Correlation of phenotypic zidovudine resistance with mutational patterns in the reverse transcriptase of human immunodeficiency virus type 1: interpretation of established mutations and characterization of new polymorphisms at codons 208, 211, and 214

Abstract

Zidovudine resistance (ZDV-R) is associated with classic genotypic changes at codons 41, 67, 70, 210, 215, and 219 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene as well as with the multinucleoside resistance (MNR) complexes (Q151M MNR complex; 6-bp insertion/A62V complex). In addition, enhanced resistance to ZDV in the context of the classic ZDV mutations plus the M184V mutation has been associated with additional mutations at positions 208, 211, 214, and 333. In this study we investigated phenotypic ZDV-R determined by a recombinant virus assay (Antivirogram; Virco) in 223 clinical samples in relation to the above genotypic changes. 150 out of 223 clinical samples had the M184V mutation. Phenotypic ZDV-R ranged from 0.3- to 5,338-fold. Sixteen samples (15 with high ZDV-R ranging from 90- to 3,571-fold) contained MNR-associated patterns. Analysis of classic mutational patterns broadly demonstrated increasing ZDV-R with increasing number of ZDV mutations. A comparable correlation was obtained when ZDV-R was analyzed only relative to the T215Y/F mutation. Site-directed mutagenesis experiments investigating the influence of the additional mutations H208Y, R211K, and L214F on ZDV-R resulted in a 7.4- or 21-fold increase in ZDV-R when the R211K/L214F or H208Y/R211K/L214F mutations, respectively, were added to a highly ZDV-R virus. In the clinical sample data set we analyzed, the combination of R211K/L214F appeared most frequently. The H208Y change was detected only in highly ZDV-R viruses, whereas the G333E/D change was distributed equally. All changes were independent of the M184V mutation. A 2.4- or 8-fold increase in ZDV-R was observed in the clinical samples with high ZDV-R containing the R211K/L214F or H208Y/R211K/L214F mutations, respectively. We have shown that the combination of the additional mutations H208Y, R211K, and L214F in HIV-1 RT may influence ZDV-R and should be considered when assessing ZDV-R.

FIG. 1.

Phenotypic resistance to ZDV dependent on mutational patterns as comparisons of IC₅₀ with that of a wild-type strain. Samples are shown as rhombi, triangles represent samples with the Q151M MNR complex, squares represent samples with the 6-bp insert/A62V, and circles represent samples with the 6-bp insert alone. Mean and median values are shown as dark and light bars, respectively. Error bars represent standard deviation values. Negative error bar values are not shown because they are below zero.

FIG. 2.

(a) Percentage of ZDV-sensitive (S), -intermediate (I), and -R samples dependent on mutational patterns and the M184V change. (b) Percentage of ZDV-sensitive (S), -intermediate (I), and -R samples dependent on the T215Y/F and the M184V change.

FIG. 3.

SDM experiments to evaluate the effect of additional polymorphisms on ZDV-R. The virus strain with four ZDV mutations had the M41L, L210W, T215Y, K219E(*) or K219N (#), and M184V changes. In addition the virus strain with six ZDV mutations had the D67N and K70R change. Mutations added by SDM are shown on top of the bars.

FIG. 4.

Distribution of additional polymorphisms H208Y (black bars), R211K/L214F (dark grey bars), and G333E/D (light grey bars) in the sample population dependent on ZDV mutational patterns. Percentages are given above each bar. Asterisks highlight statistical significance (χ²-test).

FIG. 5.

Distribution of samples carrying the R211K/L214F double mutation dependent on ZDV mutational patterns (see above) and the M184V change. Samples wild type at position 184 are shown as black bars, and samples with the M184V mutation are shown as light grey bars. Percentages are given above each bar.

FIG. 6.

Phenotypic ZDV-R for samples carrying no polymorphisms or combinations of the additional polymorphisms H208Y, R211K, and L214F dependent on ZDV mutational patterns. Black bars represent samples carrying the H208Y/R211K/L214F triple combination, dark grey bars represent samples carrying the R211K/L214F combination, and light grey bars represent samples carrying none of the above-mentioned combinations. The dotted line represents a 10-fold increase in ZDV-R.