Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing.
The substitution of a glycine for glutamic acid at position 89 in human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) confers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT.template/primer complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To characterize further 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. We now show 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. No significant differences were observed among the various enzymes in regard to Km values for the heteropolymeric RNA template used in these studies. 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. Thus, 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.
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.
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Quan
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Inouye
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Liang
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Rong
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Götte
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Wainberg
D019943:Amino Acid Substitution; D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013698:Templates, Genetic
D012367:RNA, Viral; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D018698:Glutamic Acid; D054303:HIV Reverse Transcriptase; D005998:Glycine