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

Abstract

We identified clinical isolates with phenotypic resistance to nevirapine (NVP) in the absence of known nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. This resistance is caused by N348I, a mutation at the connection subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Virologic analysis showed that N348I conferred multiclass resistance to NNRTIs (NVP and delavirdine) and to nucleoside reverse transcriptase inhibitors (zidovudine [AZT] and didanosine [ddI]). N348I impaired HIV-1 replication in a cell-type-dependent manner. Acquisition of N348I was frequently observed in AZT- and/or ddI-containing therapy (12.5%; n = 48; P < 0.0001) and was accompanied with thymidine analogue-associated mutations, e.g., T215Y (n = 5/6) and the lamivudine resistance mutation M184V (n = 1/6) in a Japanese cohort. Molecular modeling analysis shows that residue 348 is proximal to the NNRTI-binding pocket and to a flexible hinge region at the base of the p66 thumb that may be affected by the N348I mutation. Our results further highlight the role of connection subdomain residues in drug resistance.

FIG. 1.

The course of patient and drug resistance profiles of clinical isolates obtained from the patient. (A) The drug treatment history is indicated at the top of the graph. The virologic responses represented by plasma viral load and CD4 counts of peripheral blood are shown. Open triangles indicate the time points of genotypic assays. Closed triangles indicate the time points of isolation of clinical isolates for genotypic assays (also see Fig. S1 in the supplemental material) and phenotypic assays (also see Table S1 in the supplemental material showing actual EC₅₀ values as mean values and standard deviations from three independent experiments). From February to June 2000 and after October 2001, the chemotherapy was interrupted due to severe adverse effects. (B) The viruses acquired NRTI resistance mutations sequentially as shown. Susceptibility to compounds tested in at least three independent experiments is shown as the relative increase in the EC₅₀ compared to HIV-1_WT obtained from a pNL4-3-based plasmid. An increase larger than 3.0-fold is indicated in bold. NRTI or NNRTI resistance mutations were reported in the HIV drug resistance database maintained by International AIDS Society 2006, the Stanford University (Stanford, CA) and Los Alamos National Laboratory (Los Alamos, NM), http://hivdb.stanford.edu/and http://resdb.lanl.gov/Resist_DB/, respectively. RTV, ritonavir; NFV, nelfinavir; IDV, indinavir.

FIG. 2.

Viral replication kinetics. Production of p24 antigen in culture supernatant was determined with a commercially available p24 antigen kit. Profiles of replication kinetics (p24 production) of HIV-1_WT (closed diamonds), HIV-1_N348I (closed squares), HIV-1_I393L (open diamonds) and HIV-1_N348I/I393L (open squares) were determined with MT-2 (A), SupT1 (B), PM1 (C) and H9 cells (D) and PHA-stimulated PBMCs (E). Representative results from at least two (or three) independent single determinations of p24 production with newly titrated viruses are shown. A competitive HIV-1 replication assay was performed in H9 cells to compare the replication kinetics of HIV-1_WT (closed diamond) and HIV-1_N348I (closed squares) (F) and of HIV-1 N348I (closed squares) and HIV-1_N348I/I393L (open square) (G).

FIG. 3.

Location of N348I in the modeled HIV-1 RT with NVP. (A) The N348I mutation (blue Van Der Waals volume) is shown in the connection subdomains of both p66 (purple) and p51 (cyan) subunits. The 348 residue of the p51 subunit is distant from the nucleic acid, shown as yellow Van Der Waals surfaces. In the p66 subunit (purple) the 348 residue is in a position to affect the flexibility of the p66 thumb, which in turn might affect binding of the nucleic acid. NVP is shown bound at the NNRTI binding pocket (red Van Der Waals volume). Magnification of the frame area of the enzyme is shown in panel B. (B) The main chain C=O of N348 is shown to interact with the N-H of 317 (yellow broken line) through a hydrogen bond interaction. Binding of NVP (white ball) repositions the p66 thumb subdomain with respect to (i) the polymerase active site (β6-β9-β10) that contains the three catalytic aspartates and the YMDD motif and (ii) the primer grip (β12-β13) of p66. The movement of the thumb subdomain is in a hinge-like motion that is based at the position where residue 348 interacts with residue 317.