No abstract

BackgroundHIV-1 protease (PR) activation is triggered by Gag-Pol dimerization. Premature PR activation results in reduced virion yields due to enhanced Gag cleavage. A p6* transframe peptide located directly upstream of protease is believed to play a modulating role in PR activation. Previous reports indicate that the C-terminal p6* tetra-peptide prevents premature PR activation triggered by a leucine zipper (LZ) dimerization motif inserted in the deleted p6* region. To clarify the involvement of C-terminal p6* residues in mitigating enhanced LZ-incurred Gag processing, we engineered constructs containing C-terminal p6* residue substitutions with and without a mutation blocking the p6*/PR cleavage site, and created other Gag or p6* domain-removing constructs. The capabilities of these constructs to mediate virus maturation were assessed by Western blotting and single-cycle infection assays.Resultsp6*-PR cleavage blocking did not significantly reduce the LZ enhancement effect on Gag cleavage when only four amino acid residues were present between the p6* and PR. This suggests that the potent LZ dimerization motif may enhance PR activation by facilitating PR dimer formation, and that PR precursors may trigger sufficient enzymatic activity without breaking off from the PR N-terminus. Enhanced LZ-induced activation of PR embedded in Gag-Pol was found to be independent of the Gag assembly domain. In contrast, the LZ enhancement effect was markedly reduced when six amino acids were present at the p6*-PR junction, in part due to impaired PR maturation by substitution mutations. We also observed that a proline substitution at the P3 position eliminated the ability of p6*-deleted Gag-Pol to mediate virus maturation, thus emphasizing the importance of C-terminal p6* residues to modulating PR activation.ConclusionsThe ability of HIV-1 C-terminal p6* amino acid residues to modulate PR activation contributes, at least in part, to their ability to counteract enhanced Gag cleavage induced by a leucine zipper substituted for a deleted p6*. Changes in C-terminal p6* residues between LZ and PR may affect PR-mediated virus maturation, thus providing a possible method for assessing HIV-1 protease precursor activation in the context of virus assembly.

Background HIV-1 pol, which encodes enzymes required for virus replication, is initially translated as a Gag-Pol fusion protein. Gag-Pol is incorporated into virions via interactions with Gag precursor Pr55gag. Protease (PR) embedded in Gag-Pol mediates the proteolytic processing of both Pr55gag and Gag-Pol during or soon after virus particle release from cells. Since efficient Gag-Pol viral incorporation depends on interaction with Pr55gag via its N-terminal Gag domain, the prevention of premature Gag cleavage may alleviate Gag-Pol packaging deficiencies associated with cleavage enhancement from PR. Results We engineered PR cleavage-blocking Gag mutations with the potential to significantly reduce Gag processing efficiency. Such mutations may mitigate the negative effects of enhanced PR activation on virus assembly and Gag-Pol packaging due to an RT dimerization enhancer or leucine zipper dimerization motif. When co-expressed with Pr55gag, we noted that enhanced PR activation resulted in reduced Gag-Pol cis or trans incorporation into Pr55gag particles, regardless of whether or not Gag cleavage sites within Gag-Pol were blocked. Conclusions Our data suggest that the amount of HIV-1 Gag-Pol or Pol viral incorporation is largely dependent on virus particle production, and that cleavage blocking in the Gag-Pol N-terminal Gag domain does not exert significant impacts on Pol packaging.

A transframe region within HIV-1 Gag-Pol (referred to as p6* or p6pol), directly linked to the protease (PR) N-terminus, plays a pivotal role in modulating PR activation. To identify specific p6* residues involved in PR activation, we created a series of p6* mutants by making substitutions for conserved p6* residues. Our results indicate that some p6* mutants were defective in terms of virus infectivity, despite displaying a wild-type virus particle processing pattern. Mutations at p6* F8 reduced virus infectivity associated with insufficient virus processing, due in part to impaired PR maturation and RT packaging. Our data strongly suggest that conserved Phe (F) residues at position 8 of p6* are involved in the PR maturation process.