Role of the reverse transcriptase tryptophan repeat motif in HIV-l replication

The HIV-l enzyme, reverse transcriptase (RT), is a successful target for antiretroviral agents used for the treatment of HI VIA IDS. Ongoing research indicates that RT may yet reveal further targets for the development of novel chemotherapeutic agents that act to inhibit HIV -1 replication. R T is an asymmetric heterodimer composed of the p66 (66 kDa) and pSI (51 kDa) subunits. Since characterisation ofRT as an obligate dimer, subunit dimerisation has been suggested as a novel drug target. Further, the ability ofRT to dimerise is suggested to playa regulatory role in Gag-Pol processing and proteolytic generation of the mature R T heterodimer. Thus targeting R T dimerisation could potentially disturb both the early and late phases of HIV -1 replication, achieving dual modes of inhibition with a single compound. The present study validates this concept, and provides insight into the role of the RT domain in the late phase of HIV -1 replication. These observations were made upon examining the effect of mutating residues belonging to or associated with the highly conserved RT tryptophan repeat motif (TRM). The TRM is comprised of a cluster of six tryptophan residues at RT codons 398, 401, 402, 406 and 410, and includes a tyrosine at codon 405. The motif is located in the connection subdomain, falling within secondary structural elements aL, f320 and the intervening loop linking these structures. The p66 aL-f320 loop protrudes into the pSI subunit, contributing to a major point of interaction at the RT heterodimer interface. Non-conservative mutation of the TRM residue W40I to alanine or leucine significantly diminishes RT subunit interaction, and accordingly RT activity. The dimerisation defect conferred by these mutations is predominantly mediated through the p66 subunit. In the p66 subunit W40I does not directly contribute to the heterodimer interface. Rather, W401 is located at the C-terminal of aL where it is proposed to have an essential role in maintaining the position of the p66 aL-f320 loop, and hence the interactions contributed by aL-f320 loop residues to the heterodimer interface. In contrast, the conservative substitution, W401F, does not affect the ability of the RT subunits to dimerise, indicating the importance of an aromatic residue at this location. The role of the p66 aL-f320 loop in RT subunit interaction is supported by mutagenic analysis of other residues in this region. Non-conservative mutation of the TRM residue W4I4, which anchors the C-terminal of the loop in f320, and ofK331, which interacts with the p66 aL-f320 loop in the pSI subunit, also perturb RT subunit dimerisation. A second-site compensatory mutation, T4091, which partially suppresses the dimerisation defected conferred by the W401A mutation, is located within the aL-f320 loop. These data strongly support the proposal of an RT dimerisation defected mediated by the position of the aL-~20 loop at the heterodimer interface. It was proposed that mutation of these residues in HIV -1 would allow examination of the role of RT subunit interaction in HIV -1 replication in the absence of pleiotropic effects that have been described for other R T mutations. Expression of TRM mutants that abrogate R T dimerisation (W 401 AIL) resulted in HIV -1 with significant decreases in levels of virion R T. Variation in the p5I :p66 ratio in viral and producer cell lysates suggested that these mutations affected proteolytic generation of mature R T. These defects were RT specific with no accompanying decrease in virion Gag-Pol or IN. Virion particle production and Gag processing resembled that of wild-type HIV -1, indicating PR activation was not affected. A similar range of defects was apparent upon expression of the dimerisation defective K33IA mutation in HIV-l. In contrast expression of the W40IF mutation, which does not adversely affect RT subunit dimerisation, conferred no significant defects when expressed in HIV -1. Furthermore, generation of a replication competent virus by passage of the W 401 A HIV -1 mutant identified three second-site mutations, CI62Y, K366R and A534T. These mutations also restored RT dimerisation to the W40IA mutant in a recombinant protein binding assay. These observations strongly suggested that the defects observed upon expression of dimerisation defective mutations were representative of the role of R T dimerisation in HIV -1 replication. While the effect of the W 401 mutations in HIV -1 replication corresponded well with their role in RT heterodimerisation, examination of other TRM residues indicated an independent role in HIV-1 replication. Expression of TRM mutants W398A and W 414A in HIV -1 resulted in reduced levels of virion Gag-Pol, RT and IN. These mutations were also found to change the Gag-Pol processing profile in a recombinant Gag-Pol expression system. The W401A second-site compensatory mutation identified in recombinant protein studies, T4091, also resulted in a similar profile when expressed in HIV -1 and recombinant Gag-Pol. Accordingly, this mutation did not act to suppress the defects conferred by W401A in HIV-1 replication. Interestingly, neither T409I nor W398A inhibit RT subunit dimerisation or RT activity in recombinant RT. The contrasting effect of these mutations in recombinant RT compared to HIV-I demonstrates the dynamic nature of the RT domain during maturation. This may be representative of a role for the TRM in regulation of GagPol cleavage events performed by the embedded PR either directly, or by interaction with regulatory host cell factors. These findings confirm that dimerisation of the HIV -1 RT domain has an important role in both the early and late phases of HIV -1 replication. Late phase defects were restricted to the proteolytic generation of mature RT, and no adverse effect on PR activation was observed. However this study identified the TRM as a key region in two independent stages of the late phase of HIV -1 replication, that being a defect in generation of a mature RT heterodimer upon disturbing RT dimerisation, and a dimerisation independent Gag-Pol processing defect. However, mutational analysis ofW414, and the combined defect of the W401A and T4091 mutations indicate that these defects can occur simultaneously. These data confirm the role of the TRM as a key region for RT interactions necessary for RT maturation and activity.