Structural Basis for Distinctions between Substrate and Inhibitor Specificities for Feline Immunodeficiency Virus and Human Immunodeficiency Virus Proteases

Abstract

We used feline immunodeficiency virus (FIV) protease (PR) as a mutational framework to define determinants for the observed substrate and inhibitor specificity distinctions between FIV and human immunodeficiency virus (HIV) PRs. Multiple-substitution mutants were constructed by replacing the residues in and around the active site of FIV PR with the structurally equivalent residues of HIV-1 PR.

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

“…To verify that the increase in nuclear fluorescence was due to the transport of intact Gag and not of the CA subdomain detected by the monoclonal antibody, we mutated the critical aspartic acid at position 30 of the protease catalytic domain (35) to a glycine residue in the FIV vector packaging plasmid pFP93 (44), generating pFP93Pr m (Fig. 4A).…”

“…pFIVGag⌬MA-CFP, pFIVGag⌬CA-CFP, pFIVGag⌬NCp2-CFP, pFIV-MA-CFP, pFIV-CA-CFP, and pFIV-NCp2-CFP, used to study domain contributions and assess potential FIV Gag nuclear localization signals (NLSs) and nuclear export signals (NESs), were made by standard PCR-based mutagenesis, using pFIVGag-CFP as a template. pFP93Pr m was constructed by overlap extension PCR that introduced an Asp-to-Gly change in the LLDTG motif (35) in the protease active site of pFP93 (the D30 GAC codon was changed to GAG). The deletion of pol from pFP93 (to yield pFP93⌬Pol) was achieved with an EcoRI-XcaI adaptor that was ligated between the EcoRI site near the gag C terminus and an XcaI site located at nucleotide (nt) 232 of the integrase gene.…”

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

“…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.…”

“…Various combinations of substitutions, including I37 32 V, N55 46 M, V59 50 I, I98 81 P, Q99 82 V, and P100 83 N, were further placed in an FIV PR background and analyzed. Other notable mutations, including I35 30 D, located in the active core, I57 48 G in the flaps, and L101 84 I in the "90s loop," yielded inactive PRs, as determined by the fluorogenic assay in vitro (36,37), and thus were not included in the present study.…”

“…Our approach in studying substrate and inhibitor specificities has been to exchange amino acid residues in and around the active site of FIV PR with structurally equivalent residues of HIV-1 PR. 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).…”