Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
PURIFICATION AND CHARACTERIZATION OF THE HIV-1 REVERSE TRANSCRIPTASE
Diss Abstr Int [B]; 52(5):2391 1991. Unique Identifier : AIDSLINE ICDB/92679486 West AB; Harvard Univ.
Abstract:
This thesis examines in detail some of the features of the primary replicative enzyme of HIV-1, the reverse transcriptase. In order to achieve this, the products of the entire pol open reading frame were overexpressed in the eukaryotic baculoviral expression system. Included in the products made, were both subunits of the reverse transcriptase as well as the integrase. Various monoclonal antibodies were made to the reverse transcriptase and they had unique characteristics with regard to ability to differentiate the two domains of the 64-kD subunit. An immunoaffinity purification step was developed that provided high yields of very pure enzyme with stoichiometric subunit ratios and with the proper amino terminal sequence. Analysis of the subunit structure by sucrose gradient velocity centrifugation indicated that the enzyme exists in several forms in equilibrium and calls into question the notion that the enzyme functions as a dimer. In addition, it was also found that the 51-kD subunit appears to be inactive as has been demonstrated elsewhere. When dNTP was the variable substrate, the enzyme kinetics deviated from standard Michaelis-Menten kinetics in that both substrate inhibition and negative cooperativity occur. Further examination demonstrates that dNTPs may act both as substrate and as noncompetitive inhibitors. A model is presented that accounts for the substrate inhibition and that is consistent with the negative cooperativity. In this model, the reverse transcriptase is a multisubunit holoenzyme where noncompetitive inhibition is allosterically mediated by one 51-kD subunit, binding nucleotide and downregulating the activity of the polymerizing 64-kD subunit. The processivity of the reverse transcriptase on an M13 ssDNA template was found to be severalfold higher, at about 150 nucleotides/association event, than that observed in previous studies. In addition, it was clearly demonstrated that the reverse transcriptase has a tendency to halt on ssDNA of random sequence when it encounters a string of As and Ts and that this was not a function of nucleotide concentration. The new features described in this thesis have important implications and provided new insights into understanding the HIV life cycle. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD91-32063).
Keywords: HIV-1/*ENZYMOLOGY Kinetics RNA-Directed DNA Polymerase/CHEMISTRY/*ISOLATION & PURIF THESIS 920730
M9271101
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Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
