N348I in HIV-1 reverse transcriptase decreases susceptibility to tenofovir and etravirine in combination with other resistance mutations.
Sluis-Cremer, Nicolasa; Moore, Katieb; Radzio, Jessicaa; Sonza, Secondob,c; Tachedjian, Gildab,c,d
aUniversity of Pittsburgh School of Medicine, Department of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA
bMolecular Interactions Group, Centre for Virology, Burnet Institute, Melbourne, Australia
cDepartment of Microbiology, Monash University, Clayton, Australia
dDepartment of Medicine, Monash University, Melbourne, Victoria, Australia.
Received 8 June, 2009
Revised 19 July, 2009
Accepted 29 July, 2009
Correspondence to Gilda Tachedjian, PhD, Molecular Interactions Group, Centre for Virology, Burnet Institute, GPO Box 2284, Melbourne, VIC 3001, Australia. Tel: +61 3 9282 2256; fax: +61 3 9282 2100; e-mail: email@example.com
We previously demonstrated that N348I in HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance. However, both of these inhibitors are currently infrequently used in developed countries, and the impact of N348I on newer reverse transcriptase inhibitors, such as tenofovir and etravirine, is unknown. In this study, we demonstrate that N348I alone confers no resistance to tenofovir and low-level resistance to etravirine. However, N348I significantly decreases tenofovir susceptibility when combined with thymidine analogue mutations and etravirine susceptibility when combined with Y181C.
We recently identified the N348I mutation in the connection domain of the HIV-1 reverse transcriptase that confers resistance to both zidovudine (AZT) and nevirapine . N348I is highly prevalent in reverse transcriptase inhibitor (RTI)-experienced patients [1–5], occurs early in therapy usually prior to the appearance of recognized thymidine analogue mutations (TAMs)  and is associated with an increase in viremia . In our study, N348I was selected by antiretroviral treatments (ARTs) that included AZT or the combination of AZT and nevirapine . N348I has also been reported to confer resistance to didanosine and delavirdine, and its emergence in a Japanese cohort was primarily associated with AZT or didanosine-containing therapies, or both .
The use of AZT, didanosine and nevirapine in ARTs in the developed world has been largely replaced with more potent and less toxic RTIs . For example, the International AIDS Society-USA panel recommends either tenofovir/emtricitabine (Truvada) or abacavir/lamivudine in combination with efavirenz or ritonavir-boosted protease inhibitor for initial combination therapy . Truvada is also used in the treatment of antiretroviral-experienced patients, as is the new nonnucleoside reverse transcriptase inhibitor (NNRTI), etravirine . The genotypic determinants of tenofovir and etravirine resistance have been established. Decreased susceptibility to tenofovir in vitro and in vivo is associated with the K65R mutation or the presence of three or more TAMs (e.g. M41L, L210W and T215Y) [7–10]. Decreased etravirine susceptibility requires at least three NNRTI-resistant mutations [11–14]. Surprisingly, etravirine activity is not compromised by the K103N mutation . To date, it has not been established whether N348I can reduce susceptibility to tenofovir or etravirine and compromise drug activity in treatment-experienced patients. Accordingly, in this study, we determined whether N348I alone, or in combination with TAMs or Y181C, decreased susceptibility to tenofovir or etravirine.
N348I was introduced by site-directed mutagenesis into the background of wild-type, K103N, Y181C, M41L/L210Y and M41L/L210W/T215Y expressing reverse transcriptase genes of the pNL4.3 (NL) or HXB-2 (HX) infectious molecular clones [15,16]. HIV was recovered by transfection of 293T cells, and drug susceptibility assays were performed in the TZM-bl indicator cell line, as described previously  with the exception that HIV replication was determined by measuring luciferase activity using the Steady-Glo Luciferase Assay System according to manufacturer's instructions (Promega, Madison, Wisconsin, USA). Statistically significant differences in the 50% effective dose (EC50) were determined using the Wilcoxon rank sum test .
Our data (Table 1) demonstrate that N348I (NL/348) alone conferred a 1.6-fold decrease (P = 0.019, n = 4) in etravirine susceptibility as compared with the corresponding wild-type strain. By comparison, Y181C conferred 2.2-fold resistance to etravirine (P = 0.02, n = 4), whereas K103N did not confer a significant change in etravirine susceptibility as compared with wild-type. When combined with K103N, no decrease in etravirine susceptibility was observed as compared with K103N alone, whereas a small decrease in etravirine susceptibility was seen as compared with wild-type (P = 0.019, n = 5) (Table 1). By contrast, when N348I was combined with Y181C, etravirine susceptibility was decreased 6.4-fold (P = 0.02, n = 4) relative to wild-type virus and 2.9-fold (P = 0.03, n = 4) relative to Y181C HIV-1 (NL/181) (Table 1). Consistent with this finding, the Y181C/N348I double mutation also significantly decreased etravirine susceptibility at the enzyme level (data not shown). Taken together, these data demonstrate that N348I confers a small decrease in susceptibility to etravirine and significantly potentiates etravirine resistance in the context of Y181C but not K103N.
As reported previously , HIV-1-containing N348I conferred no significant increase in tenofovir EC50 as compared with the corresponding wild-type strain (Table 1). However, when combined with M41L and T215Y (NL/2AZT), N348I decreased tenofovir susceptibility by 1.7-fold (P = 0.014, n = 4) as compared with wild-type. By contrast, the NL/2AZT strain was susceptible to tenofovir (Table 1). N348I also increased tenofovir resistance when combined with M41L, L210W and T215Y (HX/3AZT) by six-fold as compared with wild-type (P = 0.009, n = 5) and three-fold as compared with the HX/3AZT strain (P = 0.008, n = 4). In this regard, A371V and Q509L in the connection and RNase H domains, respectively , and mutations located at residues that form part of the RNase H primer grip [20,21] potentiate resistance to tenofovir in cell culture-based assays when combined with TAMs. Taken together, these data demonstrate that N348I decreases tenofovir susceptibility in the presence of TAMs, and notably, this effect is observed with less than three TAMs.
According to the International AIDS Society-USA panel drug-resistant mutations update, the presence of three or more TAMs inclusive of M41L and L210W is expected to give a reduced in-vivo response to tenofovir . Therefore, in current genotyping algorithms, tenofovir could be prescribed in the presence of two TAMs (e.g. M41L and T215Y) and N348I, which may result in reduced in-vivo drug efficacy. Similarly, we have shown that N348I enhances resistance to etravirine in the context of Y181C, a mutation that is associated with reduced virological response in vivo [13,14]. As the presence of three or more NNRTI mutations V90I, A98G, L100I, K101E/P, V106I, V179D/F, Y181C/I/V and G190A/S results in no response to etravirine treatment [13,14], the presence of two of these NNRTI mutations and N348I at baseline may also reduce etravirine efficacy in vivo.
N348I is not a polymorphism. It is found in treatment-experienced but rarely in treatment-naive individuals infected with HIV-1 clades A, B, AE, AG, C, D, F and G (http://hivdb.stanford.edu/cgi-bin/AgMutPrev.cgi). Furthermore, the prevalence of Y181C and K103N is 11% and 22%, respectively. Accordingly, at least one in 10 HIV-infected patients will have Y181C prior to etravirine exposure, particularly in patients failing first-line ARTs in resource-poor settings due to the continued use of nevirapine . Accordingly, the acquisition of N348I in HIV-1 reverse transcriptase may significantly impact both first and second-line ARTs in resource-poor settings.
Taken together, our in-vitro data warrant studies to determine the clinical significance of the appearance of a preexisting N348I mutation in regimens containing tenofovir or etravirine.
This study was supported by the National Health and Medical Research Council of Australia (NHMRC) Senior Research Fellowship 543105 and NHMRC Project Grant 433903 awarded to G.T. N.S-C. was supported by a grant (R01 AI081571) from the United States National Institutes of Health. We thank the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute for Allergy and Infectious Diseases, NIH for the supply of etravirine, tenofovir and the TZM-bl indicator cell line. We also thank P. Richard Harrigan for critically reading the manuscript.
N.S-C., S.S. and G. T. designed the study, analyzed the data and wrote the manuscript. K.M. and J. R. performed all experiments described in the study.
There are no conflicts of interest.
1. Yap SH, Sheen CW, Fahey J, Zanin M, Tyssen D, Lima VD, et al
. N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance. PLoS Med 2007; 4:e335.
2. Hachiya A, Kodama EN, Sarafianos SG, Schuckmann MM, Sakagami Y, Matsuoka M, et al
. Amino acid mutation N348I in the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase confers multiclass resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors. J Virol 2008; 82:3261–3270.
3. Cane PA, Green H, Fearnhill E, Dunn D. Identification of accessory mutations associated with high-level resistance in HIV-1 reverse transcriptase. AIDS 2007; 21:447–455.
4. Santos AF, Lengruber RB, Soares EA, Jere A, Sprinz E, Martinez AM, et al
. Conservation patterns of HIV-1 RT connection and RNase H domains: identification of new mutations in NRTI-treated patients. PLoS ONE 2008; 3:e1781.
5. Waters JM, O'Neal W, White KL, Wakeford C, Lansdon EB, Harris J, et al
. Mutations in the thumb-connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients. Antivir Ther 2009; 14:231–239.
6. Hammer SM, Eron JJ Jr, Reiss P, Schooley RT, Thompson MA, Walmsley S, et al
. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA 2008; 300:555–570.
7. Wainberg MA, Miller MD, Quan Y, Salomon H, Mulato AS, Lamy PD, et al
. In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA. Antivir Ther 1999; 4:87–94.
8. Miller MD, Margot N, Lu B, Zhong L, Chen SS, Cheng A, et al
. Genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients. J Infect Dis 2004; 189:837–846.
9. Barrios A, de Mendoza C, Martin-Carbonero L, Ribera E, Domingo P, Galindo MJ, et al
. Role of baseline human immunodeficiency virus genotype as a predictor of viral response to tenofovir in heavily pretreated patients. J Clin Microbiol 2003; 41:4421–4423.
10. Gallant JE, Staszewski S, Pozniak AL, DeJesus E, Suleiman JM, Miller MD, et al
. Efficacy and safety of tenofovir DF vs stavudine in combination therapy in antiretroviral-naive patients: a 3-year randomized trial. JAMA 2004; 292:191–201.
11. Andries K, Azijn H, Thielemans T, Ludovici D, Kukla M, Heeres J, et al
. TMC125, a novel next-generation nonnucleoside reverse transcriptase inhibitor active against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus type 1. Antimicrob Agents Chemother 2004; 48:4680–4686.
12. Vingerhoets J, Azijn H, Fransen E, De Baere I, Smeulders L, Jochmans D, et al
. TMC125 displays a high genetic barrier to the development of resistance: evidence from in vitro selection experiments. J Virol 2005; 79:12773–12782.
13. Madruga JV, Cahn P, Grinsztejn B, Haubrich R, Lalezari J, Mills A, et al
. Efficacy and safety of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-1: 24-week results from a randomised, double-blind, placebo-controlled trial. Lancet 2007; 370:29–38.
14. Lazzarin A, Campbell T, Clotet B, Johnson M, Katlama C, Moll A, et al
. Efficacy and safety of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-2: 24-week results from a randomised, double-blind, placebo-controlled trial. Lancet 2007; 370:39–48.
15. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, et al
. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986; 59:284–291.
16. Fisher AG, Collalti E, Ratner L, Gallo RC, Wong-Staal F. A molecular clone of HTLV-III with biological activity. Nature 1985; 316:262–265.
17. Bhattacharyya GK, Johnson RA. Statistical concepts and methods. New York: John Wiley and Sons; 1977.
18. Hachiya A, Shimane K, Sarafianos SG, Kodama EN, Sakagami Y, Negishi F, et al
. Clinical relevance of substitutions in the connection subdomain and RNase H domain of HIV-1 reverse transcriptase from a cohort of antiretroviral treatment-naive patients. Antiviral Res 2009; 82:115–121.
19. Brehm JH, Koontz D, Meteer JD, Pathak V, Sluis-Cremer N, Mellors JW. Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type 1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine. J Virol 2007; 81:7852–7859.
20. Delviks-Frankenberry KA, Nikolenko GN, Barr R, Pathak VK. Mutations in human immunodeficiency virus type 1 RNase H primer grip enhance 3'-azido-3'-deoxythymidine resistance. J Virol 2007; 81:6837–6845.
21. Sarafianos SG, Das K, Tantillo C, Clark AD Jr, Ding J, Whitcomb JM, et al
. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. EMBO J 2001; 20:1449–1461.
22. Johnson VA, Brun-Vezinet F, Clotet B, Gunthard HF, Kuritzkes DR, Pillay D, et al
. Update of the drug resistance mutations in HIV-1. Top HIV Med 2008; 16:138–145.
23. Keiser O, Anastos K, Schechter M, Balestre E, Myer L, Boulle A, et al
. Antiretroviral therapy in resource-limited settings 1996 to 2006: patient characteristics, treatment regimens and monitoring in sub-Saharan Africa, Asia and Latin America. Trop Med Int Health 2008; 13:870–879.
This article has been cited 3 time(s).
Etravirine: A unique non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1
Antiviral ResearchMolecular basis of human immunodeficiency virus type 1 drug resistance: Overview and recent developmentsAntiviral Research
Current Opinion in VirologyDrug resistance in HIV-1Current Opinion in Virology
© 2010 Lippincott Williams & Wilkins, Inc.