency of nonstandard base pair formation is the ratio of second-order rate constants (k2) for the nonstandard (ns) pairing relative to the competing Watson-Crick (WC) base pair at the same template position. This insertion frequency (fins) is thus WC defined as kns 2 /k2 .

Results

Figure 3. DNA primers and 35-mer RNA/DNA templates used in these studies. The template sequences are derived from the hypervariable region of the extra-cellular portion of the envelope gene (env) of the WMJ-1 strain of HIV-1 (23). Arrows indicate bases where dNTP/dNoxoTP insertion kinetics were evaluated; letters indicate the corresponding primers used. DNA sequence is identical to the RNA sequence shown, with T replacing U. terminate immediately before the targeted insertion site. DNA oligonucleotide primers were purified by 20% polyacrylamide (1.5 mm) gel electrophoresis (PAGE) and radiolabeled using standard methods. HIV-1 RT Primer Extension Reactions. HIV-1 RT was expressed from plasmid pHIV-RT(His)Prot transformed into E. coli BL21(DE3)pLysS cells (Novagen, Madison, WI) and purified as described (2). The plasmid was a generous gift of Dr. Paul Boyer (National Cancer Institute). The final enzyme concentration was determined spectrophotometrically using an extinction coefficient (280) of 520 mM-1cm-1 (19). The unit activity was determined by measurement of the incorporation of [R-32P]TTP into a poly(rA)‚p(dT)12-18 template using the tricholoroacetic acid precipitation method (2). Single nucleotide primer extension reactions were carried out using the method of Goodman (16). Reactions containing RNA/DNA template, 5′-32P end-labeled DNA primers, varying concentrations of dNoxoTP/dNTP and a fixed concentration of HIV-1 RT were allowed to proceed into the linear reaction phase prior to quenching and analysis by 20% PAGE (∼ 5 min). Gels were dried and analyzed using a PhosphorImager workstation with the ImageQuant software package (Molecular Dynamics). The reaction velocities were determined from the autoradiograpic data using the relationship ν ) I1[primer]/I0 + 1/2I1)t, where ν is the velocity in pM/s, I0 is the intensity of the primer band, I1 is the intensity of the extended band(s) and t is the reaction time (16). Velocities were

Watson-Crick Base Pair Forming Efficiencies on RNA and DNA Templates. The sequences of DNA primers and RNA/DNA templates used in this study are provided in Figure 3. The steady-state kinetic parameters for all primer extension reactions on RNA and DNA templates are shown in Tables 1 and 2, respectively. The efficiencies of standard base pair formation on DNA and RNA templates were evaluated by comparing k2 values, the apparent second-order rate constant. We observed decreased efficiencies for Watson-Crick base pair formation by HIV-1 RT on RNA templates in all cases (Tables 1 and 2). The k2 values for formation of G‚C base pairs on RNA templates were diminished by 4-fold (G19 + dCTP) and 8-fold (C17 + dGTP) relative to formation on DNA. For A‚T(U) base pairs, the formation efficiencies on DNA templates exceeded those for the corresponding RNA template by 13 and 5-fold (T/U25 + dATP and A20 + dTTP, respectively). Incorporation of 5-HO-dCTP and dCTP opposite purines in RNA and DNA. The insertion frequency WC values (fins ) kns 2 /k2 ) for 5-HO-dCTP and dCTP are graphed separately in Figure 4 for RNA (A) and DNA (B) templates. These values provide a direct indication of the ability of the nonstandard base pair to compete against Watson-Crick base pairing assuming equimolar concentrations of dNTP and dNoxoTPs. Watson-Crick base pairing reactions are characterized by an insertion frequency of one by definition. 5-HO-dCTP was synthesized from 5-bromo-dCTP using a modification of a published procedure (22) and purified twice by HPLC prior to characterization and subsequent use as a substrate for HIV-1RT (Figure 1). Neither brominated precursor nor deaminated products were detected by ESI-O-TOF mass spectrometry. The characteristic UV spectrum of the purified material was consistent with that previously reported for 5-hydroxycy-

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Table 1. Kinetic Data for Insertion of 5-HO-dCTP on RNA by HIV-1 RT template basea U25 U25 U25 G19 G19 C17 C17 C17 A20 A20 A20 c

dNTP 5-HO-C A C 5-HO-C C 5-HO-C C G 5-HO-C C T

KM (µM) 90 0.14 54 0.038 0.039 ND 200 0.099 5 6.2 0.052

Vmax (pM/s) 15 84 14 38 39 ND 9 30 106 32 44

kcat (×104 s-1)

k2b

finsc

1.2 6.7 1.1 3 3.1 ND 0.72 2.4 8.5 2.6 3.5

1.3 × 4.8 × 103 2.0 × 100 7.9 × 103 7.9 × 103 ND 3.6 × 10-1 2.4 × 103 1.7 × 102 4.2 × 101 6.7 × 103

2.8 × 10-4

100

4.3 × 10-4 9.9 × 10-1 ND 1.5 × 10-4 2.5 × 10-2 6.2 × 10-3

a Subscripts denote template positions according to the numbering in Figure 3. b The apparent second-order rate constant k ) k /K . 2 cat M WC Defined as kns 2 /k2 , the ratio of rates for nonstandard and Watson-Crick base pairs.

Table 2. Kinetic Data for Insertion of 5-HO-dCTP on DNA by HIV-1 RT template base

dNTP

KM (µM)

Vmax (pM/s)

kcat (×104 s-1)

k2

fins

T25 T25 T25 G19 G19 C17 C17 C17 A20 A20 A20

5-HO-C A C 5-HO-C C 5-HO-C C G 5-HO-C C T

167 0.027 240 0.11 0.026 ND 3100 0.075 0.12 52 0.056

15 205 110 50 104 ND 9 190 50 95 250

1.2 16 8.8 4 8.3 ND 0.74 15 4 7.6 20

7.2 × 10-1 5.9 × 104 3.7 × 100 3.6 × 103 3.2 × 104 ND 2.4 × 10-2 2.0 × 104 3.3 × 103 1.5 × 101 3.3 × 104

1.2 × 10-5 6.2 × 10-5 1.1 × 10-1 ND 1.2 × 10-6 9.3 × 10-2 4.1 × 10-4

Figure 4. Graph of insertion frequencies for dCTP and 5-HO-dCTP at the base positions indicated in Figure 2 for (A) RNA and (B) DNA.

tosine at neutral pH (21). The ability of 5-HO-dCTP to substitute for dCTP as a substrate for HIV-1RT on RNA and DNA templates was first examined. Determination of the steady-state values for incorporation of 5-HO-dCTP opposite guanine in RNA surprisingly revealed an apparent kcat/KM value that was indistinguishable from that measured for dCTP insertion at the same base position (Figure 4A). Thus the frequency of incorporation during first strand synthesis is only limited by the availability of 5-HO-dCTP in dNTP precursor pools. When the corresponding reaction was examined on the homologous DNA template (Figure 4B) the insertion frequency of 5-HO-dCTP was observed to be 10-fold lower than that for dCTP; thus, insertion of the modified base is discriminated against only when the template is DNA. This suggests that incorporation of 5-HO-dCTP takes place preferentially on RNA templates and is more likely to occur during first strand HIV-1 RT catalyzed cDNA synthesis. Comparison of KM and kcat in Table 1 for the base pairing reactions of dCTP and 5-HO-dCTP at G19 in the RNA template reveals indistinguishable values. For the DNA template reactions, the observed 10-fold

lower insertion frequency for 5-HO-dCTP at G19 relative to dCTP is due to the combined effects of a higher KM and a lower kcat. Incorporation of 5-HO-dCTP at adenine is favored over dCTP for both RNA and DNA templates. For insertion opposite template adenines in RNA, 5-HO-dCTP was preferred by 4-fold. Examination of the KM and Vmax components for these respective reactions at adenine reveals that this difference is nearly entirely due to differences in Vmax. The values of KM from Table 1 are virtually identical (5 and 6.2 µM) whereas the corresponding Vmax values were found to be 106 and 32 pM/s for insertion of 5-HO-dCTP and dCTP, respectively. For most if not all polymerases, steady-state values of Vmax(kcat) describe the rate-limiting step of primer-template dissociation from the enzyme (22). Thus for the RNA template, the dissociation of HIV-1 RT from a primer/ template complex with a terminal 5-HO-C‚A base pair is faster than the dissociation from the analogous complex containing a C‚A terminal pair. For the corresponding DNA template, the formation of 5-HO-C‚A base pairs was favored over C‚A by more than 200-fold. Comparison

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Table 3. Comparison of Fidelities (F) for Insertion of dITP and 5-HO-dCTP dNTP dITP

5-HO-dCTP

RNA template

Fa

DNA template

F

U25 G19 C17 A20 U25 G19 C17 A20

1700 1200 0.125b 4200 3600 1 ND 40

T25 G19 C17 A20 T25 G19 C17 A20

3800 24 000 1 14 925 8300 9 ND 11

a Values for dITP were calculated from data in ref 8. b In order must be greater than kns for F g 1, kWC 2 2 . A unique exception to this trend is the insertion of dITP opposite C17 in RNA, where ns WC k2 is 8-fold greater than k2 .

of KM and Vmax components for these reactions reveals strong discrimination based upon KM. Whereas the value of Vmax is larger for insertion of dCTP opposite A in DNA (95 vs 50 pM/s), reaction of 5-HO-dCTP is characterized by a favorable KM value which is 433-fold lower than for dCTP (0.12 vs 52 µM, Table 2). A clear physical interpretation of KM from steady-state determinations is usually not possible, since this value may reflect the contributions of several parameters including kp, the rate constant for polymerization, and koff, the rate of primer/ template dissociation (22). Incorporation of 5-HO-dCTP and dCTP Opposite Pyrimidines in RNA and DNA. The insertion reactions opposite pyrimidines were observed to occur with generally lower efficiencies for both 5-HO-dCTP and dCTP. Examination of Figure 4, panels A and B, reveals fins values of less than 10-3 for pairing opposite pyrimidines on both DNA and RNA templates. HIV-1 RT did not appear to discriminate to any significant degree for the insertion of 5-HO-dCTP or dCTP opposite uridine (U25). Preferential insertion (5-fold) for dCTP over 5-HO-dCTP was observed however for the corresponding DNA template reaction at T25. Mispair formation at uridine in the RNA template was preferred relative to the DNA template reaction for both dCTP and 5-HO-dCTP. The ratio of fins values 5-HO-C‚U/5-HO-C‚T is 23, while for C‚U/C‚T it is 7 (Tables 1 and 2). We were unable to detect any HIV-1 RT catalyzed incorporation of 5-HO-dCTP opposite C for either DNA or RNA templates. The formation of C‚C mispairs by HIV-1 RT occurred with an extremely low frequency on the DNA template (1.2 × 10-6). This is consistent with what has been usually observed with other polymerases, owing to the unstable (extrahelical) arrangement of C‚C mispairs in DNA (23). In contrast, the frequency of C‚C mispair formation on the RNA template was more than 2 orders of magnitude greater, suggesting a potential role in HIV base substitution mutations during first strand synthesis. Comparison with HIV-1 RT Insertion of dITP on DNA and RNA Templates. The incorporation of dITP by HIV-1 RT was examined using the primers and templates shown in Figure 3 and compared to the results obtained with 5-HO-dCTP. These results are expressed as fidelity values (F ) 1/fins) in Table 3 (24). Fidelity values denote the number of Watson-Crick base pairing events which occur at a particular site per nonstandard pairing event. Thus, the fidelity value in Table 3 for the incorporation of dITP opposite U25 denotes one dITP incorporation for every 1700 HIV-1 RT catalyzed dATP insertions. The pairing of dITP with cytosine on both DNA and RNA templates was observed to be highly

preferred. The fidelity for the reaction at C17 in DNA is 1, demonstrating that HIV-1 RT incorporated dITP or dGTP at the same frequency. The HIV-1 RT catalyzed reaction of dITP at C17 in this RNA template is highly unusual in that the apparent second-order rate constant (kns 2 ) exceeds the reaction with dGTP by 8-fold. This is the only example of which we are aware where HIV-1 RT forms a nonstandard base pair in preference to the normal Watson-Crick pair. The corresponding reaction at C17 of the DNA template was characterized by an apparent second order rate which was equivalent to dGTP insertion. Aside from these striking examples, base pairing reactions of dITP were typically characterized by substantially larger fidelity values relative to 5-HOdCTP. The fidelities for reactions of dITP on both DNA and RNA templates for other bases were typically greater than 1000, suggesting lower probabilities for incorporation.

Discussion The principal route for the generation of adaptive retroviral mutations appears to be base substitutions introduced during the conversion of the single-stranded (+) RNA genome into a DNA provirus by HIV-1 RT. The first intermediate produced during retroviral replication is a DNA:RNA heteroduplex. The cDNA produced during this step is copied by HIV-1 RT to generate the doublestranded DNA provirus and is the template for the resynthesis of retroviral genomic RNA by host cell RNA polymerase. Only base substitutions introduced in this (-) proviral strand can give rise to mutations. Nonstandard base pairing events such as incorporation of dNoxoTPs or mispair formation by HIV-1 RT during this step of the replication cycle cannot be corrected, since HIV-1 RT does not possess proofreading activity, and an efficient repair mechanism for mispairs or oxidative damage in heteroduplexes appears to be absent (25). Thus, miscoding events which occur during first strand cDNA synthesis by HIV-1 RT may account for the majority of base substitutions observed in the HIV genome. It is of interest to determine whether the utilization of RNA templates by HIV-1 RT is inherently more error prone relative to DNA templated replication. This would further reinforce the importance of first strand cDNA synthesis events in retroviral hypermutation phenomena. Our data address this issue for the incorporation of two dNoxoTPs. The measured insertion frequencies for 5-HOdCTP opposite G and U in RNA were 9- and 23-fold higher, respectively, compared to the analogous reactions on a DNA template of identical sequence. However, insertion of 5-HO-dCTP opposite A occurred preferentially on the DNA template. Insertion of dITP was favored on the RNA template for all pairing reactions. The insertion frequencies for dITP opposite G, C, A, and U in RNA were 20-, 8-, 4-, and 2-fold greater, respectively. These differences in HIV-1 RT insertion frequencies suggest that polymerases discriminate between DNA‚ DNA homoduplexes and RNA‚DNA heteroduplexes. The different topological constraints presented by the mixed conformation RNA‚DNA (26, 27) and B form DNA‚DNA duplexes could conceivably influence HIV-1RT/dNTP binding as well as the phosphodiester bond forming step. Detailed speculation is difficult since structural information is currently only available for HIV-1 RT/DNA‚DNA primer/template complexes (28, 29). Significant differ-

5-HO-dCTP and dITP Incorporation by HIV-RT

ences in hydration between DNA‚DNA and RNA‚DNA duplexes, owing to the presence of the 2′-OH in the latter, have been identified by NMR (30). Differential hydration may conceivably affect the kinetics of binding events or subsequent chemistry. Pre-steady-state experiments are in progress to address these issues. Lower efficiencies for Watson-Crick base pair formation (kWC 2 ) on RNA templates can result in more effective competition from nonstandard base pairing. The kinetic data suggest that the enhanced insertion frequencies observed for dITP and 5-HO-dCTP on RNA relative to DNA templates are due in part to this effect. The insertion of dITP opposite C is favored on RNA by 8-fold, yet the kns 2 values are indistinguishable (2). The fins value for incorporation of 5-OH-dCTP opposite G is 10fold greater for RNA. The difference in kns 2 values favors the RNA template by only 2-fold, while the smaller value on RNA exerts the dominant influence on the of kWC 2 insertion frequency. A similar trend was observed for 5-HO-C‚T/U base pair formation. The pKa for the hydroxy group of 5-HO-dC is 7.4, thus it is substantially enolized at physiological pH. Resonance Raman data suggests that the enolate form stabilizes the imino base tautomer (31). In this form, N4 is a hydrogen bond acceptor and N3 becomes a hydrogen bond donor, the reverse of the normal arrangement in cytosine. This configuration is isosteric with uracil, resulting in the formation of base pairs with adenine. These base pairing arrangements are depicted in Figure 5. Perturbation of the equilibrium between enolized and protonated forms of 5-OH-dCTP by binding to the polymerase and/or the primer/template would be expected to modify the frequency of 5-OH-dCTP insertion opposite A. Steady-state kinetic studies of the incorporation of 5-HO-dCTP by E. coli DNA polymerase I (KF, Klenow fragment) have also been described (32). Whereas HIV-1 RT catalyzed the insertion of 5-HO-dCTP opposite G or A in DNA with equivalent insertion frequencies, pairing opposite G by DNA polymerase I was preferred over A by a factor of 1000. HIV-1 RT favors the creation of 5-HOC‚A over C‚A base pairs by more than 200-fold, while this preference is only 2-fold for the bacterial enzyme. Enhanced mispair incorporation efficiency for 5-OHdCTP relative to 8-oxo-dGTP and 5-OH-dUTP by DNA polymerase I in vitro (32) and in vivo (33) has been reported. The kinetics (19) and template preference (15) for incorporation of 8-oxo-dGTP by HIV-1 RT has also been described. Reported DNA mispair insertion frequencies were lower (19) compared to 5-OH-dCTP, and there appears to be no obvious discrimination in favor of RNA templates for 8-oxo-dGTP (15). The HIV mutation rate hovers near the theoretical maximum permissible for viability, and any perturbation which can increase this rate even slightly might be expected to induce lethality by creating a large mutation burden in essential genes (34). The use of dNoxoTPs as therapeutic agents to drive hypermutagenesis over this threshold error level has been proposed by Loeb and coworkers, who examined the ability of several modified dNTPs to inhibit retroviral replication by this mechanism (35). Significantly, passage of HIV infected human CEM cells in the presence of 5-HO-dC, but not several other nucleic acid analogues, resulted in a loss of viral replicative potential. A sharp decline in virus production was evident despite only a modest increase in mutation

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Figure 5. Hydrogen bonding arrangements for 5-HO-C‚G, 5-HO-C‚A and 5-OH-U‚A base pairs. Hydrogen bonds are indicated by block arrows in the direction of H-bond donor to acceptor. 5-HO-C is predominantly in the enolate form at neutral pH (31). Cytosine in the imino form is capable of forming base pairs with adenine which are isosteric with 5-OH-U‚A and U‚A base pairs.

frequency over untreated controls, consistent with the threshold mutagenesis hypothesis. Sequence analysis of the HIV-1 RT gene after passaging HIV infected cells 16 times in the presence of 5-HO-dC revealed a large increase in G to A transitions relative to untreated controls. The number of C to U and U to C transition mutations were also observed to increase. Figure 6 summarizes the data for incorporation of 5-OH-dCTP on DNA and RNA templates determined in this study. Although all the factors governing mutational output are complex and difficult to evaluate entirely, the unusually high 5-OH-dCTP incorporation frequencies observed in our studies may help explain the mutagenesis results of Loeb et al. We suggest that the over representation of G to A transitions observed in those studies may result from the high frequency of incorporation of 5-OHdCTP opposite G in RNA during first strand cDNA synthesis (Figure 6A). The duplex stoichiometries shown in Figure 5 reflect the fidelity values of Table 3, assuming retroviral replication in the presence of equimolar 5-OHdCTP, dCTP, and dTTP. Under these conditions, a 1:1 distribution of 5-OH-C‚G to C‚G base pairs in heteroduplex intermediates would be produced during RNA templated replication. In contrast 5-OH-C‚A pairs would be formed only once for every 40 A‚T base pairs; consistent with the lack of a dramatic increase in A to G mutations upon administration of 5-OH-dCTP. Base pairing op-

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Figure 6. Summary of heteroduplex and homoduplex stoichiometries arising from replication in the presence of equimolar 5-OH-dCTP, dCTP and dTTP for first (A) or second (B) strand cDNA synthesis by HIV-1 RT (RT). Only duplexes arising from reactions with fidelities of e40 are shown. RNA templates are denoted by wavy lines; DNA primers and templates by solid lines. Possible mutations arising from 5-OH-dCTP incorporation and subsequent replication of the indicated intermediates by either RT or DNA and RNA polymerases are also shown. See text for details.

posite 5-OH-C in template DNA by HIV-1 RT likely involves insertion of dGTP or dATP, although these frequencies were not explicitly measured in these studies. This assumption is reasonable in light of the low frequencies of pyr‚pyr base pairing observed on DNA templates for different polymerases. The available evidence indicates that insertion of dATP opposite 5-OH-C in DNA is generally preferred. In bacteria, virtually the only mutations arising from 5-OH-C substitution in template DNA are C to T transitions (36, 37). Formation of these base pairs by HIV-1 RT during second strand synthesis will contribute to G to A transitions in RNA. Possible mutagenic outcomes arising from incorporation of 5-OH-dCTP by HIV-1 RT during the DNA template directed replication step are shown in Figure 6B. Incorporation opposite A or G by HIV-1 RT occurs approximately once for every ten Watson-Crick pairing events. Integration of these 5-OH-C containing DNA duplexes into the host genome and subsequent DNA polymerase mediated replication of this DNA may alter the identity of the (-) strand A or G due to the ambiguous pairing potential of 5-OH-C. Assuming only a measurable contribution from purine base pair insertion opposite 5-HO-C by host DNA polymerases, Figure 6B suggests a rationale for the increases in U to C and C to U substitutions observed when HIV infected cells are passaged in the presence of 5-OH-dCTP. The greater increase (2-fold) of C relative to U (1.4-fold) transitions may reflect more favorable incorporation frequencies of dATP opposite 5-OH-C in DNA by host polymerase. The incorporation of dITP during first strand synthesis by HIV-1 RT might be expected to be of significance in light of evidence which suggests that the intracellular concentration of dITP in nucleotide precursor pools may be substantial (38). The fidelity data in Table 3 indicate, however, that with the exception of incorporation opposite cytosine, measured dITP insertion frequencies are

Wuenschell et al.

relatively low. If dITP incorporation by HIV-1 RT results almost exclusively in I‚C base pair formation, then dITP will merely substitute for dGTP during replication and no changes in the transcribed strand can occur. In contrast to 5-HO-dCTP, dITP should not be mutagenic in HIV. The “lethal mutagenesis” strategy represents a distinct departure from the typical deployment of nucleotide analogues as chain-terminating inhibitors of retroviral replication. The high incorporation observed in RNA and DNA for dITP may account in part for the efficacy of the retroviral analogue ddI (dideoxyinosine, didanosine) in HIV treatment protocols. It is interesting that exogenously added 5-OH-dCTP enhances a class of mutation which is already over represented in the HIV genome. G to A transitions account for ∼20% of all “spontaneous” base substitution mutations in env and TAR sequences in HIV (2). Factors contributing to this particular hypermutation have yet to be clearly defined. It still remains to be established whether dNTP precursor pools of HIV infected cells possess concentrations of 5-OH-dCTP sufficient to bias the mutation spectrum in this manner. However, the unusually high incorporation frequency of this base on RNA by HIV-1 RT and the ambiguous pairing potential exhibited during DNA templated replication provide a rationale for investigating a role in HIV mutagenesis.

Acknowledgment. We would like to thank Dr. Punna Lim and Vanessa Holland for discussion and comments on the manuscript. This work was supported by NIH Grant GM 53962 to J.T.

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