Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system

Abstract

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.

FIG. 1.

(A) Amino acid sequence alignment of FIV and HIV-1 PRs. FIV PR monomer is composed of 116 residues, as opposed to 99 residues for HIV-1 PR. There are 27 identical residues between the FIV and HIV-1 PRs. *, catalytic aspartic acid, D25 for FIV PR and D30 for HIV-1 PR. The substitutions made in this study are in boldface. (B) Schematic representation of FIV and HIV-1 Gag and Gag-Pol polyprotein. Cleavage sites and individual mature proteins are shown. HIV-1 has an additional spacer protein, p1, between NC and p6 in the Gag polyprotein, whereas FIV has an additional enzyme, DU, between RH and IN compared to HIV-1. TF, transframe region.

FIG. 2.

Structural locations of substitutions in FIV PR. Substituted residues are shown on one chain of dimeric PR. Substitutions include I37³²V in the active core; N55⁴⁶M, M56⁴⁷I, I57⁴⁸G, V59⁵⁰I, G62⁵³F, and K63⁵⁴I in the flap; and L97⁸⁰T, I98⁸¹P, Q99⁸²V, P100⁸³N, and L101⁸⁴I in the 90's loop.

FIG. 3.

(A) Expression and processing of FIV Gag polyprotein in viral particles produced by 293T cells transfected with pCFIV constructs are shown in a Western blot assay probed with anti-MA antibodies. (B) Expression and processing of Gag polyprotein in mutants D30²⁵N, I35³⁰D, and I57⁴⁸G are shown in a Western blot assay probed with anti-MA antibodies. wt, wild type. The values on the left are molecular sizes in kilodaltons.

FIG. 4.

(A) Processing efficiency of Gag polyprotein in wild-type and mutant pCFIV constructs. Average percent MA (percent processed MA/total unprocessed Gag) was quantified by densitometry. (B) Average percent RT activity (fraction of wild-type activity) of virus-associated supernatant in FIV mutants. wt, wild type. SQV, saquinovir.

FIG. 5.

Chemical structures of HIV-1 PR inhibitors used to assess the inhibitor specificities of mutants in vitro and ex vivo.

FIG. 6.

(A) Processing of Gag polyprotein by wild-type and mutant PRs in the presence and absence of 2.5 μM TL-3. Processed bands are shown by a Western blot assay probed with anti-MA antibodies. (B) Processing of Gag polyprotein by F4s and F9s PRs in the presence of additional inhibitors including APV-1, AB-2, AB-6, AB-8, and RTV at 2.5 μM. Western blot assays were probed with anti-MA antibodies. wt, wild type. The values on the left are molecular sizes in kilodaltons.

FIG. 7.

Inhibition of Gag polyprotein processing in the presence and absence of different inhibitors (2.5 μM). Inhibition (% MA) of processing in the wild type (wt), F4s, and F9s was quantified by densitometry.

FIG. 8.

(A) Pattern of FIV Gag polyprotein processing by wild-type FIV PR in pCFIV constructs, shown by Western blot assays probed with anti-CA antibodies. Various concentrations of TL-3 (1 to 5 μM) were used (note the increase in inhibitor sensitivity) to temper processing and reveal Gag polyprotein processing intermediates. Altered patterns of Gag polyprotein processing in F4s (B), F9s (C), and F12s (D) are shown in Western blot assays probed with anti-CA antibodies. Various concentrations of TL-3 (25 to 100 nM) were used (note the increase in inhibitor sensitivity). Four Gag polyprotein processing intermediates are indicated. The values on the left are molecular sizes in kilodaltons.

FIG. 9.

Comparison of processing of FIV Gag polyprotein generated by wild-type (wt) FIV PR, “HIVinized” FIV mutants, and HIV-1 PR expressed in the context of FIV Gag-Pol. Processed bands are revealed by Western blot assays with anti-CA antibodies. F4s, F9s, and F12s represent FIV mutants containing 4, 9, and 12 HIV-equivalent substitutions, respectively. FHIV-5A represents the FIV Gag polyprotein expressing wild-type HIV-1 PR, with the entire N-terminal PR cleavage junction composed of HIV residues; FHIV-5B also expresses wild-type HIV-1 PR, but the N-terminal half (P4 to P1) of the N-terminal PR cleavage junction is FIV derived. The efficiency of cleavage of FHIV-5A is greater than of FHIV-5B, consistent with more-efficient release of PR activity from the former construct. The values on the left are molecular sizes in kilodaltons.