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Mutating P 2 and P 1 residues at cleavage junctions in the HIV‐1 pol polyprotein Effects on hydrolysis by HIV‐1 proteinase
Author(s) -
Jupp Ray A.,
Phylip Lowri H.,
Mills John S.,
Le Grice Stuart F.J.,
Kay John
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80583-o
Subject(s) - scissile bond , cleavage (geology) , rnase h , chemistry , reverse transcriptase , integrase , hydrolysis , stereochemistry , residue (chemistry) , rnase p , peptide bond , peptide , biochemistry , enzyme , protease , biology , dna , rna , gene , paleontology , fracture (geology)
Mutations were introduced into the P 2 and P 1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (‐Leu*Phe‐) and (b) between the p51 and RNase H domain (‐Phe*Tyr‐) within p66 of RT in the HIV‐1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli . Whereas the presence of Leu or Phe in P 1 permitted rapid cleavage at either junction, substitution of a β‐branched (He) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a β‐branched (Val) residue in the P 1 position flanking such ‐Hydrophobic*Hydrophobic‐ junctions resulted in effective cleavage of the scissile peptide bond. Gly in P 2 , however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.