Low-Level Resistance to ( Immunodeficiency Virus ...

A New Point Mutation (P157S) in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Confers Low-Level Resistance to ( −)-β-2′,3′ -Dideoxy-3′-Thiacytidine Robert A. Smith, George J. Klarmann, Kirsten M. Stray, Uta K. von Schwedler, Raymond F. Schinazi, Bradley D. Preston and Thomas W. North Antimicrob. Agents Chemother. 1999, 43(8):2077.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1999, p. 2077–2080 0066-4804/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 8

A New Point Mutation (P157S) in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Confers Low-Level Resistance to (⫺)-␤-2⬘,3⬘-Dideoxy-3⬘-Thiacytidine ROBERT A. SMITH,1 GEORGE J. KLARMANN,1 KIRSTEN M. STRAY,1 UTA K. VON SCHWEDLER,1 RAYMOND F. SCHINAZI,2,3 BRADLEY D. PRESTON,1* AND THOMAS W. NORTH4 Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah 841121; Georgia VA Research Center for AIDS and HIV Infections2 and Department of Pediatrics, Emory University School of Medicine,3 Decatur, Georgia 30033; and Center for Comparative Medicine, University of California, Davis, California 956164 Received 18 November 1998/Returned for modification 8 March 1999/Accepted 13 May 1999

similarity with HIV-1 RT (35). The corresponding amino acid in HIV-1, P157, is predicted to reside in the “template grip” region of the enzyme and is proximal to M184, which is located in the active site of RT (10, 13, 15). In the present study, we examined changes in drug susceptibility resulting from the P157S mutation in HIV-1 RT. Virus containing the M184V mutation, which is commonly found in 3TC-resistant HIV-1 (33), was also constructed and used as a reference strain in these experiments. Drug susceptibilities were examined in cell culture, and inhibition constants for drug triphosphates were determined in kinetic assays with purified recombinant RTs. Infectivity of the P157S mutant. To determine if HIV-1 containing the P157S mutation in RT is replication competent, molecular clones containing P157S, M184V, or wild-type RT were assayed for the ability to produce infectious virions in a single round of replication. Mutations were constructed in the R9⌬Apa proviral clone (37) by using oligonucleotide-mediated mutagenesis (Muta-Gene phagemid mutagenesis kit; Bio-Rad) and the subcloning strategy of Iversen et al. (12). The presence of the desired mutations and the absence of additional changes were confirmed by automated DNA sequencing of the RTencoding region of the pol gene. The R9⌬Apa clone contains the gag, pol, and env genes from HIV-1NL4-3, with 5⬘ and 3⬘ long terminal repeats derived from HIV-1HXB2. Molecular clones were transfected into 293tsA1609neo (293T) cells for the production of virus (28). Genetic heterogeneity in the resulting stocks was minimal (⬍10⫺4 mutations per nucleotide [27]), as the 293T cultures do not express the CD4 receptor and therefore cannot be reinfected by progeny virions. Viral titers were quantitated by plating supernatants from 293T cultures onto P4 (HeLa-CD4-LTR-␤-galactosidase) indicator cells and staining with 5-bromo-4-chloro-3-indolyl-␤-Dgalactopyranoside (X-Gal) to develop blue foci (4). Titers from the focal assay were normalized against the p24 concentration (DuPont HIV-1 p24 enzyme-linked immunosorbent assay) to determine the infectivity of the mutants relative to wild-type virus (Table 1). In this single-cycle assay, P157S did not substantially differ from M184V or wild-type clones with respect to p24 production and infectivity of the resulting particles. Studies of the M184V mutant in spreading infections show that replication fitness is cell type dependent. Thus, M184V virus

The drugs 3TC [(⫺)-␤-2⬘,3⬘-dideoxy-3⬘-thiacytidine, also known as lamivudine] and AZT (3⬘-azido-3⬘-deoxythymidine, also known as zidovudine) are nucleoside analogs commonly used in the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1). The efficacy of these drugs in both monotherapy and combination therapy is limited by the emergence of drug-resistant variants (7, 17, 23, 32). Resistance to 3TC or AZT alone has been shown to result from specific mutations in the reverse transcriptase (RT)-encoding region of the pol gene (19, 33). However, the mechanism of resistance to the combination of 3TC plus AZT has remained somewhat elusive. Dual drug resistance in virus isolates derived from patients receiving 3TC plus AZT combination chemotherapy has recently been reported (23, 25). In one study, dual resistance was attributed to the combined presence of M184V (which alone confers ⬎100-fold resistance to 3TC [2, 9, 20, 34, 42]), the mutations commonly associated with AZT resistance, and several additional mutations in the N-terminal portion of RT (25). Passage of virus in culture in the presence of 3TC and AZT has also provided HIV-1 isolates resistant to both inhibitors (14). In this case, dual resistance is attributed to the presence of a previously undocumented mutation at codon 333 in genomes containing other AZT and 3TC resistance mutations. In addition, virus isolates containing either the Q151M V75I F77L F116Y mutation series or a threonine-to-serine mutation followed by an insertion of two amino acids at codon 69 of RT are resistant to AZT and dideoxynucleosides and possess 5- to 40-fold-decreased susceptibility to 3TC in vitro (12, 46). Our investigation of dual AZT-3TC resistance stems from previous work with the feline immunodeficiency virus (FIV). We recently reported the selection of 3TC-resistant mutants of FIV that contained a novel P156S mutation in RT (35). In addition to conferring 3TC resistance, the P156S mutation conferred low-level resistance to AZT alone and eightfold resistance to the combination of 3TC plus AZT (35). P156 is highly conserved in RTs from retroviruses and retroelements (8) and is located in a region which has 87% amino acid * Corresponding author. Mailing address: Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112. Phone: (801) 585-6342. Fax: (801) 585-3501. E-mail: [email protected]. 2077


A P157S mutation in the reverse transcriptase (RT) of human immunodeficiency virus type 1 conferred fivefold resistance to (ⴚ)-␤-2ⴕ,3ⴕ-dideoxy-3ⴕ-thiacytidine in cell culture. Interestingly, the P157S mutation resulted in increased sensitivity (two- to threefold) to 3ⴕ-azido-3ⴕ-deoxythymidine (AZT) and to (R)-9-(2phosphonylmethoxypropyl)adenine (PMPA). A similar increase in susceptibility to AZT and to PMPA was also conferred by the M184V mutation in RT.

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TABLE 1. Infectivity of M184V and P157S mutants relative to wild-type HIV-1 in CD4⫹ HeLa cellsa Virus

FFU/ml (105)

ng of p24/ml (103)

FFU/ng of p24 (102)

Wild-type M184V P157S

2.1 ⫾ 0.2 2.5 ⫾ 0.3 2.4 ⫾ 0.3

1.4 ⫾ 0.4 1.4 ⫾ 0.2 1.8 ⫾ 0.2

1.5 ⫾ 0.6 1.8 ⫾ 0.5 1.3 ⫾ 0.3

a Values are the means ⫾ standard errors of the means of at least three experiments. Each experiment included two determinations of focus-forming units (FFU) per milliliter and one determination of nanograms of p24 per milliliter.

Virus

Wild-type M184V P157S

EC50 (␮M)a 3TC

AZT

PMPA

1.3 ⫾ 0.2 (1) ⬎100 (⬎100) 6.1 ⫾ 1.8 (5)

0.16 ⫾ 0.07 (1) 0.07 ⫾ 0.02 (0.4) 0.06 ⫾ 0.01 (0.4)

2.2 ⫾ 0.4 (1) 1.0 ⫾ 0.2 (0.5) 0.9 ⫾ 0.1 (0.4)

a EC50s were obtained from the linear portion of dose-response plots (Fig. 1) by using computer-generated regression lines. Numbers in parentheses indicate EC50s relative to those of the wild-type virus. Values are the means ⫾ standard errors of the means from two or more experiments, with four determinations per experiment.

M184V variants displayed a fivefold increase in susceptibility to AZT when assayed in MT-2 cells (34). In addition, recent studies demonstrate that two- to fivefold AZT hypersensitivity is conferred by M184V both in the HXB2D clonal background (44) and in recombinant constructs containing patient-derived RTs (22, 24, 41). Variability in reports of the AZT susceptibility of M184V HIV-1 may reflect differences in the genetic backgrounds used to select or construct the mutant or differences in the sensitivities of the particular assays used to quantitate drug susceptibility. Cell culture assays with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) revealed that the M184V and P157S mutations also confer a twofold increase in sensitivity to PMPA (Table 2). This result is consistent with recent reports that virus isolates containing the M184V mutation display increased susceptibility to PMPA (22, 44). Taken together, these data suggest that P157S and M184V mutants may be more effectively suppressed than wild-type virus by PMPA and AZT in vivo. AZT hypersensitivity may in part account for the observed delay in the appearance of AZT resistance mutations in viruses containing M184V (41). A comparison of HIV-1 P157S and FIV P156S drug susceptibility reveals important similarities and differences between these two closely related lentiviruses. The resistance of both variants to 3TC demonstrates the utility of the FIV model for discovering candidate drug-resistant mutants of HIV-1 which

FIG. 1. Susceptibility of a wild-type strain (■) and M184V (E) and P157S (‚) mutants of HIV-1 to inhibition by 3TC (A) and AZT (B) in CD4⫹ HeLa cells. Foci were detected as described in the text. Data are plotted as the percentage of control foci (no drug) versus inhibitor concentration. Results are from two or more assays, with four determinations per assay. Bars represent standard errors of the means.


exhibits reduced fitness relative to wild-type HIV-1 in peripheral blood mononuclear cells but not in a T-cell line (1). Our data indicate that both the M184V and P157S mutants replicate at near wild-type levels in CD4⫹ HeLa cells. However, subtle differences in replication capacity that are magnified over multiple rounds of replication in a spreading infection (5, 30) would not be detected in our single-cycle assay. Drug susceptibility in culture. To examine the potential role of the P157S mutation in dual AZT-3TC resistance, we first determined the relative susceptibility of P157S virus to inhibition by 3TC (Fig. 1A). Drug susceptibility was determined by measuring the dose-dependent reduction of focus formation in CD4⫹ HeLa indicator cell cultures. Concentrations of drug required to inhibit focus formation by 50% (50% effective concentrations [EC50s]) are summarized in Table 2. P157S conferred fivefold resistance to 3TC, compared to the ⬎100fold resistance resulting from the M184V mutation. The lowlevel 3TC resistance conferred by P157S in HIV-1 is comparable to that observed in P156S mutants of FIV (eightfold) (35). We also determined the susceptibility of P157S, M184V, and wild-type virus to AZT (Fig. 1B). Surprisingly, both P157S and M184V were slightly hypersensitive to AZT, with EC50s twoto threefold lower than that for wild-type virus (Table 2). Initial reports describe little or no change in AZT sensitivity for M184V relative to wild-type virus (2, 9, 20, 34, 42), with one exception: a virus isolate selected with (⫺)-␤-2⬘,3⬘-dideoxy-5fluoro-3⬘-thiacytidine and containing a mixture of M184I and

TABLE 2. Susceptibilities of wild-type, M184V, and P157S HIV-1 to antiviral compounds in CD4⫹ HeLa cells

VOL. 43, 1999

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TABLE 3. Kinetic constants for wild-type, M184V, and P157S HIV-1 RTsa Fold increase in Ki/Km

RT

Substrate

Km (␮M)

Inhibitor

Ki (␮M)

Ki/Km

Wild-type

dTTP dCTP

20 ⫾ 5.2 13 ⫾ 1.4

AZTTP 3TCTP

0.015 ⫾ 0.0047 0.280 ⫾ 0.034

0.00075 0.022

M184V

dTTP dCTP

25 ⫾ 2.5 17 ⫾ 1.9

AZTTP 3TCTP

0.037 ⫾ 0.0059 61 ⫾ 8.1

0.0015 3.7

2 200

P157S

dTTP dCTP

25 ⫾ 12 20 ⫾ 2.1

AZTTP 3TCTP

0.064 ⫾ 0.018 2.3 ⫾ 0.22

0.0026 0.12

3 5

a Values are the means ⫾ standard errors of the means of at least two experiments, with three determinations per experiment. All three RTs were expressed in E. coli and purified as p66-p51 heterodimers (36). The mode of inhibition by 3TCTP or AZTTP was competitive with respect to the substrate (data not shown). Template-primers were poly(rA)-oligo(dT)12–18 for dTTP and AZTTP and poly(rI)-oligo(dC)21 for dCTP and 3TCTP. Specific reaction conditions (45) and analysis of the resulting data (6, 26) are described elsewhere.

frequencies prior to and/or during the course of therapy (5) and may contribute to the development of high-level drug resistance in 3TC-treated individuals (5, 30). Contribution of RT template grip to drug susceptibility. Cocrystal structures of HIV-1 RT bound to template-primer show that P157 is located near the N terminus of helix ␣E, which contributes to the “template grip” functionality of the enzyme (10, 13). P157 interacts with the minor groove of the template strand, making van der Waals contacts with both the sugar and base moieties of the nucleotide located two residues from the catalytic active site. P157 does not directly interact with the incoming deoxynucleoside triphosphate. Thus, the altered drug susceptibility conferred by P157S must be due to an indirect effect on drug triphosphate recognition at the active site. This idea is supported by the observation that other mutations in the template grip region of RT resulted in resistance to phosphonoformic acid (a pyrophosphate analog) and altered susceptibility to a variety of nucleoside analogs (11, 21, 29, 38–40, 43). Boyer et al. (3) propose that the template grip mutant E89G confers drug resistance by repositioning the template-primer at the active site, thereby perturbing the precise relative position of protein and nucleic acid required for normal substrate recognition. This mechanism might also explain the alterations in drug susceptibility resulting from P157S and other mutations in the template grip of RT. Additional biochemical, biophysical, and structural studies are required to directly test this model. This work was supported by Public Health Service grants R01 AI34834, R01 AI38755, and P30 CA42014 to B.P., R01 AI28189 to T.N., and F32 AI10139 to R.A.S. from the National Institutes of Health and by the Department of Veterans Affairs and the Georgia Research Center on AIDS and HIV Infection (R.F.S.). REFERENCES 1. Back, N. K., M. Nijhuis, W. Keulen, C. A. Boucher, B. O. Oude Essink, A. B. van Kuilenburg, A. H. van Gennip, and B. Berkhout. 1996. Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO J. 15:4040–4049. 2. Boucher, C. A. B., N. Cammack, P. Schipper, R. Schuurman, P. Rouse, M. A. Wainberg, and J. M. Cameron. 1993. High-level resistance to (⫺) enantiomeric 2⬘-deoxy-3⬘-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob. Agents Chemother. 37:2231–2234. 3. Boyer, P. L., C. Tantillo, A. Jacobo-Molina, R. G. Nanni, J. Ding, E. Arnold, and S. H. Hughes. 1994. Sensitivity of wild type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug resistant mutants does not. Proc. Natl. Acad. Sci. USA 91:4882–4886. 4. Charneau, P., G. Mirambeau, P. Roux, S. Paulous, H. Buc, and F. Clavel. 1994. HIV 1 reverse transcription. A termination step at the center of the genome. J. Mol. Biol. 241:651–662. 5. Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic


otherwise may not have been identified. However, the P157S variant of HIV-1 does not share the AZT-resistant phenotype of the FIV mutant and is instead slightly hypersensitive to the drug. This result should be considered in future experiments when FIV is used for studying resistance to the combination of 3TC plus AZT. Inhibitor susceptibilities of purified RTs. Changes in drug susceptibility resulting from the P157S mutation were further characterized in cell-free RT assays. P157S, M184V, and wildtype RTs were expressed in Escherichia coli and purified as p66-p51 heterodimers (36). The resulting enzyme preparations contained equal ratios of each subunit and were approximately 95% pure as judged by Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels (data not shown). Sensitivities of the RTs to the 5⬘-triphosphate forms of 3TC (3TCTP) and AZT (AZTTP) were compared in kinetic assays (Table 3). Wild-type and mutant RTs exhibited similar apparent Km values for dCTP and dTTP, ranging from 13 to 25 ␮M. Based on Ki/Km ratios (45), the P157S and M184V RTs had 5- and 200-fold-increased resistance to 3TCTP, respectively. These results parallel the trend in 3TC sensitivity observed with cultured virus (wild-type ⬎ P157S ⬎⬎ M184V) (Fig. 1A and Table 2). Although the M184V and P157S viruses showed increased sensitivity to AZT in culture (Fig. 1B and Table 2), the purified RTs containing these mutations both showed modest AZTTP resistance (two- and threefold, respectively) (Table 3). Similar discrepancies between virus susceptibility to AZT and inhibition of purified RTs by AZTTP have been noted in other studies (18, 31). The biochemical basis of this discordance is not fully understood, but it is clear that conventional assays used to measure AZTTP susceptibilities of purified RTs do not accurately reflect the susceptibilities of virus isolates to AZT. Existence of low-level 3TC-resistant variants in vivo. The P157S mutation described here was detected in a significant proportion of cloned RT sequences from a patient (C0034b) after 2 years of AZT-plus-3TC combination chemotherapy (25); M184V, five AZT resistance mutations (at amino acid positions 41, 67, 70, 215, and 219), and other mutations were also present in the sequences from this individual. A search of 912 viral sequences deposited in the Los Alamos HIV database also revealed the presence of the P157S mutation in the pol gene sequence from a German isolate of HIV-1 (clone NH51, accession no. LO7423) submitted in 1992 (16). Other point mutations conferring low-level resistance to 3TC (e.g., K70E, K65R, and V75T) are also occasionally observed in isolates from patients receiving nucleoside analog therapy (33). The clinical significance of these rare variants is not known. It is likely that such mutations exist in viral populations at low

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26.

27. 28. 29.

30. 31. 32. 33. 34.

35.

36. 37. 38. 39. 40. 41.

42.

43.

44.

45.

46.

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