Impact of human leukocyte antigen-B*51-restricted cytotoxic T-lymphocyte pressure on mutation patterns of nonnucleoside reverse transcriptase inhibitor resistance
Gatanaga, Hiroyukia,b; Ode, Hirotakad; Hachiya, Atsukoa,c; Hayashida, Tsunefusaa,b; Sato, Hironorid; Takiguchi, Masafumic; Oka, Shinichia,b
aAIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan
bDivision of Infectious Disease, Japan
cDivision of Viral Immunology, Center for AIDS Research, Kumamoto University, Kumamoto, Japan
dLaboratory of Viral Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
Received 9 November, 2009
Revised 28 December, 2009
Accepted 19 January, 2010
Correspondence to Hiroyuki Gatanaga, MD, AIDS Clinical Center, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Tel: +81 3 3202 7181; fax: +81 3 5273 6483; e-mail: firstname.lastname@example.org
Objective: The objective of this study is to determine the impact of human leukocyte antigen (HLA)-B*51-restricted cytotoxic T-lymphocyte (CTL) pressure on the development of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance.
Design: The prevalence of HIV-1 harboring an escape mutation, I135X, in a major epitope of HLA-B*51-restricted CTL located in reverse transcriptase is increasing worldwide. We analyzed the effects of escape mutations on the emerging mutation patterns of NNRTI resistance.
Methods: Monoclonal HIV-1 sequences harboring each of the escape mutations, including I135L (HIV-1I135L), I135V (HIV-1I135V), I135T (HIV-1I135T), and I135R (HIV-1I135R) in reverse transcriptase, and a wild-type monoclonal HIV-1 (HIV-1WT) were cultured in the presence of increasing concentrations of efavirenz. Induced mutations during culture passages of the culture were analyzed.
Results: E138K emerged during the cultural passages of HIV-1I135V, HIV-1I135T, and HIV-1I135R, but not during the passages of HIV-1WT. The combination of I135T, the most frequent escape mutation, and E138K (HIV-1I135T/E138K) conferred significant resistance to efavirenz, nevirapine, and etravirine. The HIV-1I135L/E138K and HIV-1I135R/E138K were significantly resistant to nevirapine and etravirine, respectively, though each solo of escape mutations and E138K did not confer significant resistance to NNRTI. Computational analysis indicated that I135T and E138K cooperatively extend the gap between the binding site of reverse transcriptase and NNRTI.
Conclusion: HLA-B*51-restricted CTL can induce novel mutation patterns of NNRTI resistance by selecting escape mutations. The spread of CTL escape variants may alter the mutation patterns of drug resistance.
Cytotoxic T lymphocytes (CTLs) are one of the antiretroviral host factors that can modify the clinical course of HIV-1 infection . However, HIV-1 evades these cells by acquiring escape mutations in recognized epitopes, and some of the CTL-escape variants remain stable without reversion even in the absence of such selective pressure . TAFTIPSI (reverse transcriptase 128–135) is a major epitope recognized by human leukocyte antigen (HLA)-B*51-restricted CTL , and we recently reported that its escape mutation, I135X, is detected in the majority of HLA-B*51-positive infected individuals and also in a significant proportion of HLA-B*51-negative individuals, and that I135X can exist persistently even in HLA-B*51-negative individuals probably because it does not cause a significant fitness cost . Consequently, I135X can spread as a polymorphic mutation among infected individuals and has in fact accumulated in the HIV-positive populations, especially among the Japanese, in whom HLA-B*51 is highly prevalent. Previous studies reported that I135X was associated with low-level resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) [5–7] and suggested that I135X may be a determinant of evolutional patterns of NNRTI resistance [8,9], though it has also been reported that there is no correlation between the presence of I135X at baseline and efficacy of NNRTI . To determine whether CTL escape mutations alter the development of drug resistance, we focused on I135X and induced NNRTI resistance from I135X-harboring HIV-1s by cultural passages in the presence of increasing concentrations of efavirenz (EFV).
Materials and methods
HIV-1 sequences and human leukocyte antigen types in treatment-naive patients
We recently reported the frequent prevalence of I135X mutations in Japan . To confirm the same and to determine the frequency of each mutation, we used another cohort that included 575 treatment-naive newly diagnosed HIV/AIDS patients recruited from across Japan between January 2003 and December 2004 . Among them, data of HLA typing were available for 97 patients.
Generation of recombinant HIV-1 sequences
The desired mutations were introduced into the XmaI-NheI region of pTZNX, which encodes Gly-15 to Ala-267 of HIV-1 reverse transcriptase (strain BH10) . The XmaI-NheI fragment was inserted into pNLH219Q, which was modified from pNL101 and encoded the full genome of HIV-1. Each molecular clone was transfected into COS-7 cells, and the obtained virions were harvested 48 h after transfection and stored at −80°C until use.
Induction of efavirenz-resistant HIV-1
The infectious HIV-1 clones were propagated in MT-2 cells in the presence of increasing concentrations of EFV . Briefly, MT-2 cells (1 × 105) were exposed to 500 blue cell-forming units (BFUs) in MAGIC-5 cells (CCR5-expressing and CD4-expressing HeLa-LTR-β-D-gal cells) of each monoclonal HIV-1 and cultured in the presence of EFV at an initial concentration of 3 nmol/l. The culture supernatant was harvested on day 7 of culture and used to infect fresh MT-2 cells for the next round of culture. When the virus began to propagate in the presence of the drug, the drug concentration was increased by half-log fold. This selection was carried out until the EFV concentration reached 1000 nmol/l. Proviral HIV-1 reverse transcriptase gene in the infected MT-2 cells was amplified and sequenced at several passages.
Drug susceptibility assay
EFV and nevirapine (NVP) were generously provided by Merck Co., Inc. (Rahway, New Jersey, USA) and Boehringer Ingelheim Pharmaceutics Inc. (Ridgefield, Connecticut, USA), respectively. Etravirine (ETR) was purchased from Toronto Research Chemicals Inc. (North York, Ontario, Canada). Recombinant HIV-1 susceptibility to EFV, NVP, and ETR was determined in triplicate using MAGIC-5 cells . The drug susceptibility assay was performed in triplicate and repeated three times. Fold resistance was calculated by comparing viral IC50 with that of monoclonal wild-type HIV-1 (HIV-1WT). Drug resistance was considered significant when it was higher than three-fold.
We constructed structural models of the HIV-1 reverse transcriptase and NNRTI complex by computational analysis. First, we constructed the initial models of wild-type reverse transcriptase with one of the three NNRTIs by homology modeling using Molecular Operating Environment (MOE) 2007.09.02 (http://www.chemcomp.com/). The crystal structures of reverse transcriptase with NNRTI (PDB code: 1IKW , 1VRT , and 1SV5 ) were used for template structures. The ff94 force field and distance-dependent electrostatic energy function were applied in the modeling. Next, we refined the initial models by energy minimization using sander module of AMBER9 software package through two steps. In the first step, energies for the NNRTI in the complex models were minimized at the gas phase by the conjugated gradient method. In the second step, energies of whole structures were converged up to 0.5 kcal/mol/Å by 50 steps of the steepest descent method and the subsequent conjugated gradient method at implicit water solvent condition. In each minimization, the AMBER ff03 [16,17], the general AMBER force field (gaff) , and the generalized Born implicit solvent surface area (GBSA) method (IGB = 2)  were applied for potential energy calculations. The charges and atom types of every atom in NNRTI were automatically assigned using the AMBER9 Antechamber module. We also constructed the respective mutant reverse transciptases with the NNRTI by considering every possible conformer of the respective mutant models. The possible conformers were generated from the wild-type homology models using PyMOL version 0.99rc6 (http://www.pymol.org). The structural model of each conformer was refined by a method similar to that used in the wild-type models. Among the refined conformers, we selected those with the lowest energy as each mutant model.
The 135th amino acid in HIV-1 reverse transcriptase and human leukocyte antigen-B*51
We analyzed the relationship between HLA-B*51 and the 135th amino acid of HIV-1 reverse transciptase in 97 infected individuals newly diagnosed in Japan between January 2003 and December 2004 (Table 1). As expected, CTL escape mutations I135X, including I135T, I135L, and I135V, were observed in all but one HLA-B*51-positive patient (94.1%), representing a significantly higher prevalence than in the HLA-B*51-negative patients (Fisher's exact test; P = 0.01). However, in the HLA-B*51-negative patients, escape mutations were still observed at a high frequency (62.5%), indicating that I135X variants can transmit from HLA-B*51-positive patients to HLA-B*51negative individuals and can persist even in the absence of HLA-B*51-restricted CTL pressure. Overall, I135X mutations were observed at a high frequency in the treatment-naive patients in Japan, and the most frequent amino acid was I135T (35.1%), which was more frequent than the wild-type I135 (32.0%).
Induction of efavirenz-resistant HIV-1
As described above, I135L, I135V, I135T, and I135R mutations were detected in treatment-naive patients. In order to analyze their effects on the mutation pattern for NNRTI resistance, EFV resistance was induced from monoclonal HIV-1s harboring each of these mutations by culturing them in the presence of increasing concentrations of EFV. These induction experiments were performed independently in triplicate. In one of the three induction experiments on HIV-1I135L, V179D emerged when EFV concentration reached 100 nmol/l, as well as emergence of K103R in the presence of EFV at 1000 nmol/l (Fig. 1a). We previously reported that the combination of K103R and V179D confers significant resistance to NNRTIs . In another experiment, V108I emerged at an EFV concentration of 100 nmol/l and L100I at an EFV of 1000 nmol/l (Fig. 1b). Both L100I and V108I are listed in the International AIDS Society (IAS)-USA Resistance Table  as EFV resistance mutations. In the last experiment on HIV-1I135L, G190A emerged followed by V106A (Fig. 1c). The latter two are also listed in the IAS-USA Table. In one of the three induction experiments on HIV-1I135V, E138K emerged at an EFV of 100 nmol/l and L100I at an EFV of 1000 nmol/l (Fig. 1d). E138K is a rare mutation and not listed as a resistance mutation in the IAS-USA Table. It was reported that E138K alone did not alter drug susceptibility significantly, though it emerged during resistance induction experiments with ETR and other experimental NNRTIs (Brillant et al. 13th International HIV Drug Resistance Workshop, 2004; Su et al. 16th International HIV Drug Resistance Workshop, 2007) [21–23]. L100I emerged first followed by Y188H in another experiment, and L100I emerged first followed by V108I in the last experiment (Figure 1e and f). In one of the three induction experiments on HIV-1I135T, V108I emerged at an EFV of 100 nmol/l and K101E at an EFV of 1000 nmol/l (Fig. 2a). In another experiment, V106I emerged first followed by V179D (Fig. 2b). The combination of V106I and V179D was confirmed to confer a significant NNRTI resistance by our group (unpublished data). In the last experiment, V108I emerged first followed by E138K and L100I (Fig. 2c). In one of the three induction experiments on HIV-1I135R, L100I emerged at an EFV of 100 nmol/l followed by E138K at an EFV of 1000 nmol/l (Fig. 2d). In another experiment, E138K emerged first then G190A and V108I (Fig. 2e). In the last experiment, L100I emerged first followed by K101E (Fig. 2f). In summary, during the induction experiments, all the induced mutations were already known NNRTI-resistance mutations except for E138K, which emerged in one of the three induction experiments on HIV-1I135V, in one of the three induction experiments on HIV-1I135T, and in two of the three induction experiments on HIV-1I135R. We also performed EFV-resistance induction experiments on HIV-1WT in triplicate using the same procedure. All the induced mutations were already known NNRTI-resistance mutations, whereas E138K did not emerge in any of the three induction experiments on HIV-1WT (data not shown).
Nonnucleoside reverse transcriptase inhibitor resistance conferred by E138K combined with I135X
During the induction experiments on HIV-1s harboring I135X, the emergence of E138K, which is usually a rare mutation, was often observed. To analyze the effects of E138K alone and its combination with I135X on NNRTI susceptibility, a panel of recombinant HIV-1 clones was constructed and their IC50 values for EFV, NVP, and ETR were determined. As expected, I135X alone did not confer significant NNRTI resistance (Table 2). The combination of I135T and E138K (I135T/E138K) conferred significant resistance to EFV, NVP, and ETR, though E138K alone did not change NNRTI susceptibility as reported previously (Su et al. 16th International HIV Drug Resistance Workshop, 2007) [22,23]. I135L/E138K and I135R/E138K conferred significant resistance to NVP and ETR, respectively. In summary, E138K conferred significant resistance when combined with some of the I135X mutations, especially I135T, which is the most prevalent in treatment-naive individuals in Japan (Table 1).
Structural modeling of reverse transcriptase harboring I135T and E138K
The in-vitro drug susceptibility assay described above showed that I135T/E138K conferred the most efficient resistance to EFV and significant resistance to NVP and ETR. To analyze the molecular mechanisms by which E138K combined with I135T alter NNRTI susceptibility, we conducted a structural analysis that included computational methods. A total of 12 structural models of reverse transcriptase-NNRTI complexes were constructed with four reverse transcriptases (wild-type, I135T, E138K, and I135T/E138K) and three NNRTIs (EFV, NVP, and ETR). We first calculated the binding energies between reverse transcriptase and NNRTI. Differences in the binding energies between mutant and wild-type complexes (ΔΔGb) were calculated using the models. The ΔΔGb value correlated positively with the logarithm of fold resistance value obtained by our in-vitro drug susceptibility assay described above: a greater reduction in the binding energy correlated with a greater resistance (r = 0.77, P < 0.02) , suggesting that our modeling appropriately reflects the actual binding mode between the reverse transcriptase molecule and NNRTI. In the 12 models tested, the ΔΔGb value of the I135T/E138K RT-NNRTI complex was persistently larger than wild-type and single mutation reverse transcriptases, indicating that I135T/E138K caused a larger loss of interactions between reverse transcriptase and NNRTI than the single mutations. We then examined the structural changes in the loss of interactions by I135T/E138K. In the wild-type reverse transcriptase, the E138 positioned relatively closely to the EFV, which could contribute to the generation of van der Waals and electrostatic interactions between reverse transcriptase and NNRTI (Fig. 3a). The I135T single substitution caused no significant changes in the steric position of the E138 side chain (Fig. 3b). E138K substitution caused significant changes in the steric position of the E138 side chain (Fig. 3c), whereas the calculated van der Waals energy was similar to that of wild-type reverse transcriptase. I135T/E138K also caused significant changes in the steric position of the K138 side chain, but the orientation of the side chain was different from that of the E138K single mutant reverse transcriptase, possibly due to the interactions between T135 and K138 (Fig. 3d). The K138 conformation in the RTI135T/E138K generated a steric gap between K138 and EFV, and significantly reduced van der Waals energy. In addition, the conformational change necessitated increased electrostatic energy of the reverse transcriptase–EFV complex. These data suggest that an appropriate steric position of the 138th residue is critical for the generation of an optimal EFV binding pocket, and that I135T/E138K, but not the single mutations, effectively break the binding pocket for EFV.
As the HIV-1 pandemic progresses, viral genetic diversity is increasing and becoming geographically heterogeneous [25,26]. We recently indicated that HIV-1 adapts to CTL by acquiring escape mutations in the CTL epitopes, and that such escape variants are increasing in the populations at an alarming high rate of corresponding HLA alleles . When escape mutations occur in drug target proteins, they may alter the mutation patterns of drug resistance even if they do not confer drug resistance themselves. In this study, we focused on I135X in reverse transcriptase, which are escape mutations of HLA-B*51-restricted CTL, because I135X are the prevailing mutations and accumulating in Japan, where the frequency of HLA-B*51 is high (∼20%). Cultural passages of HIV-1 sequences harboring I135X in the presence of increasing concentrations of EFV induced the emergence of E138K, which is not listed as a resistance mutation in the IAS-USA Table. The analysis of recombinant HIV-1 sequences showed that the combination of E138K and some of the I135X, especially I135T, which is most frequent, conferred significant resistance to NNRTI, though solo E138K did not alter drug susceptibility significantly. However, E138K did not always emerge in triplicate experiments of EFV-resistance induction from HIV-1 sequences harboring I135X, whereas the already known NNRTI-resistance mutations emerged. Importantly, variable mutation patterns emerged under the same conditions of resistance induction experiments, indicating that the drug selective pressure is one of the driving forces making the genetic diversity of HIV-1 at population levels as CTL pressure does (HLA-B*51-restricted CTL pressure selects not only I135T but also other I135Xs).
In clinical data, Richard et al.  examined HIV-1 reverse transcriptase sequences in treated Ugandans. In their longitudinal cohort, the HIV-1 infecting one patient (JLT05) acquired I135T/E138K during EFV-containing treatment without any other NNRTI resistance-associated mutations (GenBank: AY556834). Marconi et al.  performed genotypic resistance testing in patients who experienced virologic failure during their first antiretroviral therapy, and the HIV-1 in one patient (SW065) was found to have I135T/E138K after the failure of EFV-containing treatment (EU308076). In tipranavir clinical trials, the HIV-1s of seven cases who experienced NNRTI treatment failure harbored I135T/E138K (DQ880123, DQ880358, DQ879290, DQ880378, DQ877823, DQ878145, and DQ878874) . These data indicate that I135T/E138K confers significant NNRTI resistance in vivo also, suggesting that HLA-B*51-restricted pressure may alter the mutation patterns of NNRTI resistance by inducing escape mutations.
Evidences for the interactions between CTL and drug resistance mutations are accumulating [30–34]. Considering that HIV-1 adapts to particular human HLA alleles and evolves among infected individuals, drug mutation patterns may be affected and altered in currently prevailing viruses. Analysis of drug resistance mutations and development of new antiretroviral agents against laboratory HIV-1 strains derived from isolates obtained decades ago may not always be a suitable strategy. The use of recently obtained clinical isolates may be critical and indispensable in some studies.
This work was supported in part by a Grant-in Aid for AIDS research from the Ministry of Health, Labor, and Welfare (H20-AIDS-002), and the Global Center of Excellence Program (Global Education and Research Center Aiming at the Control of AIDS) from the Ministry of Education, Science, Sports and Culture of Japan.
H.G. designed and executed the study, analyzed the data and wrote the manuscript. H.O. and H.S. performed computational analysis and wrote the manuscript. A.H. and T.H. executed the study and collected data. M.T. provided the hypothesis and participated in discussion and review. S.O. participated in discussion and review and supervised the study.
There are no conflicts of interest.
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