Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli.
We expressed the gag and proteinase regions of human immunodeficiency virus (HIV) type 1 by transcription and translation in vitro. A synthetic RNA spanning the gag and pro domains gave primarily the unprocessed capsid precursor pr53. Efficient cleavage of this precursor was observed when the gag and pro domains were placed in the same translational reading frame, yielding equimolar amounts of the gag protein and of proteinase (PR). Expression of HIV type 1 PR in Escherichia coli as a fusion protein gave rapid autocatalytic processing to an HIV-specific protein of approximately 11 kilodaltons. HIV PR generated in E. coli specifically induced cleavage of the HIV capsid precursor, whereas deletion of the carboxy-terminal 17 amino acids of the proteinase rendered it inactive. Inhibitor studies showed that the enzyme was insensitive to inhibitors of serine and cysteine proteinases and metalloproteinases and was inhibited only by a very high concentration (1 mM) of pepstatin A.
D003001:Cloning, Molecular; D010450:Endopeptidases / Q000096:biosynthesis / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag; D005822:Genetic Vectors; D016333:HIV Protease; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011480:Protease Inhibitors; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000138:chemical synthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D012191:Retroviridae Proteins / Q000096:biosynthesis*
D015683:Gene Products, gag; D011480:Protease Inhibitors; D011498:Protein Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D010450:Endopeptidases; D016333:HIV Protease