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 huma n immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) c onfers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggest ed that this mutation may modulate the conformation of the RT.template /primer complex. in addition, certain mutations in RT that confer resi stance to nucleoside analogs, such as M184V, ape located near the poly merase active site. To characterize further these substitutions, we pe rformed processivity assays alongside era analysis of pausing profiles with wild-type (nt) RT and recombinant RTs containing substitutions a t E89G, M184V, or both, We now show that E89G RT has higher processivi ty thats wt enzyme as well as a different pattern of pausing sites. Si milar findings were obtained with the doubly mutated RT, although enzy me containing only the M184V mutation had lower processivity thats wt. Consistent with these observations, and from a mechanistic standpoint , both E89G-containing as well as doubly mutated RT had decreased diss ociation constants from a complex consisting of RT and template-primer , in comparison with either mt RT or M184V-containing RT. No significa nt differences were observed among the various enzymes in regard to K- m values for the heteropolymeric RNA template used in these studies. V iruses containing the E89G mutation synthesized longer strand DNA prod ucts than either wt viruses or viruses containing only the M184V mutat ion in endogenous RT assays. Thus, the E89G substitution is a dominant determinant in regard to each of the k(off) values from an RT.templat e/primer complex, RT processivity, and specific patterns of pausing du ring DNA polymerization.