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.

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

“…The cell-free supernatant was harvested 48 h after transfection with the pCFIV⌬orf2⌬env clone. The virus-like particles were pelleted, lysed, and clarified as described previously (38). The clear lysate was loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the separated protein bands were transferred to a nitrocellulose membrane.…”

“…The sequence of the 3Ј primer containing the NsiI site was 5Ј-CAAGAGG AATGGTGAAATATGCATCCCCTATATC-3Ј. Sequences of mutagenic primers for FIV PR have been described in detail previously (37,38). Single substitutions for structurally equivalent residues of HIV-1 PR (FIV numbering with equivalent HIV-1 numbering in superscript) included I37 32 V, N55 46 M, M56 47 I, V59 48 I, L97 80 T, I98 81 P, Q99 82 V, and P100 83 N, and multiple substitutions included I37 32 V N55 46 M V59 50 I, I98 81 P Q99 82 V P100 83 N, and I37 32 V N55 46 M V59 50 I I98 81 P Q99 82 V P100 83 N. The structural locations of the substitutions listed above in FIV PR are shown in Fig.…”

“…Both cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 1 mM sodium pyruvate. The pCFIV expression plasmids were transfected into 293T cells for expression of FIV Gag and Gag-Pol polyprotein as described previously (38). HIV-1 PR inhibitors, including fosamprenavir (FPV), ATV, RTV, LPV, TPV, DRV, and TL-3 (31,32), and other compounds, including APV-1 (35) and AB-2 (8, 9), were used to evaluate the inhibitor sensitivity of PRs in the context of Gag-Pol polyprotein expression in 293T cells.…”

“…However, the generation of infectious chimeric viruses for ex vivo and in vivo assays has proven more difficult. Viruses encoding chimeric PRs with 4, 9, or 12 substitutions have been tested and are not infectious in cell culture, probably due to deleterious changes in the temporal cleavage of the FIV Gag polyprotein (38). In the present study, we address this issue by examining the effects of single substitutions in FIV PR on FIV Gag processing and infectivity.…”

“…Earlier studies showed that the drug specificity of FIV/HIV-1 chimeric PR could be dramatically changed to HIV-1-like specificity by the substitution of as few as 4 amino acids (I37 32 V, N55 46 M, M56 47 I, and V59 48 I), causing the K i values for the HIV-1 PR inhibitors to drop from the millimolar range down to around 20 nM (36,37). The use of a cell-based FIV Gag-Pol expression system (25) further allowed ex vivo assessment of the processing efficiency and the order of FIV Gag polyprotein processing by chimeric PRs in the context of the natural polyprotein substrate (38). In addition to confirming the drug sensitivities observed in vitro, the results showed that the substrate specificity of these mutants was also altered, with cleavage of the FIV Gag polyprotein displaying a signature more similar to that of HIV-1 PR than to that of WT FIV PR.…”