A recombinant virus assay using full-length envelope sequences to detect changes in HIV-1 co-receptor usage

The clinical management of HIV-1 infection has benefited enormously from mo lecular characterization of drug resistance as well as determination of the viral phenotype in vitro. HIV-1 infected individuals on HAART are currentl y monitored for the development of drug resistance variants allowing clinic ians to redesign drug regimens. An understanding of the molecular basis of the evolution of drug resistance in vivo allows the improvement of the drug s as well as in vitro evaluation of new antiviral compounds alone or in com bination with those currently approved. New findings suggest that viral env elopes could be a target to inhibit infection and replication. Therefore th e generation of a recombinant virus assay (RVA) to allow the phenotypic det ermination of drug resistance against entry inhibitors (EI) is anticipated. We constructed an env-deleted clone of HIV-1 using the molecular clone NL- 4.3. PCR amplified complete envelope genes (NL-4.3, BaL, primary envelope-g enes) were ligated in vitro with a deletion clone (pNL-DeltaK) and PM1-cell s, supporting the replication of R5- and X4-tropic viruses, were transfecte d. Determination of co-receptor usage of the harvested recombinant virus-sw arm revealed no difference compared to the molecular clones derived individ ually from three different patients. These results clearly show that an env elope-based RVA is practicable to monitor HIV-co-receptor usage at a given time point. Furthermore, this assay will allow to monitor resistance develo pment against existing and future entry inhibitors and will aid to improve the management of HIV-therapy.