Genotypic Resistance in Protease

Nucleotide sequences were translated and aligned to facilitate analysis of amino acids in PR by type and position. Amino acid substitutions associated with resistance to PR inhibitors were summarized for each patient (Fig. 7). Patient A virus levels in plasma were reduced to undetectable, even though pretreatment PR alleles displayed one of three amino acids that contribute to reduced sensitivity to various inhibitors (M361, ritonavir; L63P, indinavir; V771, other inhibitors). Even though HIV-1 was undetectable in plasma, virus persisted in peripheral blood cells and contained a subset of PR alleles that displayed an additional resistant mutation at position 82.

Patient B, whose pretherapy virus population contained only one indinavir-resistant L63P substitution, displayed no sustained virus response to ritonavir therapy. Viral PRS developed amino acid substitutions at multiple positions, including 20, 36, 54, 71, and 82. Viruses with similar PR alleles displayed about 40-fold increased resistance to ritonavir in culture.10

In contrast, viruses in patient D, who displayed only transient response to therapy, contained pretreatment PR alleles that uniformly contained four amino acids associated with resistance to various inhibitors. Two changes, L10I and A71V, are associated specifically with resistance to indinavir.27 PR alleles with some additional, resistance-associated substitutions appeared during the course of therapy. Nonetheless, virus in this patient appeared "primed" to resist indinavir.

Amino Acid Positions in Protease

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FIGURE 7. Summary of amino acid substitutions in HIV-1 protease in children receiving therapy with PR inhibitor. Amino acid substitutions in key positions are associated with, or contribute directly to, protease resistance to inhibitors. Protease sequences were determined in plasma virus RNA (dotted triangles) and in cell-associated viral DNA (black triangles) at baseline (B), and at indicated times in weeks for each of three patients.

FIGURE 7. Summary of amino acid substitutions in HIV-1 protease in children receiving therapy with PR inhibitor. Amino acid substitutions in key positions are associated with, or contribute directly to, protease resistance to inhibitors. Protease sequences were determined in plasma virus RNA (dotted triangles) and in cell-associated viral DNA (black triangles) at baseline (B), and at indicated times in weeks for each of three patients.

3.5. Genetic Distance in Protease over Time

Considerable insight into response to therapy was apparent when genetic changes over time were analyzed relative to baseline (Fig. 8). PR distance declined over time in patient A, who responded to therapy. In contrast, virus from patient B exhibited a distinct increase in distance over the course of 28 weeks of therapy. Failure of therapy to suppress virus was not invariably related to increased distance relative to baseline. For example, little change appeared in viral PR in patient D, whose virus contained multiple resistance-associated amino acids at the start of therapy (Fig. 8).

Even though all proteases contained specific amino acid substitutions related to resistance, none of the PR populations exhibited a preponderance of replacement versus silent nucleotide substitutions, i.e., non-synonymous or synonymous changes, respectively (Fig. 8). In each case, silent substitutions comprised the majority of nucleotide changes and made the greater contribution to the variability. Thus, strong selection by inhibitor for amino acid substitutions in PR was insufficient to tip the balance of nucleotide changes so that nonsynonymous substitutions exceeded silent substitutions.

3.6. Models for Evolution of Virus with Inhibitor

Based upon genetic analysis of PR resistance during therapy, we developed three models to account for the types of changes found in the patients

FIGURE 8. Change in PR alleles during PR inhibitor therapy. Virus populations in patients A (top panel), B (middle panel), and D (lower panel) were evaluated in PR for total distance and nonsynonymous or synonymous substitutions (x axis) over time (y axis). Symbols: filled diamonds, total distance; triangles, nonsynonymous (replacement) substitutions; filled squares: synonymous (silent) substitutions.

FIGURE 8. Change in PR alleles during PR inhibitor therapy. Virus populations in patients A (top panel), B (middle panel), and D (lower panel) were evaluated in PR for total distance and nonsynonymous or synonymous substitutions (x axis) over time (y axis). Symbols: filled diamonds, total distance; triangles, nonsynonymous (replacement) substitutions; filled squares: synonymous (silent) substitutions.

(Fig. 9). Model 1, the suppression model, accounts for the results in patient A, whose virus levels were reduced to undetectable in plasma. Variants of the virus that were detected in plasma at entry were suppressed. Infection persisted in cells, suggesting that there may be reservoirs of long-lived infected cells or continued viral replication within tissues inaccessible to protease inhibitors. Failure to achieve sustained suppression of virus levels or prolonged increase in CD4 T cells, as in patient D, was associated with emergence of multiple lineages of virus variants (model 2) that were directly related to the baseline population. Noncompliance, subtherapeutic drug levels, preexisting resistance, or resistance mapping outside of protease may account for this type of evolutionary pattern. Model 3 represents

FIGURE 9. Models for emergence of virus based on protease variability. Model 1 —suppression is represented by patient A, whose viral load became undetectable early in therapy. Model 2— progression reflects incompletely suppressed viral replication in the presence of inhibitor and the outgrowth of a highly genotypically resistant subpopulation, as in patient B. Model 3— multiple lineages demonstrates concurrent evolution of multiple populations without the outgrowth or selection for a specific population, as in patient D.

FIGURE 9. Models for emergence of virus based on protease variability. Model 1 —suppression is represented by patient A, whose viral load became undetectable early in therapy. Model 2— progression reflects incompletely suppressed viral replication in the presence of inhibitor and the outgrowth of a highly genotypically resistant subpopulation, as in patient B. Model 3— multiple lineages demonstrates concurrent evolution of multiple populations without the outgrowth or selection for a specific population, as in patient D.

selective outgrowth of a minor population from the multiple variants present at entry, as in patient B.

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