Identification of a Potential Inhibitor of the FIV p24 Capsid Protein and Characterization of Its Binding Site.

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.

Neurologic disease in feline immunodeficiency virus infection: disease mechanisms and therapeutic interventions for NeuroAIDS.

Feline immunodeficiency virus (FIV) is a lentivirus that causes immunosuppression through virus-mediated CD4+ T cell depletion in feline species. FIV infection is complicated by virus-induced disease in the nervous system. FIV enters the brain soon after primary infection and is detected as FIV-encoded RNA, DNA, and proteins in microglia, macrophages, and astrocytes. FIV infection activates neuroinflammatory pathways including cytokines, chemokines, proteases, and ROS with accompanying neuronal injury and loss. Neurobehavioral deficits during FIV infection are manifested as impaired motor and cognitive functions. Several treatment strategies have emerged from studies of FIV neuropathogenesis including the therapeutic benefits of antiretroviral therapies, other protease inhibitors, anti-inflammatory, and neurotrophic compounds. Recently, insulin's antiviral, anti-inflammatory, and neuroprotective effects were investigated in models of lentivirus brain infection. Insulin suppressed HIV-1 replication in human microglia as well as FIV replication of lymphocytes. Insulin treatment diminished cytokine and chemokine activation in HIV-infected microglia while also protecting neurons from HIV-1 Vpr protein-mediated neurotoxicity. Intranasal (IN) insulin delivery for 6 weeks suppressed FIV expression in the brains of treated cats. IN insulin also reduced neuroinflammation and protected neurons in the hippocampus, striatum, and neocortex of FIV-infected animals. These morphological and molecular effects of IN insulin were confirmed by neurobehavioral studies that showed IN insulin-treated FIV-infected animals displayed improved motor and cognitive performance compared to sham-treated FIV-infected animals. Thus, FIV infection of the nervous system provides a valuable comparative in vivo model for discovering and evaluating disease mechanisms as well as developing therapeutic strategies for NeuroAIDS in humans.

Novel fused tetrathiocines as antivirals that target the nucleocapsid zinc finger containing protein of the feline immunodeficiency virus (FIV) as a model of HIV infection.

A novel series of fused tetrathiocines were prepared for evaluation of activity against the nucleocapsid protein of the feline immunodeficiency virus (FIV) in an in vitro cell culture approach. The results demonstrated that the compounds display potent nanomolar activity and low toxicity against this key model of HIV infection.

Selection of drug-resistant feline immunodeficiency virus (FIV) encoding FIV/HIV chimeric protease in the presence of HIV-specific protease inhibitors.

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 (IC50s) 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 IC50 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.

Evaluation of the antiviral efficacy of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives against the nucelocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV infection.

A diverse library of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives were prepared for evaluation of activity against the nucleocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV, using an in vitro cell culture approach, yielding nanomolar active compounds with low toxicity.

Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

9-[2-(R)-(Phosphonomethoxy)propyl]-2,6-diaminopurine (R)-PMPDAP and its prodrugs: optimized preparation, including identification of by-products formed, and antiviral evaluation in vitro.

New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH(2)P(O)(OiPr)(2) or BrCH(2)P(O)(OiPr)(2) followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using ß-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

Generation of infectious feline immunodeficiency virus (FIV) encoding FIV/human immunodeficiency virus chimeric protease.

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.

Design and synthesis of membrane fusion inhibitors against the feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a pathogenic virus that causes an AIDS-like syndrome in the domestic cats. For viral entry and infection, fusion between the virus and the cell membrane is the critical process and this process is mediated by an envelope glycoprotein gp40. We have identified fusion inhibitory peptides from the heptad repeat-2 (HR2) of gp40. Remodeling of the original sequences using alpha-helix-inducible motifs revealed the interactive residues of gp40. Comparative analysis of HR2 peptides derived from four FIV strains demonstrated that the interactive surface of the Shizuoka strain-derived HR2 peptides provides the highest affinity of all the FIV strains examined.

Antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein.

For the entry of lentivirus into target cells, fusion between its viral membrane and cellular membrane is essential. The present study was conducted to examine the inhibitory effect of modified peptides corresponding to heptad repeats (HR) 1 and 2 of feline immunodeficiency virus (FIV) envelope gp40 on the fusion between the viral and cellular membranes. FIV-N36 and FIV-C35 were synthesized as authentic peptides of the N-terminal HR1 domain and C-terminal HR2 domain of FIV gp40, respectively. FIV-C35EK1, FIV-C35EK2, and FIV-C35EK3 were peptides synthesized by modifying FIV-C35 as the X-EE-XX-KK concept to increase their solubility in water and the stability of their alpha-helicity. FIV-C35 and FIV-C35EK1 inhibited the cell membrane fusion mediated by FIV-infected cells and the replication of FIV. FIV-N36, FIV-C35EK2, and FIV-C35EK3 did not show any apparent inhibitory effect. These results indicated that the newly developed membrane fusion inhibitors could facilitate the development of novel anti-lentiviral chemotherapies.

Follow-Up of Viral Parameters in FeLV- or FIV-Naturally Infected Cats Treated Orally with Low Doses of Human Interferon Alpha.

Specific treatments for the long-life infections by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are either toxic, expensive or not too effective. Interferon α (IFN-α) is an immunomodulatory molecule which has been shown in vitro to decrease the release of infective particles. The aim of this study was to follow the progress of the clinical score and viral parameters of FeLV- and FIV-naturally infected privately owned cats treated with recombinant human IFN-α (rHuIFN-α, Roferon-A). Twenty-seven FeLV-infected cats (FeLV+) and 31 FIV-infected cats (FIV+) were enrolled in the study. Owners were instructed to orally administer 1 mL/day of 60 IU rHuIFN-α/mL in alternating weeks for four months. Blood samples were taken at the beginning of the study (M0), mid-treatment (M2), end of treatment (M4), and 6-10 months later (M10). Clinical status at these time points improved notably with rHuIFN-α treatment, regardless of the initial severity of the disease, an effect which lasted throughout the study in most animals (15 of the 16 FeLV+ symptomatic cats; 20 of the 22 FIV+ symptomatic cats) improved markedly their clinical situation. In FeLV+ cats plasma antigenemia (p27CA), reverse transcriptase (RT) activity, and proviral load decreased at M2 and M4 but increased again at M10 ("rebound effect"). The level of antigenemia or RT activity was below the detection limits in FIV+ cats, and the effect on proviral load was less marked than in FeLV+ cats. Taken together, these results indicate that rHuIFN-α is a good candidate for treating FeLV+ cats, but the "rebound effect" seen when treatment was discontinued suggests that additional studies should be conducted to clarify its effect on progression of the infection in cats.

Immunopathologic Effects of Prednisolone and Cyclosporine A on Feline Immunodeficiency Virus Replication and Persistence.

Feline immunodeficiency virus (FIV) induces opportunistic disease in chronically infected cats, and both prednisolone and cyclosporine A (CsA) are clinically used to treat complications such as lymphoma and stomatitis. However, the impact of these compounds on FIV infection are still unknown and understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective therapies. To determine the immunologic and virologic effects of prednisolone and CsA during FIV infection, FIV-positive cats were administered immunosuppressive doses of prednisolone (2 mg/kg) or CsA (5 mg/kg). Both prednisolone and CsA induced acute and transient increases in FIV DNA and RNA loads as detected by quantitative PCR. Changes in the proportion of lymphocyte immunophenotypes were also observed between FIV-infected and naïve cats treated with CsA and prednisolone, and both treatments caused acute increases in CD4+ lymphocytes that correlated with increased FIV RNA. CsA and prednisolone also produced alterations in cytokine expression that favored a shift toward a Th2 response. Pre-treatment with CsA slightly enhanced the efficacy of antiretroviral therapy but did not enhance clearance of FIV. Results highlight the potential for drug-induced perturbation of FIV infection and underscore the need for more information regarding immunopathologic consequences of therapeutic agents on concurrent viral infections.

Investigation of the Pentathiepin Functionality as an Inhibitor of Feline Immunodeficiency Virus (FIV) via a Potential Zinc Ejection Mechanism, as a Model for HIV Infection.

A small diverse library of pentathiepin derivatives were prepared to evaluate their efficacy against the nucleocapsid protein function of the feline immunodeficiency virus (FIV) as a model for HIV, using an in vitro cell culture approach. This study led to the development of nanomolar active compounds with low toxicity.

Co-infection with feline retrovirus is related to changes in immunological parameters of cats with sporotrichosis.

Feline sporotrichosis due to Sporothrix brasiliensis is frequently severe and often correlated to zoonotic transmission. Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) cause immunodeficiency in cats; no association has been identified with critical cases of sporotrichosis. Moreover, the cytokine profile in Sporothrix-infected cats and a potential impact of retrovirus co-infections on their immunity is unknown. This study assessed immunological parameters in cats with sporotrichosis with and without FIV or FeLV co-infection. FeLV infection was detected by antigen ELISA and by provirus PCR. FIV infection was investigated through ELISA and Western blot. Cytokine transcription (IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α) was quantified using RT-qPCR and lymphocyte subpopulations (CD4, CD8, CD5 and CD21) were assessed by flow cytometry. Thirty cats with sporotrichosis were recruited to the study, including three FIV-positive and five FeLV-positive (progressive infection) cats. One cat with regressive FeLV infection was excluded from statistics. In comparison to retrovirus-negative cats, FIV-positive cats and FeLV-positive cats had higher IL-10 levels, FeLV-positive cats had lower IL-4 levels and FIV-positive cats had lower IL-12 levels and a lower CD4+/CD8+ ratio. Remarkably, all cats with poor general condition were FeLV (progressive infection) or FIV-positive, but the retrovirus status was not associated with the sporotrichosis treatment length or outcome. The immunological changes and the more severe clinical presentation observed in cats with retrovirus co-infections encourage future prospective studies that address the impact of these changes on prognostic determinants of feline sporotrichosis and the development of new therapy strategies that control disease spread.

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems.

P59 is the Trp-rich 20-mer peptide ((767)L-G(786)), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BACKGROUND: Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. METHODS: Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. RESULTS: The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. CONCLUSION: We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

Comparative evaluation of the activity of antivirals towards feline immunodeficiency virus in different cell culture systems.

Influences of the cell system on observed EC(50) values of different agents against feline immunodeficiency virus (FIV) were assessed. The activity of various nucleoside reverse transcriptase inhibitors (NRTI) against a lymphotropic FIV strain was evaluated using monocultured thymocytes and a DC-thymocyte coculture. In the second set of experiments activity of carbohydrate binding agents (CBA) towards FIV strains derived from different cell lines (e.g. Crandall feline kidney cells (CRFK) and thymocytes) was compared. We examined three different FIV-based antiviral evaluation systems and obtained marked differences in EC(50) values, especially for CBA entry inhibitors. Our study confirms and extends earlier observed differences between cell systems used for the evaluation of the activity of antivirals towards FIV.

Application of RNA interference for inhibiting the replication of feline immunodeficiency virus in chronically infected cell lines.

RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. The present study was carried out to apply the RNAi technology to inhibit the replication of feline immunodeficiency virus (FIV). Four small interfering RNAs (siRNAs) homologous to the FIV gag gene were synthesized and transfected into a feline fibroblastic cell line chronically infected with FIV (CRFK/FIV). These synthetic siRNAs efficiently inhibited the replication of FIV. Next, we examined the effect of retroviral vector-mediated transfer of FIV-specific short hairpin RNA (shRNA) on the replication of FIV in a feline T-cell line chronically infected with FIV (FL4). The retroviral vector-mediated transfer of FIV-specific shRNA was shown to markedly inhibit the replication of FIV in the FL4 cells. These results provide useful information for the development of RNAi-based gene therapy strategy to control FIV infection.