Background:  The reverse transcriptase of HIV-1 is a heterodimeric enzyme comprised of 51-kDa and 66-kDa subunits. Since the subunits are derived from the same coding region and the relative arrangement of reverse transcriptase subdomains differs markedly between p51 and p66, a mutation in 1 subunit is structurally and functionally not equivalent to that of the other subunit. Detailed molecular analysis of the individual subunits has therefore not been feasible in the context of infectious virus, impeding the development of dimerization antagonists.

Methods:  By co-transfecting cells with reverse transcriptase–defective proviral DNA and an LTR-vpr-p51-IRES-p66 expression cassette, we demonstrated that Vpr-p51 interacts with p66 and mediates virion incorporation of a Vpr-p51/p66 heterodimeric complex. Cleavage by the viral PR liberates Vpr, generating functional heterodimeric reverse transcriptase (p51/p66) and infectious virions. Using this novel strategy for subunit-specific mutagenesis, the Trp-motif at the connection subdomain dimer interface of reverse transcriptase was analyzed in infectious virus.

Results:  Mutagenic analysis of p51 indicated that a cluster of Trp residues (W398, W402, W406, and W414) proximal to the p51/p66 interface is important for subunit interaction. Independent mutagenesis of p51^W401, p51^Y405, p51^N363, and p66^W410 residues within interacting distance at the dimer interface, did not have significant effects on subunit association or viral infectivity. However, simultaneous mutagenesis of 2 residues positioned within interacting distance of each other reduced infectivity by at least 50%. Likewise, the p66^W401A also reduced virus infectivity by approximately 50%. Mutation of W401 in p51 and p66 simultaneously caused a severe defect in both reverse transcriptase subunit dimerization and virus infectivity.

Conclusions:  Detailed examination of our results, reverse transcriptase crystal structure, and previous reports on the Trp-motif suggests that mutating p66^W401/p51^W401 causes repositioning of the aL-b20 loop in p66 and disruption of specific intersubunit interactions, resulting in a loss of reverse transcriptase dimerization and virus infectivity. This analysis of the reverse transcriptase connection subdomain identifies a putative “hot spot” for p51-p66 interaction in the Trp-motif and provides new insights relevant to targeted drug design.

Keywords: reverse transcriptase; subunit-specific mutagenesis; dimerization