HIV-1 reverse transcriptase stability correlates with Gag cleavage efficiency: reverse transcriptase interaction implications for modulating protease activation

Proteolytic processing of human immunodeficiency virus type 1 particles mediated by viral protease (PR) is essential for acquiring virus infectivity. Activation of PR embedded in Gag-Pol is triggered by Gag-Pol dimerization during virus assembly. We previously reported that amino acid substitutions at the RT tryptophan repeat motif destabilize virus-associated RT and attenuate the ability of efavirenz (EFV, an RT dimerization enhancer) to increase PR-mediated Gag cleavage efficiency. Furthermore, a single amino acid change at RT significantly reduces virus yields due to enhanced Gag cleavage. These data raise the possibility of the RT domain contributing to PR activation by promoting Gag-Pol dimerization. To test this hypothesis, we investigated the putative involvement of a hydrophobic leucine repeat motif (LRM) spanning RT L282 to L310 in RT/RT interactions. We found that LRM amino acid substitutions led to RT instability and that RT is consequently susceptible to degradation by PR. The LRM mutants exhibited reduced Gag cleavage efficiencies while attenuating the EFV enhancement of Gag cleavage. In addition, an RT dimerization-defective mutant, W401A, reduced enhanced Gag cleavage via a leucine zipper (LZ) motif inserted at the deleted Gag-Pol region. Importantly, the presence of RT and integrase domains failed to counteract the LZ enhancement of Gag cleavage. A combination of the Gag cleavage enhancement factors EFV and W402A markedly impaired Gag cleavage, indicating a disruption of W402A Gag-Pol dimerization following EFV binding to W402A Gag-Pol. Our results support the idea that RT modulates PR activation by affecting Gag-Pol/Gag-Pol interaction. IMPORTANCE A stable reverse transcriptase (RT) p66/51 heterodimer is required for HIV-1 genome replication in host cells following virus entry. The activation of viral protease (PR) to mediate virus particle processing helps viruses acquire infectivity following cell release. RT and PR both appear to be major targets for inhibiting HIV-1 replication. We found a strong correlation between impaired p66/51RT stability and deficient PR-mediated Gag cleavage, suggesting that RT/RT interaction is critical for triggering PR activation via the promotion of adequate Gag-Pol dimerization. Accordingly, RT/RT interaction is a potentially advantageous method for anti-HIV/AIDS therapy if it is found to simultaneously block PR and RT enzymatic activity.