Complete mutagenesis of the HIV-1 protease.
Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.
Loeb
D D
DD
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599.
Swanstrom
R
R
Everitt
L
L
Manchester
M
M
Stamper
S E
SE
Hutchison
C A
CA
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015683:Gene Products, gag; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011498:Protein Precursors / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism; D014169:Transformation, Bacterial
D015683:Gene Products, gag; D011498:Protein Precursors; D012191:Retroviridae Proteins; D010450:Endopeptidases; D016333:HIV Protease