Effects of human immunodeficiency virus type 1 resistance to protease inhibitors on reverse transcriptase processing, activity, and drug sensitivity

Abstract

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

FIG. 1

Virus particle-associated material in the supernatant of transfected HeLa cells was analyzed by Western blotting with monoclonal antibodies that recognize the RT subunits p66 and p51 (A) or the gag-encoded MA and CA proteins (B). The top half of the Western blot shown in panel A was deliberately overexposed to allow the detection of the Gag-Pol precursor molecules. Arrows point to Gag-Pol cleavage intermediates. The molecular mass marker (in kilodaltons) is indicated for both panels A and B. Pre and post indicate that the viral protease was cloned from a pretherapy or postresistance patient isolate, respectively. neg, mock-transfected cells.

FIG. 2

Virus particle-associated RT activity measured in a poly(rA)-oligo(dT) assay. The RT activity of each pair of pretherapy and resistant (-post) viruses was expressed as a percentage of the pretherapy virus. Open columns represent viruses carrying pretherapy protease alleles, and hatched columns represent viruses carrying resistant proteases. The measurements were repeated at least three times, and the averages and standard deviations are indicated.

FIG. 3

Effect of impaired processing by resistant protease on AZT resistance on viruses carrying wild-type (A) or mutated (B) RT sequences. IC₉₀ values for AZT were calculated in a single-cycle assay, measurements were repeated at least three times, and the averages and standard deviations are indicated. Open columns represent viruses carrying pretherapy protease alleles, and hatched columns represent viruses carrying resistant proteases. The mutated residues in the RT coding region are indicated.

FIG. 4

Partial rescue of infectivity by AZT resistance mutations. Infectivity of 402-derived viral clones with and without mutations in the RT was analyzed in a single-cycle assay and expressed as percent of infectivity of the 402-pre virus (pretherapy protease and wild-type RT) (A). Open columns represent viruses carrying pretherapy protease alleles, and hatched columns represent viruses carrying resistant proteases. Three independent experiments were performed, and the averages and standard deviations are shown. (B) Replication kinetics of the 402-derived viral clones in MT4 cells. Squares represent 402-pre, circles correspond to 402-pre-41/215, triangles correspond to 402-post-41/215, and diamonds represent 402-post.