AEGiS-12IAC: Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing.

12th International AIDS Conference

Geneva, Switzerland - June 28-July 3, 1998

Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing.

Int Conf AIDS 1998 Jun 28-Jul 3; 12:7 (abstract no. 11125)

Wainberg MA, Quan Y
McGill AIDS Center, Montreal, Canada.

OBJECTIVES: To compare various form of wild-type (wt) and mutated recombinant reverse transcriptase (RT) molecules, containing drug-resistance-conferring mutations, in regard to pausing, processivity, and dissociation from an RNA template/primer.

METHODS: The substitution of a glycine for glutamic acid at position 89 in HIV-1 RT (E89G) confers resistance to several nucleoside and nonnucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT primer/template complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To further characterize these substitutions, we performed processivity assays alongside an analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing substitutions at E89G, M184V, or both.

RESULTS: We found that E89G RT has higher processivity than wt enzyme as well as a different pattern of pausing sites. Similar findings were obtained with the doubly mutated RT, although enzyme containing only the M184V mutation had lower processivity than wt. Consistent with these observations, and from a mechanistic standpoint, both E89G-containing as well as doubly mutated RT had decreased dissociation constants from a complex consisting of RT and template:primer, in comparison with either wt RT or M184V-containing RT. However, all of the wt and mutant enzymes tested had similar K(m) values for the RNA template primer used in this work. Viruses containing the E89G mutation synthesized longer strand DNA products than either wt viruses or viruses containing only the M184V mutation in endogenous RT assays.

CONCLUSION: The E89G substitution is a dominant determinant in regard to each of the koff values from an RT template/primer complex, RT processivity, and specific patterns of pausing during DNA polymerization.

Keywords: AEGIS, HIV-1 Reverse Transcriptase, HIV-1, RNA-Directed DNA Polymerase, Mutation, RNA, DNA Replication, Glycine, DNA, RNA primers, genetics, ICA12
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