Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease H as substrates of the viral protease.
A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483-Leu484 and Tyr532-Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436-Ala437 and at Phe440-Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.
Tomasselli
A G
AG
Biochemistry Unit, Upjohn Laboratories, Kalamazoo, Michigan 49001.
Sarcich
J L
JL
Barrett
L J
LJ
Reardon
I M
IM
Howe
W J
WJ
Evans
D B
DB
Sharma
S K
SK
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D016333:HIV Protease / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D016333:HIV Protease