Yeast-based assay for measuring the functional activity of an hiv-1 protease in response to an antiviral agent

Abstract

A method of determining sensitivity or resistance of isolates of HIV retroviruses to a molecule includes a) amplifying sequences coding for a protease of a retrovirus to be studied, with or without the or some of amino acid sequences situated upstream and downstream of a cleavage site of a precursor in which the amino acid sequences are situated, b) recombining fragments of DNA, a final product of the amplification, and an expression vector allowing expression of sequence coding for the protease of the retrovirus to be studied under control of a known inducible promoter through co-transformation of the vector and the DNA fragments with at least one yeast cell, c) culturing co-transformed yeast cell or cells to obtain a sufficient number of transformants to perform a sensitivity or resistance test, and recovering transformants issuing from the co-transformed cell, on any suitable medium, d) incubating the transformants in the presence of a molecule to be tested, e) qualitatively or quantitatively analyzing the living cells, and f) deducing the sensitivity or resistance phenotype.