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An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro , and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC 50 s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99 82 A, I59 50 V, and V37 32 I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC 50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.

The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been co-crystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel ‘inhibition of nucleation’ crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5–5 °C as assayed by differential scanning fluorimetry (DSF), and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.

FIV is a significant pathogen in the cat and is, in addition, the smallest available natural model for the study of lentivirus infections. Although divergent at the amino acid level, the cat lentivirus has an abundance of structural and pathophysiological commonalities with HIV and thus serves well as a model for development of intervention strategies relevant to infection in both cats and man. The following review highlights both the strengths and shortcomings of the FIV/cat model, particular as regards development of antiviral drugs.

Treatment with HIV-1 protease inhibitors, a component of highly active antiretroviral therapy (HAART), often results in viral resistance. Structural and biochemical characterization of a 6X protease mutant arising from in vitro selection with compound 1, a C 2-symmetric diol protease inhibitor, has been previously described. We now show that compound 2, a copper(I)-catalyzed 1,2,3-triazole derived compound previously shown to be potently effective against wild-type protease (IC 50 = 6.0 nM), has low nM activity (IC 50 = 15.7 nM) against the multidrug-resistant 6X protease mutant. Compound 2 displays similar efficacy against wild-type and 6X HIV-1 in viral replication assays. While structural studies of compound 1 bound to wild type and mutant proteases revealed a progressive change in binding mode in the mutants, the 1.3 A resolution 6X protease-compound 2 crystal structure reveals nearly identical interactions for 2 as in the wild-type protease complex with very little change in compound 2 or protease conformation.

We report a new simple method that allows rapid preparation in solution of a library of compounds for in situ high-throughput screening to identify new inhibitors of HIV-1 protease. The method is based on the amide-forming reaction of a C(2)-symmetrical diamino diol core with various carboxylic acids, followed by a direct assay of the inhibition activity without product isolation. Sixty-two compounds were made and screened in less than 1 hr. The utility of this method is demonstrated by the identification of new P3-P3' residues that convert a transition state analog core from a poor binding molecule (1, K(i) > 2 microM) to a potent inhibitor (AB1, K(i) = 2 nM) against the wild-type, and the inhibition activities against resistant mutants are better than those of two existing drugs. This method reduces the time required for synthesis and testing of a large number of characterized inhibitors and should find useful applications in other enzyme systems.

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH 2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH 2 NH)VVNGL-NH 2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.

A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC(50) values in the nanomolar range.

ts1 is a highly neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus TB (MoMuLV-TB). We previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-->Ile, in precursor envelope protein gPr80env. This Val-25-->Ile substitution apparently renders gPr80env inefficient for transport from the endoplasmic reticulum to the Golgi apparatus. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to the accumulation of gPr80env in the endoplasmic reticulum. Since endothelial and glial cells are targets of ts1 infection in the central nervous system, we established primary endothelial and astrocyte cultures to investigate the mechanism of cell killing caused by ts1. A continuous cell line, TB, was used as a control. Our results showed that both ts1 and MoMuLV-TB replicated and induced a cytopathic effect in astrocyte cultures, albeit to different degrees; ts1 appeared to be more lethal than MoMuLV-TB. On the other hand, ts1 and MoMuLV-TB infections of endothelial or TB cells were not cytopathic. The cytopathic effect in infected astrocytes correlated with the inefficiency of gPr80env transport and the intracellular accumulation of gPr80env as well as aberrant virus particles.

With various recombinant DNA and protein engineering techniques now available, enzyme-bad technologies are emerging as practical methods for large-scale synthesis of chiral intermediates and bioactive molecules, especially carbohydrates, oligosaccharides, their conjugates and related substances. This paper describes recent developments in the synthesis of novel monosaccharides and aza sugars based on aldolases, and the synthesis of oligosaccharides and analogs based on glycosyltransferases coupled with in sins regeneration of sugar nucleotides. As many enzymes are available for the stereocontrolled synthesis of chiral synthons (l), attention has been extended to the development of more effective and stable enzymes for the synthesis of molecules with increasing complexity (2). One class of such complex molecules are carbohydrates and their conjugates, especially those that exist on cell surfaces (3). These molecules are involved in many types of recognition phenomena (3-6); however, most of their precise functions have not been clearly identified at the molecular level. Part of the reason is that these molecules have been difficult to isolate, characterize and synthesize. Enzyme-based technology seems to be well suited for the synthesis of glycoconjugates and related substances for the study of their functions as these molecules are multifunctional and highly soluble in polar solvents, and many enzymes are available for the transformation of these molecules (7). The following describes some new technologies developed for the synthesis of sugar- and peptide- related substances based on recombinant or engineered enzymes.

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 (IC 50 ) 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.

Feline immunodeficiency virus (FIV) is an important viral pathogen worldwide in the domestic cat, which is the smallest animal model for the study of natural lentivirus infection. Thus, understanding the molecular mechanisms by which FIV carries out its life cycle and causes an acquired immune deficiency syndrome (AIDS) in the cat is of high priority. FIV has an overall genome size similar to HIV, the causative agent of AIDS in man, and shares with the human virus genomic features that may serve as common targets for development of broad-based intervention strategies. Specific targets include enzymes encoded by the two lentiviruses, such as protease (PR), reverse transcriptase (RT), RNAse H, and integrase (IN). In addition, both FIV and HIV encode Vif and Rev elements essential for virus replication and also share the use of the chemokine receptor CXCR4 for entry into the host cell. The following review is a brief overview of the current state of characterization of the feline/FIV model and development of its use for generation and testing of anti-viral agents.

We have obtained the 1.7 Å crystal structure of FIV protease (PR) in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR). The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants 1234. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively 234. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

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.

In vivo tests were performed to assess the influence of the protease inhibitor TL-3 on feline immunodeficiency virus (FIV)-induced central nervous system (CNS) deficits. Twenty cats were divided into four groups of five animals each. Group 1 received no treatment, group 2 received TL-3 only, group 3 received FIV strain PPR (FIV-PPR) only, and group 4 received FIV-PPR and TL-3. Animals were monitored for immunological and virological status, along with measurements of brain stem auditory evoked potential (BAEP) changes. Groups 1 and 2 remained FIV negative, and groups 3 and 4 became virus positive and seroconverted by 3 to 5 weeks postinoculation. No adverse effects were noted with TL-3 only. The average peak viral load for the virus-only group 3 animals was 1.32 × 10 6 RNA copies/ml, compared to 6.9 × 10 4 copies/ml for TL-3-treated group 4 cats. Group 3 (virus-only) cats exhibited marked progressive delays in BAEPs starting at 2 weeks post virus exposure, which is typical of infection with FIV-PPR. In contrast, TL-3-treated cats of group 4 exhibited BAEPs similar to those of control and drug-only cats. At 97 days postinfection, treatments were switched; i.e., group 4 animals were taken off TL-3 and group 3 animals were treated with TL-3. BAEPs in group 3 animals returned to control levels, while BAEPs in group 4 animals remained at control levels. After 70 days on TL-3, group 3 was removed from the drug treatment regimen. Delays in BAEPs immediately increased to levels observed prior to TL-3 treatment. The findings show that early TL-3 treatment can effectively eliminate FIV-induced changes in the CNS. Furthermore, TL-3 can counteract FIV effects on the CNS of infected cats, although continued treatment is required to maintain unimpaired CNS function.

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. Mutants included combinations of three critical regions (FIV numbering, with equivalent HIV numbering in superscript): I37 32 V in the active core region; N55 46 M, M56 47 I, and V59 50 I in the flap region; and L97 80 T, I98 81 P, Q99 82 V, P100 83 N, and L101 84 I in the 90s loop region. Significant alterations in specificity were observed, consistent with the involvement of these residues in determining the substrate-inhibitor specificity distinctions between FIV and HIV PRs. Two previously identified residues, I35 and I57 of FIV PR, were intolerant to substitution and yielded inactive PRs. Therefore, we attempted to recover the activity by introducing secondary mutations. The addition of G62 53 F and K63 54 I, located at the top of the flap and outside the active site, compensated for the activity lost in the I57 48 G substitution mutants. An additional two substitutions, D105 88 N and N88 74 T, facilitated recovery of activity in mutants that included the I35 30 D substitution. Determination of K i values of potent HIV-1 PR inhibitors against these mutants showed that inhibitor specificity paralleled that of HIV-1 PR. The findings indicate that maintenance of both substrate and inhibitor specificity is a function of interactions between residues both inside and outside the active site. Thus, mutations apparently peripheral to the active site can have a dramatic influence on inhibitor efficacy.

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

ts1, a mutant of MoMuLV, selectively kills T cells and neurons in the infected host resulting in neuroimmunodegeneration. In the infected thymus there is an early increase in mitosis of thymocytes followed by rapid death, suggesting that thymocyte death may be induced by viral mitogenic activation. Studies on thymocytes obtained fromts1-infected mice indicated that thets1-induced depletion of thymocytes is mediated through activation-induced death by apoptosis. To further investigate the interaction betweents1 and thymocytes, we have established long-term primary murine thymocyte cultures by placing the thymocytes together with thymic remnants in culture medium containing IL-2 and IL-7. These thymocytes retained their immature phenotype and were susceptible to infection byts1 and its parental wild-type MoMuLV.ts1-infected thymocytes proliferated initially at accelerated rate but subsequently produced more infectious virus and died much faster than control or MoMuLV-infected thymocytes. Thesein vitrostudies to some extent reflect ourin vivostudies reported previously.

The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection.