Date Published: March 21, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Liyan Jiao, Hanping Li, Lin Li, Daomin Zhuang, Yongjian Liu, Zuoyi Bao, Siyang Liu, Jingyun Li.
Objective. To clarify the impact of H221Y mutation on drug resistance to NVP. Methods. 646 bp HIV-1 pol gene fragments (from 592 to 1237 nucleotide) with different NNRTIs mutation profiles from AIDS patients receiving antiretroviral therapy containing NVP regimens were introduced into pNL4-3 backbone plasmid. H221Y and (or) Y181C mutations were reverted to wild type amino acids by site-directed mutagenesis, then strains containing various mutation patterns were packaged. Phenotypic drug resistance was analyzed on TZM-bl cells. Results. 12 strains containing different drug-resistant mutation profiles were constructed, including the K101Q series (K101Q/Y181C/H221Y, K101Q/Y181C, K101Q/H221Y, and K101Q), the V179D series (V179D/Y181C/H221Y, V179D/Y181C, V179D/H221Y, and V179D), and the K103N series (K103N/Y181C/H221Y, K103N/Y181C, K103N/H221Y, K103N). For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by 2.1 ± 0.5 to 3.6 ± 0.5 fold. To the mutation profiles K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, and K103N, the presence of Y181C reduced NVP susceptibility by 41.9 ± 8.4 to 1297.0 ± 289.1 fold. For the strains containing K101Q, V179D, and K103N, the presence of Y181C/H221Y combination decreased NVP susceptibility by 100.6 ± 32.5 to 3444.6 ± 834.5 fold. Conclusion. On the bases of various NNRTIs mutation profiles, Y181C remarkably improved the IC50 to NVP, although H221Ymutation alone just increases 2.1 ∼ 3.6-fold resistance to NVP, the mutation could improve 100.6 ∼ 3444.6-fold resistance to NVP when it copresent with Y181C, the phenotypic drug resistance fold was improved extremely. For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by 2.1 ± 0.5 to 3.6 ± 0.5 fold.
The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are small molecules which can bind to a nonactive hydrophobic site pocket close to the catalytic domain of RT (termed NNRTI-binding pocket). The binding of NNRTIs to RT could alter the structure of RT and block the polymerase activity of RT. As the results, the replications of HIV-1 were inhibited. However, despite their high efficiencies and low toxicities, the use of NNRTIs is hampered owing to the rapid selection of drug-resistant HIV-1 strains [1, 2]. Therefore, a deeper understanding of resistance is necessary in order to effectively prevent and manage HIV-1 drug resistance.
Selection of Plasma SamplesPlasma samples were collected from 3 AIDS patients in clinical cohort receiving antiretroviral therapy [nevirapine (NVP) + zidovudine (AZT) + didanosine (ddI)]. All patients have written informed consent. Genotype-resistant results of these patients showed all samples contained three NNRTIs mutations (Y181C/H221Y plus another mutation) which conferred resistance to NVP.
Construction 12 HIV-1 Virus Containing Different Mutation ProfilesThese viruses were named 101Q-1, 101Q-2, 101Q-3, 101Q-4, 179D-1, 179D-2, 179D-3, 179D-4, 103N-1, 103N-2, 103N-3, and 103N-4; the mutation patterns they contained were K101Q/Y181C/H221Y, K101Q/Y181C, K101Q/H221Y, K101Q, V179D/Y181C/H221Y, V179D/Y181C, V179D/H221Y, V179D, K103N/Y181C/H221Y, K103N/Y181C, K103N/H221Y, and K103N. As the control virus, Con-1 was pNL4-3 without any drug-resistant mutations. The virus name and corresponding drug resistance mutation profiles were indicated at Table 1.
H221Y was first reported related to NRTI treatment in 2003 , but in recent study, H221Y was concerned associated with exposed to NNRTIs [6, 10]. Ceccherini-Silberstein et al. found the prevalence rate of H221Y in isolates from patients failing NVP treatment was 10.3% and the mutation was included in the top 10 and 15 determinants for NVP and EFV resistance, respectively, ranking even above some classical NNRTI resistance mutations, such as K101E, V108I, and G190E . The group found H221Y was strongly associated with the use of NVP and showed positive interactions with Y181C and was also negatively associated with the use of ZDV and with TAMs (particularly TAMs-2, such as D67N, K70R, K219Q/E, and T215F) and was then associated with an increased susceptibility to ZDV. When H221Y was copresent with TAMs-2, the ZDV susceptibility was even greater than that observed when TAMs-2 were copresent with Y181C. The presence of H221Y along with Y181C was associated with a 12.4-fold increase in NVP resistance. Jiang et al. found that the occurrence of the H221Y was 19.8% in 91 patients receiving nevirapine and lamivudine plus stavudine (57.1%) or zidovudine (42.9%) . Reuman et al. analyzed viruses from 13039 individuals with sequences containing at least one of 52 published NNRTI-selected mutations; the frequency of H221Y among 1510 viruses from individuals who received nevirapine but no other NNRTI was 12%. H221Y occurred along with Y181C in our study (data not shown) and the virus with mutation pattern K103N/Y181C/H221Y can replicate stably in vitro without drug pressure . Our study showed that as for the mutation profiles K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N, the presence of H221Y, respectively lead to 2.2 ± 0.9, 3.2 ± 0.3, 3.6 ± 0.5, 3.0 ± 0.4, 2.1 ± 0.5, and 2.2 ± 0.1-fold increase in NVP resistance.
The success of HAART (Highly Active Antiretroviral Therapy) in treating AIDS patients is hampered by the emergence of drug-resistant strains, and more and more studies indicate that there are more drug-resistant mutations than we have known. The roles of these novel mutations in drug resistance have still been indistinct. In this study, we found that Y181C remarkably improved the IC50 to NVP, although the novel mutation H221Y only slightly conferred resistance to NVP, when it combined with Y181C, the phenotypic drug resistance folds were improved extremely.