Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Abstract

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease's specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1' substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1' site. Second site substitutions have also been designed to produce "revertant" substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1' substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable "revertants" showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the "revertant" mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

Keywords: HIV-1; human immunodeficiency virus; nucleocapsid protein; protease; retroviruses; specificity; substrate specificity; viral proteases; viral proteins.

Figure 1

The N-terminal sequence of the first (proximal) zinc-finger of the HIV-1 nucleocapsid protein. The sequence is numbered, the Cys and His residues involved in zinc binding are coloured by dark blue. The black arrow indicates the natural cleavage site of PR.

Figure 2

Fitting of Asn and Phe residues into the S1 substrate binding site of HIV-1 protease. (a) The structure of HIV-1 PR complexed with KIVKCFFGCK oligopeptide substrate. The S1′ and P1′ residues are shown by blue and red colours, respectively. (b) In contrast to the wild-type P1′-Asn, the P1′-Phe residue of mutant NC-1 sequence was expected to provide favourable hydrophobic interactions at the S1′ subsite.

Figure 3

Plotting experimentally determined relative activation energies and calculated S1′-P1′ relative interaction energies. (a) Volumes of non-charged P1′ residues (solid circles) correlated well (r² = 0.76) with the experimentally determined relative activation energies, while charged P1′ residues (open circles) were kinetically substantially less favourable than non-charged residues having similar sizes and were excluded from the correlation. (b) Interaction energies calculated for nonpolar (solid circles) and polar/charged (open circles) residues gave separate correlations with the specificity constants (r² = 0.88 and r² = 0.73, respectively). (c) Correlation of calculated S1′-P1′ relative interaction energies corrected for the solvation energy with the experimentally determined relative activation energies. Calculated relative interaction energies were multiplied by an empirical value (m = 0.25) and corrected with the relative solvation energies as described in the text gave a good correlation (r² = 0.85) with the relative activation energies.

Figure 4

Predicted subsite interactions that lead to the shifted cleavage of the Ile for Lys-substituted peptide. Modified residues are highlighted in red. Favourable subsite interactions are indicated by purple arrows. The red arrow shows within the sequence if cleavage occurs. (a) The figure represents the productive binding mode of the peptide representing the naturally occurring proximal zinc-finger cleavage site in HIV-1 (peptide 1 in Table 3), leading to hydrolysis at the Phe↓Asn site. Upon P1′Asn→Gly mutation, the peptide is not cleaved (peptide 14 in Table 3). (b) The peptide substrate is not processed by cleavage between Phe and Gly residues of the double-substituted peptide (peptide 16 in Table 3) when Ile occupies the S3 binding subsite. For this peptide, the same Ile residue occupies the S2 binding subsite in the productive binding where cleavage occurs between Cys and Phe residues.

Figure 5

The cleavage of recombinant protein substrates with HIV-1 PR. Cleavage reactions were performed with HIV-1 PR, using buffers supplemented either with DTT and EDTA (a) or ZnCl₂ (b). After the cleavage of recombinant substrates, the separation of substrates and cleavage products with PAGE was performed using non-denaturing conditions. The proteins were detected in the gel using blue-light transillumination. Arrows indicate bands of the uncleaved substrates, while asterisk indicates bands of the cleavage products containing the C-terminal mApple fluorescent protein. The dashed line (b) indicates putative oligomers.

Figure 6

A comparison of relative cleavage efficiencies of recombinant substrates. The substrate conversion was determined based on the band intensities of uncleaved substrates and fluorescent cleavage products. n = 2.