Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase

Abstract

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization. In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

“…Past studies have confirmed that most amino acid substitution mutations in RT do not significantly impact PR-mediated Gag cleavage, regardless of whether or not they affect RT dimerization. Substitution mutations at the RT primer grip residues L234, W239, and W229 (6,16,25,43,50) or at the tryptophan repeat motif residues W401 and W414 (44) that abrogate RT dimerization in vitro are not associated with impaired Gag processing and/or virion production, even though the L234D and W239A mutants are both deficient in virusassociated RT-presumably due (at least in part) to premature Gag-Pol cleavage (25,49,51). Furthermore, substitution mutations at HIV-1 RT G191 that are capable of facilitating RT dimerization in vitro (15) have no significant impact on virus particle assembly and release (23).…”

Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles

“…Thus, it is very likely that the domains responsible for RT incorporation into VLPs exist between RT residues 183 and 305. Some mutations in this region have been shown to block p66/p51 heterodimer formation (49). It remains to be determined if the truncation mutations affect RT dimerization and consequently diminish RT incorporation into VLPs.…”

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

“…Previous studies have shown enhancement of the homodimerization of RT and PR-RT fragments by EFV in yeast two-hybrid assays (27,28,30). We initially used yeast two-hybrid assay systems with pAD-GAL4 and pBD-GAL4 plasmids (Stratagene) but did not see detectable levels of RT expression (data not shown), as suggested previously (61). As an alternative method, we employed a bacterial two-hybrid assay with pBT and pTRG plasmids (Stratagene) (Fig.…”