Relative replicative fitness of zidovudine-resistant human immunodeficiency virus type 1 isolates in vitro

Abstract

Replication of mixtures of two or more human immunodeficiency virus type 1 (HIV-1) variants would be expected to result in the eventual selection of the fittest virus due to Darwinian competition among the variants. The relative proportions of known HIV-1 variants (which may differ only by a single nucleotide from a standard "wild-type" virus, HIV-1HXB2) in mixed viral cultures were quantified by analysis of automated sequence signals of reverse transcriptase PCR products. With this method, the relative levels of replicative fitness of several zidovudine (3'-azidothymidine)-resistant HIV-1HXB2 variants were estimated under controlled in vitro conditions by measuring the rate of change in the proportions of viral variants as they replicated in cell cultures both in the presence and in the absence of drug selection pressure. These variants were engineered to contain commonly observed zidovudine resistance mutations in the HIV-1 reverse transcriptase (M41L, K70R, T215Y, and M41L+T215Y). In the absence of zidovudine, all variants tested displayed reduced replicative fitness compared to wild-type HIV-1HXB2. The order of relative fitness was wild type > K70R >> T215Y = M41L+T215Y > M41L. Mixed cultures in the presence of zidovudine showed a dose-dependent selection pressure against the wild-type virus which varied according to the resistance profile of each virus. The information gathered from this approach provides insight into competition among multiple HIV-1 variants, which likely occurs in vivo with drug selection pressure, and may be applicable in more complex mathematical models for predicting the emergence of HIV-1 variants after the initiation of antiretroviral therapy.

FIG. 1

Replication competition between wild-type HIV-1_HXB2 and HIV-1_HXB2-based zidovudine-resistant clones. The relative proportions of DNA signal contributed by various zidovudine-resistant mutant viruses compared to wild-type virus over several passages in the absence of zidovudine are indicated for each of two (▪ and ⧫) independent initial starting ratios of mutant to wild type. Mutant viruses had the mutation K70R (A), M41L (B), T215Y (C), or M41L+T215Y (D). Dotted lines show the best fit.

FIG. 2

Replication competition of a mixture of several isolates in the absence of zidovudine. Nearly equal mixtures of wild-type virus (□) and three zidovudine-resistant variants, K70R (⧫), M41L (•), and T215Y (▪), were allowed to replicate in the absence of zidovudine. Observed data are indicated by the data points, while data calculated based upon the relative fitness values from Fig. 1 and the equations given in Materials and Methods are indicated by the dotted lines.

FIG. 3

Effect of zidovudine selection pressure on growth of mutant viruses with equal initial levels of fitness. (A) Virus with mutation M41L+T215Y was grown in competition with virus with mutation T215Y at nearly equal starting quantities in the absence of zidovudine. (B and C) Mixtures of wild-type and mutant viruses at an initial ratio of 9:1 were allowed to replicate in the presence of 0.2 (▪) or 2 (⧫) μM zidovudine. Mutant virus in the mixture was virus M41L+T215Y (B) or the less zidovudine-resistant virus T215Y (C). Data points represent observed values, and dotted lines represent values calculated based upon the fitness values from Fig. 1, the known IC₅₀ values, and the equations given in Materials and Methods.

FIG. 4

Titration of zidovudine selection pressure. Nine mixtures of wild-type and T215Y viruses were allowed to replicate for three passages in the presence of increasing micromolar concentrations of zidovudine: 0 (—○—), 0.0001 (—•—), 0.0002 (—□—), 0.0005 (—⋄—), 0.001 (—♦—), 0.002 (—×—), 0.005 (··•··), 0.01 (··○··), 0.05 (··▪··), and 0.2 (··♦··). (A) Relative proportions of mutant viruses. (B) IC₅₀ curves determined with a fitness value of 0.85 in the absence of zidovudine for the mutant and wild-type viruses. (C) Observed relative replicative fitness values for the two strains at each concentration (data points) and values determined by the ratio of the curves in panel B (dotted lines). Note that at concentrations higher than 0.01 μM zidovudine, the wild-type virus (⧫) had a lower relative fitness than the mutant virus, while at drug concentrations lower than this value, the wild-type virus was more fit (◊).

FIG. 5

Replication of mixtures of viruses in the presence of zidovudine. Mixtures of wild-type HIV-1_HXB2 (□) and three zidovudine-resistant variants, K70R (⧫), M41L (•), and T215Y (▪), similar to those shown in Fig. 2 were allowed to replicate in the presence of 0.2 (A) or 2 (B) μM zidovudine. Data points represent observed values, and dotted lines represent calculated values obtained with the values and methods indicated earlier. Extensive growth of the M41L+T215Y virus is indicated by the solid line.