title: Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy.
abstract: OBJECTIVE
  To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI).
  
  
  METHODS
  A non-replicative HIV-1 molecular vector (designated pdelta prodelta env) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pdelta prodelta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain).
  
  
  RESULTS
  The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P = 0.0038 and P = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P = 0.022; ritonavir, P = 0.0466) were observed.
  
  
  CONCLUSIONS
  The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.
authors.affiliation: Institute of Microbiology, University of Ancona, Italy. menzo@popcsi.unian.it
authors.forename: S
authors.initials: S
authors.lastname: Menzo
authors.forename: S
authors.initials: S
authors.lastname: Rusconi
authors.forename: A
authors.initials: A
authors.lastname: Monachetti
authors.forename: M C
authors.initials: MC
authors.lastname: Colombo
authors.forename: M
authors.initials: M
authors.lastname: Violin
authors.forename: P
authors.initials: P
authors.lastname: Bagnarelli
authors.forename: P E
authors.initials: PE
authors.lastname: Varaldo
authors.forename: M
authors.initials: M
authors.lastname: Moroni
authors.forename: M
authors.initials: M
authors.lastname: Galli
authors.forename: C
authors.initials: C
authors.lastname: Balotta
authors.forename: M
authors.initials: M
authors.lastname: Clementi
mesh_terms: D002678:Chimera; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D005838:Genotype; D015658:HIV Infections / Q000097:blood / Q000821:virology; D016333:HIV Protease / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; D017320:HIV Protease Inhibitors; D012367:RNA, Viral; D016333:HIV Protease