HIV-1 protease with leucine zipper fused at N-terminus exhibits enhanced linker amino acid-dependent activity

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.

Effects of reduced gag cleavage efficiency on HIV-1 Gag-Pol package

“…4 b; supplementary Fig. 4 b); this is consistent with previous reports [ 19 , 34 ]. We found that compared to the wt transfectant, cellular Pr55 gag was barely detectable in DWzPR and DWz/PR transfectants (Fig.…”

Effects of Reduced Gag Cleavage Efficiency on HIV-1 Gag-Pol Package

“…4b; supplementary Fig. 4b); this is consistent with previous reports [19,32]. We found that compared to the wt transfectant, cellular Pr55 gag was barely detectable in DWzPR and DWz/PR transfectants (Fig.…”

Amino acid substitutions at the HIV-1 transframe region significantly impair virus infectivity

“…Previous studies have suggested that conserved C-terminal p6* tetrapeptide mutations trigger significant PR maturation impairment [ 10 , 20 , 21 ]. For the present study we focused on analyzing the effects of other conserved p6* residue mutations on virus processing and PR maturation.…”

“…Single amino acid substitutions of the four C-terminal p6* residues can significantly impair virus maturation, strongly suggesting that the C-terminal p6* tetrapeptide is important for PR activation regulation [ 10 , 21 ]. Data from a study involving stepwise cluster substitutions for p6 codons (3–13 residues) suggest that both C- and N-terminal p6* regions are important, with the middle p6* part being largely dispensable in terms of PR activation [ 13 ].…”

“…Consistent with these data, a deletion of approximately 63% of p6* amino acid residues, or partial substitution of p6* with a heterologous peptide, does not significantly affect HIV-1 infectivity [ 23 ]. Results from our previous study, in which we performed single-cycle-infection assays of HIV-1 virions produced from Gag and Gag-Pol co-transfectants, indicate a significant reduction (20–40% of wt level) in virus infectivity following a major deletion of the p6* coding sequence [ 21 , 24 ]. The remaining C-terminal p6* tetrapeptide in the p6*-deleted mutant is insufficient for mitigating the reduced infectivity resulting from deficient virus processing [ 24 ].…”