IDENTIFICATION OF TEMPERATURE-SENSITIVE MUTANTS OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE THROUGH SATURATION MUTAGENESIS - AMINO-ACID SIDE-CHAIN REQUIREMENTS FOR TEMPERATURE SENSITIVITY

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia col i expression and processing assay system was used to screen 285 protea se mutants for temperature-sensitive activity. Fourteen protease mutan ts had a temperature-sensitive phenotype, and approximately half resul ted from conservative amino acid substitutions. Of the 14 substitution s that conferred a temperature-sensitive phenotype, 11 substitutions o ccurred at 6 positions that represent 3 pairs of residues in the prote ase that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are importa nt for enzyme activity and stability.