Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production.
The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.
Arad
G
G
Department of Molecular Genetics, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Chorev
M
M
Shtorch
A
A
Goldblum
A
A
Kotler
M
M
D001355:Alpharetrovirus / Q000201:enzymology* / Q000502:physiology; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000235:genetics* / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014771:Virion / Q000201:enzymology; D014779:Virus Replication
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D016282:Aspartic Acid Endopeptidases