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Feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteases (PRs) share only 23% amino acid identity and exhibit distinct specificities yet have very similar 3-dimensional structures. Chimeric PRs in which HIV residues were substituted in structurally equivalent positions in FIV PR were prepared in order to study the molecular basis of PR specificity. Previous in vitro analyses showed that such substitutions dramatically altered the inhibitor specificity of mutant PRs but changed the rate and specificity of Gag cleavage so that chimeric FIVs were not infectious. Chimeric PRs encoding combinations of the I37V, N55M, M56I, V59I, L97T, I98P, Q99V, and P100N mutations were cloned into FIV Gag-Pol, and those constructs that best approximated the temporal cleavage pattern generated by wild-type FIV PR, while maintaining HIV-like inhibitor specificity, were selected. Two mutations, M56I and L97T, were intolerant to change and caused inefficient cleavage at NC-p2. However, a mutant PR with six substitutions (I37V, N55M, V59I, I98P, Q99V, and P100N) was selected and placed in the context of full-length FIV-34TF10. This virus, termed YCL6, had low-level infectivity ex vivo, and after passage, progeny that exhibited a higher growth rate emerged. The residue at the position of one of the six mutations, I98P, further mutated on passage to either P98H or P98S. Both PRs were sensitive to the HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV), as well as to the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC50) of 30 to 40 nM, consistent with ex vivo results obtained using mutant FIVs. The chimeras offer an infectivity system with which to screen compounds for potential as broad-based PR inhibitors, define structural parameters that dictate specificity, and investigate pathways for drug resistance development.
Retroviral protease (PR) is responsible for the temporal processing of viral Gag and the Gag-Pol polyprotein into structural and enzymatic proteins during viral maturation (2, 50). The proper cleavage of the polyprotein by PR is required in order to produce mature, infectious virus particles. Therefore, PR has been a prime target for inhibitor development. There are currently nine FDA-approved PR inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1): saquinavir (SQV), indinavir (IDV), nefinavir (NFV), amprenavir (APV), atazanavir (ATV), ritonavir (RTV), lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). In combination with reverse transcriptase (RT) inhibitors, multidrug therapy has dramatically reduced the mortality rate and improved the quality of life for infected patients (2, 27, 44, 53). In spite of the success of drug development and chemotherapy, however, the continuous selection and emergence of viral variants resistant to these inhibitors and the generation of cross-resistant mutants remain major challenges to drug development. More than 70 mutations in 38 residues of HIV-1 PR have been identified in association with drug resistance to PR inhibitors (7, 24). Given this extreme plasticity in PR, new strategies are required for designing a new generation of drugs against these drug-resistant mutants.
Feline immunodeficiency virus (FIV) has been used as a small-animal model for the study of the lentivirus life cycle and for the development of intervention strategies against HIV-1 (14-17, 22). One focus has been to study the molecular basis of the substrate and inhibitor specificities of FIV and HIV-1 PRs in order to develop broad-based inhibitors against a wide range of retroviral PRs, including drug-resistant variants. FIV and HIV-1 PRs share 27 identical amino acids (see Fig. Fig.1A)1A) and display distinct substrate and inhibitor specificities. FIV PR cleaves FIV Gag polyprotein into 5 individual proteins, including matrix (MA), capsid (CA), p1, nucleocapsid (NC), and p2, whereas HIV-1 PR cleaves HIV-1 Gag polyprotein into 6 individual proteins, MA, CA, p2 (SP1), NC, p1 (SP2), and p6 (see Fig. Fig.1B).1B). The clinical drugs against HIV-1 PR are very poor inhibitors for wild-type (WT) FIV PR, and interestingly, eight of the drug resistance mutations in HIV-1 PR mentioned above, namely, V11I, K20I, V32I, I50V, I62V, A71I, N88D, and L90M, are already present in the structurally equivalent positions of FIV PR (1611I, 2520I, 3732I, 5950V, 7162V, 8571I, 10588D, and 10790M [FIV numbering is given, with equivalent HIV-1 numbering in superscript]) (7, 24).
Comparisons of the 3 dimensional structures of the two PRs led to the rational design of TL-3, a broad-based PR inhibitor capable of blocking infection by FIV, simian immunodeficiency virus (SIV), and HIV-1, as well as many drug-resistant HIV-1 variants (10, 12, 21, 23, 31, 32). Similar approaches comparing the structures of FIV and drug-resistant HIV-1 PRs to that of WT HIV-1 PR have led to the development of additional PR-inhibiting compounds with broadened efficacy (8, 9, 19, 29, 41, 42). 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 (I3732V, N5546M, M5647I, and V5948I), causing the Ki 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.
The studies described above demonstrated that active FIV/HIV chimeric PRs could be generated and assayed both in vitro and ex vivo for alterations in substrate and inhibitor specificities in the context of the natural polyprotein substrates. 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. We report the successful generation of infectious FIV encoding FIV/HIV chimeric PR with 6 mutations: I3732V in the active core, N5546M and V5950I in the flaps, and I9881(H/S), Q9982V, and P10083N in the “90s loop.”
The approach for examining the processing specificity of FIV Gag polyprotein by chimeric PRs was to use the pCFIVΔorf2Δenv packaging expression vector (25). The expression of FIV Gag and Gag-Pol polyprotein is driven by the cytomegalovirus (CMV) promoter. The system is useful for examining the biological relevance of PR function, since PR is expressed in the context of Gag-Pol polyprotein ex vivo. To generate mutant pCFIV clones containing chimeric PRs, PCR-mediated mega-primer mutagenesis (48) was performed on the wild-type unique PflFI/NsiI restriction fragment. The sequence of the 5′ primer containing the PflFI site was 5′-CAGGACTTTTAAATATGACGGTGTCTACTGCTGCTGC-3′. The sequence of the 3′ primer containing the NsiI site was 5′-CAAGAGGAATGGTGAAATATGCATCCCCTATATC-3′. 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 I3732V, N5546M, M5647I, V5948I, L9780T, I9881P, Q9982V, and P10083N, and multiple substitutions included I3732V N5546M V5950I, I9881P Q9982V P10083N, and I3732V N5546M V5950I I9881P Q9982V P10083N. The structural locations of the substitutions listed above in FIV PR are shown in Fig. Fig.2.2. I3732V is in the active core, whereas N5546M, M5647I, and V5950I are in the flap region. L9780T, I9881P, Q9982V, and P10083N are in the “90s loop” region. To construct the mutant FIV-34TF10 (51) viral plasmid, the unique PflFI/NsiI restriction fragments of mutant pCFIVΔorf2Δenv clones containing the corresponding PR mutations were purified and ligated into the PflFI/NsiI-restricted 34TF10 plasmid.
Human 293T cells (13) were used for transfection of pCFIVΔorf2Δenv clones to examine the processing pattern of FIV Gag polyprotein and the inhibitor sensitivity of Gag polyprotein processing by PR. Feline G355-5 astrocytic glial cells (a gift from Don Blair, NIH) were used for the transfection of FIV-34TF10 viral clones in order to study infectivity and inhibitor sensitivity ex vivo. 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. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: APV, ATV, LPV, TPV, and DRV (catalog no. 11447) from Tibotec, Inc. Fresh PR inhibitor was replenished 24 h after transfection at the same concentration. The processing of FIV Gag polyprotein and viral particle-associated reverse transcriptase (RT) activity were analyzed 48 h after transfection.
For infectivity of 34TF10 clones, plasmid DNAs carrying wild-type and chimeric 34TF10 were transfected into G355-5 glial cells as described above in the absence and presence of various inhibitors, including LPV, DRV, and TL-3, at concentrations ranging from 200 nM to 800 nM for the wild type and from 50 nM to 200 nM for mutants. The viral RT activity released into the supernatant was monitored every 3 to 4 days for as long as 4 weeks. The cells were trypsinized and split at 1:10 every 3 to 4 days.
The cell-free supernatant was harvested 48 h after transfection with the pCFIVΔorf2Δenv 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. A rabbit antiserum against FIV CA (24 kDa) was used at 1:4,000 to monitor the processing of FIV Gag polyprotein by Western blot analysis. Horseradish peroxidase-conjugated secondary antibodies at 1:10,000 and SuperSignal West Dura enhanced chemiluminescent substrate (Pierce Biotechnology, Inc.) were used to visualize the processed bands of FIV Gag polyprotein. Six CA-associated protein bands, including MA-CA-p1-NC-p2 (Gag), MA-CA-p1-NC, MA-CA-p1, CA-p1-NC-p2, CA-p1-NC, and CA (CA-p1), could be detected by Western blot analysis. Viral particle production after transfection was monitored by cell-free RT activity in the culture supernatant, as described previously (38). The specific activity of [methyl-3H]2′-deoxythymidine-5′ triphosphate salt, used for the RT activity assay, was 70.0 Ci/mmol (2.59 Tbq/mmol), and 1.25 μCi was used for each RT assay.
To characterize the extent of viral evolution that occurred during the long passage of infected cells, the PR gene and the Gag gene were cloned from the cell-free viral supernatant, and the cloned DNA was sequenced. The cell-free viral supernatant from 8 weeks posttransfection was harvested and filtered through a 0.22-μm-pore-size filter. The viral particles were pelleted using ultracentrifugation at 100,000 × g for 30 min. The viral RNA was extracted using the Qiagen viral RNA extraction kit, and complementary viral DNA was reverse transcribed and amplified using the Qiagen one-step RT-PCR kit (Qiagen Sciences). The primer pair used for amplification of the PR region consists of a 5′ forward primer encoding the unique EcoRI restriction site in the NC gene (5′-GGAAATAGAAAGAATTCGGGAAACTGGAAGG-3′) and a 3′ reverse complementary primer in the RT gene (5′-CTTTCCCTTTTTCTAGTCTTTCTAC-3′). The primer pair used for amplification of the Gag and PR regions comprises a 5′ forward primer containing the unique PflFI site in the MA gene and the reverse complementary primer containing a unique NsiI site in the RT gene, as described above. The PCR conditions were as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s, and extension at 72°C for 2 min, with a final extension at 37°C for 10 min. The PCR-amplified product was gel purified and ligated into the pCR2.1 vector using TOPO cloning (Invitrogen Corporation).
The full-length chimeric PR genes, including 6s (I37V N55M V59I I98P Q99V P100N), 6s-98H, and 6s-98S, were PCR amplified and cloned into the pET21a(+) expression vector (EMD Chemicals, Inc.) for protein expression, using unique NdeI and HindIII restriction sites. PR expression was induced with 1 mM isopropyl β-d-1-thiogalactoptranoside for 4 h at 37°C in the transformed Rosetta 2 strain of Escherichia coli (EMD Chemicals, Inc.), and PR was subsequently purified using ion-exchange chromatography and fast protein liquid chromatography (FPLC) from inclusion bodies as described previously (20, 31).
The activity of FIV PR was assayed in 50 mM sodium citrate-100 mM phosphate buffer (pH 5.25) with 200 mM NaCl and 1 mM dithiothreitol at 37°C using 50 μM fluorogenic substrate A-L-T-(2-amino benzoic) K-V-Q/(p-NO2) F-V-Q-S-K-G as described previously (18). The sensitivities of chimeric 6s, 6s-98H, and 6s-98S PRs to the inhibitors DRV, LPV, and TL-3 were measured. The data were obtained continuously at an excitation wavelength of 325 nm and an emission wavelength of 410 nm for 5 min, using an F-2000 fluorescence spectrophotometer (Hitachi Inc.). The inhibitor concentration that inhibited PR activity by 50% (IC50) was determined using the GraFit 4 program (Erithacus Software Ltd.).
One monomer of the homodimeric FIV PR consists of 116 amino acids, while each HIV-1 PR monomer contains only 99 amino acids. These two enzymes share 27 identical amino acids (Fig. (Fig.1A).1A). The two PRs display distinct substrate specificities, consistent with the divergence of the cleavage junctions in the Gag proteins and Gag-Pol polyproteins of the two viruses (Fig. (Fig.1B1B [only Gag is shown]). Most of the residues in the active sites of the two PRs are distinct (Fig. (Fig.1A,1A, residues around Asp25 in HIV-1 PR and Asp 30 in FIV PR, the flaps, and the “ 90s loop”), but the 3-dimensional structures of the two active cores are highly conserved (20, 28, 54). The structural locations of the substitutions investigated in this study are shown on one monomer of the dimeric FIV and HIV-1 PRs in Fig. Fig.2.2. In previous studies (38), dramatically different profiles for the processing of FIV Gag polyprotein were observed with certain combinations of these mutations compared to WT FIV PR, and mutants tested in the context of full-length FIV were not infectious. Specifically, two processing intermediates, MA-CA-p1-NC and CA-p1-NC, were missing under controlled cleavage conditions as a result of poor cleavage of the NC-p2 junction by the mutant PRs (38).
Current work is focused on the following questions. First, we want to further identify the molecular determinants that dictate substrate specificity and to define the basis for loss of infectivity with certain single substitution mutants. Second, we want to determine if infectious FIV/HIV-1 chimeras with multiple substitutions in PR can be generated. Last, we want to assess the feasibility of using a cell-based chimeric infectivity assay to measure sensitivity to PR inhibitors and to potentially serve as a useful means for selecting broad-based PR inhibitors. We systemically examined FIV PR mutants with single substitutions, including I3732V near the active core; N5546M, M5647I, and V5950I on the flap; and L9780T, I9881P, Q9982V, and P10083N on the 90s loop (Fig. (Fig.2).2). Substrate specificity was analyzed by determining the effects of these mutations on the processing specificity of FIV Gag and on viral infectivity in glial cells. Various combinations of substitutions, including I3732V, N5546M, V5950I, I9881P, Q9982V, and P10083N, were further placed in an FIV PR background and analyzed. Other notable mutations, including I3530D, located in the active core, I5748G in the flaps, and L10184I 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.
Since the 4s (I3732V N5546M M5647I V5950I) virus is noninfectious (Table (Table1),1), pCFIV mutants containing these individual substitutions were constructed, and FIV Gag processing patterns were examined using the Gag-Pol expression system in the presence of suboptimal concentrations of TL-3 (Fig. (Fig.3A).3A). The N55M and V59I mutants gave processing patterns essentially identical to that of WT FIV PR. However, the pattern generated by the M5647I mutant was dramatically different from that of WT FIV PR and other mutants, with a loss of temporal processing at NC-p2, as judged by the complete lack of band 2 and band 5 processing intermediates (Fig. (Fig.3A).3A). The inability of this mutant to process Gag polyprotein correctly is most likely responsible for the loss of infectivity in the 4s, 9s, and 12s mutants, all of which carry this M5647I mutation. The I3732V mutant also failed to process the NC-p2 junction efficiently (Fig. (Fig.3A),3A), consistent with the lack of infectivity of chimeras carrying this mutation.
Additional pCFIV mutants containing L9780T, I9881P, Q9982V, or P10083N in the “90s loop” were constructed, and the processing patterns of FIV Gag were analyzed in the absence and presence of suboptimal concentrations of TL-3 (Fig. (Fig.3B).3B). The L9780T mutant exhibited slightly reduced overall Gag processing and marked changes in the processing pattern from those of the other three mutants (Fig. (Fig.3B).3B). As with the M5647I mutant (Fig. (Fig.3A),3A), there was little or no processing at NC-p2 by the L9780T mutant, as judged by the lack of band 2 and band 5 in the Western blot analysis, suggesting that L9780T is detrimental to viral infectivity.
To determine if the altered FIV Gag polyprotein processing noted above correlated with infectivity and viral fitness, plasmids encoding full-length FIV-34TF10 with single and multiple mutations in PR were constructed and transfected into G355-5 glial cells. The infectivities of mutants were monitored by RT activity in culture supernatants for 3 to 4 weeks after transfection. The results of infectivity studies, summarized in Table Table1,1, show that mutant FIVs carrying M5647I in the flaps and L9780T in the “90s loop” were not infectious, consistent with the aberrant Gag processing mentioned above (Fig. 3A and B). Likewise, FIVs with other amino acid substitutions at position 56, including 5647A, 5647V, and 5647L, were not infectious, underscoring the need for conservation at this site. The rest of the single mutants investigated, including the I3732V, I3732A, N5546M, V5948I, I9881P, Q9982V, and P10083N mutants, were infectious, despite some variation in Gag processing. FIV mutants carrying 3 substitutions, excluding the intolerant M5647I and L9780T mutations, were constructed and tested for infectivity ex vivo. Two triple mutants (3s), with the I3732V, N5546M, and V5950I mutations or the I9881P, Q9982V, and P10083N mutations, were infectious (Table (Table1).1). These two triple mutants were then combined to form a mutant containing six substitutions (37V-6s). An additional version of 6s was prepared with alanine at position 37 (37A-6s), since results with single mutants indicated better viral growth with I37A. The infectivities of the two 6s mutants were monitored for several weeks posttransfection in G355-5 cells, with only low-level RT activity detected in the culture supernatant (Fig. (Fig.4A).4A). Interestingly, the infectivity of the 37A-6s mutant never recovered, despite the improved results with the single I37A mutant. In contrast, the RT activity in the 37V-6s culture started to increase after 6 weeks in culture and rose rapidly over the following 2 weeks. After 64 days, the final cell-free supernatants from 2 independent experiments were used to infect fresh G355-5 cells, and RT activity in the culture supernatant was monitored (Fig. (Fig.4B).4B). RT activity increased at an accelerated rate compared to that of the original virus, suggesting the emergence of progeny virus with improved fitness.
Viral particles from 3-week culture supernatants of two independent infections (Fig. (Fig.4B,4B, 37V-6s-1 and 37V-6s-2) were harvested, and viral RNA was extracted and purified. The gene encoding PR was then amplified from viral RNA by RT-PCR and was cloned into the pCR2.1 vector (Invitrogen Corporation), and multiple clones were sequenced in order to look for the emergence of new PR mutations. Additional mutations were identified in 6s PR, with two dominant changes apparent in the two cultures. In the progeny virus of 37V-6s-1, 24 out of 28 clones had a 98P → H mutation in PR, and 3 had a 98P → S mutation (Table (Table2).2). All clones from 37V-6s-2 contained a 98P → S mutation. Thus, the data indicated that the original I9881P mutation in the 6s mutant was not well tolerated, and further mutations at this position corresponded with better viral growth kinetics.
Using the FIV Gag-Pol expression system, pCFIV constructs carrying either 6s-98H or 6s-98S PR were prepared and transfected into 293T cells in the absence and presence of a panel of potent HIV-1 PR inhibitors at a concentration of 2.5 μM. The panel included clinical drugs such as DRV, FPV, LPV, RTV, and TPV. The Gag processing of virus-like particles by mutant FIV PR was analyzed in Western blots probed with an anti-CA antibody (Fig. 5A and B). Both the 6s-98H and 6s-98S PRs were very sensitive to TL-3, LPV, DRV, and, to a lesser degree, APV-1, as shown by the blockage of Gag processing. As with the original 37V-6s mutant, neither the 6s-98H nor the 6s-98S mutant demonstrated sensitivity to RTV or TPV in this assay, although all three 6s mutant PRs were sensitive to TPV in vitro (data not shown). The insensitivity to TPV ex vivo might be partially due to its lack of bioavailability in this assay. Of importance, these PRs with further mutations at position 98 retained HIV-like inhibitor sensitivity rather than reverting to the WT FIV character.
All three 6s PR mutants were cloned into the pET21a (+) expression vector for protein expression, and purified PRs were assessed for their relative sensitivities to DRV, LPV, and TL-3 in vitro (Table (Table3).3). The chemical structures of DRV, LPV, and TL-3 are shown in Fig. Fig.6.6. The IC50s of DRV and LPV against 6s-98H and 6s-98S PRs were 30 to 37 nM, values very similar to that for the original 6s PR. The IC50s of DRV against all three chimeric PRs were markedly decreased, about 50-fold, from that against WT FIV PR, and the IC50 of LPV was more than 100-fold lower than that against WT FIV PR. TL-3 exhibited slightly lower IC50s against both 6s-98H and 6s-98S than against WT FIV PR, consistent with the increased HIV-1 PR character in the new mutant FIV PRs that emerged.
Since the processing of FIV Gag polyprotein by mutant PRs was severely blocked by DRV, LPV, and TL-3, the sensitivities of FIVs carrying WT and chimeric PRs were further assessed. WT FIV DNA was transfected into G355-5 cells in the absence and presence of DRV, LPV, and TL-3 at concentrations of 200 nM, 400 nM, 600 nM, and 800 nM. The RT activity in culture supernatants was monitored for 3 to 4 weeks. WT FIV was not sensitive to DRV or LPV at any concentration (Fig. 7A and B). WT FIV showed sensitivity to TL-3 down to 200 nM and was completely inhibited at concentrations of 400 nM and higher (Fig. (Fig.7C),7C), consistent with previous ex vivo results (31). The infectivity of mutant FIV carrying 6s-98H or 6s-98S PRs was tested in the absence and presence of DRV (50 nM, 100 nM, and 200 nM), LPV (50 nM, 100 nM, and 200 nM), and TL-3 (50 nM, 100 nM, and 150 nM). The mutant FIV encoding 6s-98H PR was very sensitive to DRV and TL-3 (Fig. 8A and C) at all concentrations tested, but it was less sensitive to LPV, which was inhibitory at 200 nM (Fig. (Fig.8B).8B). The mutant encoding 6s-98S PR was also sensitive to both DRV and TL-3 at all concentrations and was less sensitive to LPV, like the 6s-98H mutant (data not shown).
Previous results showed that the inhibitor and substrate specificities of chimeric FIV/HIV PRs could be altered drastically and made more similar to that of HIV-1 PR by introducing multiple substitutions into the active site of FIV PR (36-38). The altered specificity of chimeric FIV mutants was shown both in in vitro PR assays and in a cell-based Gag-Pol expression system. The results also showed that chimeric mutants could be useful as an alternative to HIV-1 for the screening of compounds for their potential as broad-based inhibitors in vitro and ex vivo. We were interested in generating infectious FIV mutants carrying chimeric FIV/HIV PR to enhance the utility of the FIV model system for studies of drug susceptibility and for investigations of the mechanisms involved in the development of resistance against HIV PR inhibitors. Initial attempts to construct infectious mutant FIVs that contained FIV/HIV chimeric PR with multiple substitutions were not successful (38). Further analysis of FIV Gag polyprotein processing by the FIV/HIV chimeric PRs revealed inefficient cleavage at the NC-p2 and MA-CA junctions of FIV Gag, which was likely responsible for the loss in viral infectivity. In the present study, we identified the individual residues that were responsible for the altered processing patterns of the Gag polyprotein and then generated infectious FIV mutants encoding selected FIV/HIV chimeric PRs. Importantly for the goals of the study, these mutant FIVs demonstrated sensitivity to potent HIV-1 PR inhibitors that failed to inhibit WT FIV.
Methionine (position 56) in the flaps of FIV PR turned out to be an important structural residue that was intolerant to change. This residue resides near the tip of the flap over the S2 subsite, and thus, M5647I could affect the interactions at the P2/P2′ positions of a substrate. PRs carrying the M5647I mutation cleaved the FIV NC-p2 site poorly. This might be due to the presence of Gln at P2 and P2′ in the FIV NC-p2 cleavage junction (NQM-QQA); Gln is not present at P2 in natural HIV PR substrates. It is very likely that M5647I has drastically changed the P2 selectivity of chimeric PR to give it a more HIV-like character; consequently, it no longer recognizes Gln at P2. Although the mechanism is still unclear, position 56 may require a more flexible and less bulky residue to maintain proper interactions with Gln in the NC-p2 junction. Gln is also found at P2 in the FIV CA-p1 cleavage junction (MQL-LAE). This feature is unique to FIV substrates and has never been found in the P2 position of any known HIV-1 cleavage site or any other HIV-1 PR substrate (4-6). The possible role of processing at the FIV CA-p1 cleavage junction in viral infectivity is currently under investigation. Interestingly, the processing of the equivalent HIV-1 CA-p2 junction, which is the final cleavage step in the temporal processing of HIV-1 Gag (26, 46), is blocked by an experimental maturation inhibitor, PA-457 (bevirimat), resulting in the formation of aberrant, noninfectious HIV-1 (1, 34).
Leucine at position 97, in the “90s loop” of FIV PR, is also intolerant to change. L9780 is associated with the S1/S2 subsites, and substitutions at this position may influence the preferences at the P1/P2 positions of a substrate (20, 28, 54). In addition to poor/delayed cleavage at the FIV NC-p2 junction, inefficient processing at the FIV MA-CA cleavage junction by the L9780T mutant was also observed (Fig. (Fig.3B).3B). The hydrophobic residue L97 most likely makes stronger interactions with the hydrophobic alanine and isoleucine residues at the P2/P2′ positions of the FIV MA-CA cleavage junction (QAY-PIQ) than the polar 97T residue can form. Blockage of the cleavage of the HIV-1 MA-CA site by a mutation in P1 has been shown to strongly inhibit viral maturation, resulting in the generation of an aberrant core and the loss of viral infectivity (30). These findings underscore the critical nature of substrate processing in the generation of infectious virus particles and are consistent with the phenotype noted with mutations at position 97.
The p2 peptide of the FIV Gag polyprotein is functionally equivalent to the late domain-containing the p6 peptide of HIV-1 Gag polyprotein and contains conserved PSAP and LXXL motifs that are essential for viral assembly and production (39, 43). The production and release of viral particles in FIV mutant constructs was not affected by PR mutations, despite the processing inefficiency at the NC-p2 junction. This indicates that cleavage at NC-p2 is not required for viral release. However, efficient processing at the NC-p2 junction to produce mature NC and p2 is required for FIV infectivity. The processing efficiency of the equivalent HIV-1 NC-p1-p6 cleavage junctions has been extensively examined, and proper temporal cleavage at these sites correlates with viral fitness, replication capacity, and drug resistance (11, 40, 45, 47, 49, 52). The findings of the current study reinforce the notion that the cleavage and maturation of NC-p2 in FIV and of the equivalent NC-p1-p6 in HIV-1 offer a new potential therapeutic target.
Two predominant FIV mutants with higher replication rates emerged after transfection with the primary 37V-6s mutant, in which the Pro resulting from the I98P mutation mutated further to either His or Ser after long passage. Some other mutations were noted in a few cloned PCR products, but subsequent generation of isogenic viruses encoding 6s-98H and 6s-98S demonstrated improved replication kinetics for the chimeric 6s FIVs. The results indicate that as with the adjoining residue 9780, changes at residue 9881 were not well tolerated by FIV, and further change improved viral fitness and infectivity. Interestingly, the two corresponding HIV-1 PR residues, T80 and P81 at the tip of the 80s loop in HIV-1 PR, are conserved, and no drug resistance mutations have been reported at these sites (7, 24). Our results suggest that these two conserved residues at the tip of the 80s loop in HIV-1 PR (corresponding to the “90s loop” of FIV PR) may be a valuable target for new drug design. If a future inhibitor forms strong interactions with these two conserved residues, it may be difficult for PR to mutate in a way that allows it both to evade the drug and also to process its natural substrates in a manner that maintains infectivity.
The 6s-98H and 6s-98S mutant FIV clones also showed more HIV-1 PR-like inhibitor sensitivity, although they were still less sensitive than HIV-1 PR in vitro. Earlier studies with the inhibitor TL-3 indicated that FIV PR, which is larger than HIV-1 PR, is sensitive to a longer inhibitor, such as TL-3. Current clinical drugs are much shorter than TL-3, and that might partially explain their lower affinity for FIV PR than for HIV-1 PR. Another reason for lower affinity is that other important substitutions, such as I3530D, M5647I, and I5748G in the active site, were not tolerated in the mutants and thus were not included in these chimeras. Structural studies are in progress to investigate the molecular basis of inhibitor affinity between FIV and HIV-1 PRs. Temporal FIV Gag polyprotein processing by both chimeric FIVs was much more sensitive to the HIV-1 PR-inhibiting drugs DRV and LPV than was that by WT FIV. The chimeric FIV/HIV system demonstrated that HIV-1 PR inhibitors with broad-spectrum properties, such as LPV, DRV, and TL-3, can be distinguished using comparative studies of WT and chimeric FIVs, both in vitro and ex vivo. DRV and LPV are new FDA-approved HIV-1 PR inhibitors that are effective against WT HIV-1 and many drug-resistant HIV-1 mutants, and they have high genetic barriers (i.e., they require many mutations for drug resistance to develop) (3, 33, 53). Conversely, RTV, an older HIV-1 PR inhibitor, was not very potent against the 6s-98S/H PRs; it also displays a lower genetic barrier and is not as effective against many of the common drug-resistant mutants found in HIV-1 PR. Interestingly, LPV has the same core structure, P2 to P1′, as TL-3. DRV, derived from APV, has the same core P1-to-P2′ structure as APV. This suggests (i) similar inhibitor specificities for these two cores against the 6s FIV mutants and (ii) broad inhibitor selectivity for both. In addition, the profile of resistance of HIV-1 PR to LPV and DRV showed that two of the main resistance mutations in the active site, V32I and I50V, are identical to the equivalent residues of WT FIV PR, I3732 and V5950, respectively. This suggests that WT FIV PR should be resistant to LPV and DRV, which is consistent with our data. This observation may provide insight regarding the evolutionary pathways that HIV-1 PR uses to escape treatment with LPV and DRV, and it highlights the utility of structure-function studies of chimeric PRs.
HIV/FIV chimeric mutant FIVs offer an alternative cell-based infectivity system to (i) screen for new, broad-spectrum inhibitors and (ii) characterize the development of resistance against FDA-approved HIV PR inhibitors, without the significant biohazards involved in studies of infectious HIV-1 variants. In addition, this new chimeric system allows decoupling of the substrate and drug selectivity profiles to facilitate the definition of structural parameters that dictate PR specificity for drugs, for substrates, or for both. Future studies will aid in the design and development of new classes of broad-based PR inhibitors by defining these structural parameters and by performing high-throughput screens against these new chimeric PRs.
We thank Alex L. Perryman for making Fig. Fig.22 and for critical review of the manuscript. We also thank Arthur J. Olson, Joan Hu, and Yang Hong for review and critical discussions of the results, Meaghan Happer for technical support, and Gale Roy-Session for administrative assistance. We also acknowledge the support of the Protein Expression and Proteomics Core at the UCSD Center for AIDS Research (NIAID AI36214).
This research was supported by grants R01 AI081585 and P01 GM083658 from the National Institutes of Health.
Published ahead of print on 21 April 2010.