A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse...

The emergence of drug-resistant viruses is a major issue in the treatment of HIV-1 infections. Quite often these drug-resistant viruses have a reduced replication capacity. A novel assay was developed to study the impact of mutations selected during therapy on viral replication capacity. Two HIV-1 HXB2 reference clones were constructed for this assay based on viral competition experiments, which are identical except for the presence of two silent nucleotide changes in p24 in one of the two clones. Within these two reference clones, three different contiguous deletions were constructed: (I) the C-terminus of Gag and protease, (II) the N-terminus of RT and (III) the C-terminus of Gag and protease together with the N-terminus of RT. Using these reference clones, recombinant viruses were created and viral competition experiments were performed. The proportion of each virus during the competition experiments was determined with a real-time PCR assay based on the two silent nucleotide changes in p24 in one of the two reference clones. With this novel assay it was possible to detect accurately differences in replication capacity due to mutations in the C-terminus of Gag and protease and/or the N-terminus of RT.