Mathieu Long

François-Xavier Cantrelle

Xavier Robert

Emmanuelle Boll

Natalia Sierra

Patrice Gouet

Xavier Hanoulle

Guzmán I Alvarez
email: guzmanalvarezlqo@gmail.com

Christophe Guillon
email: christophe.guillon@ibcp.fr

Identification of a potential inhibitor of the FIV p24 capsid protein and characterization of its binding site

Keywords: Feline immunodeficiency Virus, NMR, assembly inhibitor, molecular modeling, capsid

Feline immunodeficiency virus (FIV) is a veterinary infective agent against 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 binding mechanism 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.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.1c00228

Introduction

Feline immunodeficiency virus (FIV) is a lentivirus that can infect domestic cats and wild feline species [START_REF] Pedersen | Isolation of a T-Lymphotropic Virus from Domestic Cats with an Immunodeficiency-like Syndrome[END_REF][START_REF] Brown | Feline Immunodeficiency Virus (FIV) in Wild Pallas' Cats[END_REF] . Infected animals can develop an immunodeficiency syndrome similar to human acquired immunodeficiency syndrome (AIDS), which is caused by infection with human immunodeficiency virus (HIV) [START_REF] Pedersen | Isolation of a T-Lymphotropic Virus from Domestic Cats with an Immunodeficiency-like Syndrome[END_REF][START_REF] O'brien | Emerging Viruses in the Felidae: Shifting Paradigms[END_REF][START_REF] Roelke | Feline Immunodeficiency Virus: An Interesting Model for AIDS Studies and an Important Cat Pathogen[END_REF][5] . Since its discovery in 1987, FIV has been detected worldwide in domestic and wild feline species and up to 10% of all domestic cats are thought to be infected [START_REF] Brown | Feline Immunodeficiency Virus (FIV) in Wild Pallas' Cats[END_REF][START_REF] Roelke | Feline Immunodeficiency Virus: An Interesting Model for AIDS Studies and an Important Cat Pathogen[END_REF]6 . A commercial vaccine, only used in Northern America, Australia, New Zealand, and Japan 7 , showed low efficacy against virus infectivity, and to date, no antiviral strategy has led to the efficient control of FIV viremia in infected animals 8,9 . Hence, there is a need for the development of efficient anti-FIV drugs, which must remain economically affordable not only for domestic veterinary practice but also for FIV epidemics in endangered wild feline species 10 .

In addition to its importance as a causative agent of feline AIDS, FIV has been thoroughly studied as a model of HIV-1 infection to test prophylactic and therapeutic antiretroviral drugs and vaccines [11][START_REF] Elder | Feline Immunodeficiency Virus (FIV) as a Model for Study of Lentivirus Infections: Parallels with HIV[END_REF][START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF][START_REF] Yamamoto | Feline Immunodeficiency Virus Model for Designing HIV/AIDS Vaccines[END_REF] . Strategies for repositioning efficient HIV-1 treatments to tackle FIV infection have also been studied to identify treatments suitable for these veterinary needs. For instance, nucleoside analogues, such as 3'-azido-3'-deoxythymidine (AZT), have been tested as treatments of FIV infection [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF][START_REF] Smyth | Susceptibility in Cell Culture of Feline Immunodeficiency Virus to Eighteen Antiviral Agents[END_REF][START_REF] Hartmann | Efficacy of Antiviral Drugs against Feline Immunodeficiency Virus[END_REF] . However, none have proven to have sufficient treatment efficacy against FIV 8,17 .

Therefore, FIV must be studied specifically to develop targeted treatments for use in veterinary medicine.

Lentiviruses are enveloped viruses that are characterized by a protein-based matrix below the membrane that protects the viral capsid containing the genome. The lentivirus genome is composed of a dimer of gRNA complexed with the nucleocapsid [START_REF] Abdusetir Cerfoglio | Structural Elements in the Gag Polyprotein of Feline Immunodeficiency Virus Involved in Gag Self-Association and Assembly[END_REF][START_REF] Affranchino | In Vitro Assembly of the Feline Immunodeficiency Virus Gag Polyprotein[END_REF][START_REF] González | Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding[END_REF][START_REF] Nath | In Vitro Assembly of Feline Immunodeficiency Virus Capsid Protein: Biological Role of Conserved Cysteines[END_REF] . Lentiviral Gag and Gag-Pol polyproteins assemble at the membrane to form immature, noninfectious viral particles (HIV-1 Gag and FIV Gag: UniProtKB P04591 and P16087, respectively). This assembly occurs via the homotypic multimerization of the Gag polyprotein through its p24 region [START_REF] Affranchino | In Vitro Assembly of the Feline Immunodeficiency Virus Gag Polyprotein[END_REF][START_REF] Ovejero | The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly[END_REF] to stabilize the Gag-RNA complexes [START_REF] Kutluay | Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging[END_REF] during gRNA encapsidation. After virus budding, the Gag polyproteins are cleaved by the viral protease, and several mature subunits are released, including the p24 subunit [START_REF] González | Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding[END_REF] , which then reorganize to generate the mature, infectious viral particles. The lentiviral capsid is composed of 1,500 to 5,000 copies of the p24 protein [START_REF] Kutluay | Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging[END_REF][START_REF] Ganser | Assembly and Analysis of Conical Models for the HIV-1 Core[END_REF][START_REF] Pornillos | Atomic-Level Modelling of the HIV Capsid[END_REF][START_REF] Pornillos | X-Ray Structures of the Hexameric Building Block of the HIV Capsid[END_REF][START_REF] Tomasini | Modeling the Dynamics and Kinetics of HIV-1 Gag during Viral Assembly[END_REF] . HIV-1 p24 protein has been shown to form hexamers and pentamers (capsomers) that subsequently assemble as a pseudo-icosahedral, fullerene-shaped capsid core [START_REF] Sundquist | HIV-1 Assembly, Budding, and Maturation[END_REF] . The same fullerene-shaped core is observed in FIV, suggesting that FIV p24 assembly is similar to that of HIV-1 p24 29 . Thus, p24 assembly appears to be a key step in the maturation of infectious FIV particles and thus in the FIV life cycle.

In addition to its role in the production of infectious virions, p24 assembly is critical for the postentry processes of the lentivirus life cycle. Indeed, the viral capsid of HIV-1 has been shown to be required for the early stages of infection of a new cell, including the entry of the viral genome into the nucleus and reverse transcription [START_REF] Dharan | Nuclear Pore Blockade Reveals That HIV-1 Completes Reverse Transcription and Uncoating in the Nucleus[END_REF][START_REF] Dismuke | Evidence for a Functional Link between Uncoating of the Human Immunodeficiency Virus Type 1 Core and Nuclear Import of the Viral Preintegration Complex[END_REF][START_REF] Jacques | HIV-1 Uses Dynamic Capsid Pores to Import Nucleotides and Fuel Encapsidated DNA Synthesis[END_REF][START_REF] Zhou | Transportin 3 Promotes a Nuclear Maturation Step Required for Efficient HIV-1 Integration[END_REF] . The reverse transcription stage occurs inside the viral capsid, transforming the gRNA into a double-stranded viral DNA (dsDNA) [START_REF] Dharan | Nuclear Pore Blockade Reveals That HIV-1 Completes Reverse Transcription and Uncoating in the Nucleus[END_REF][START_REF] Selyutina | Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating[END_REF] . The capsid is transported to the nucleus via the cytoskeleton [START_REF] Dharan | Role of Microtubules and Microtubule-Associated Proteins in HIV-1 Infection[END_REF][START_REF] Fernandez | Microtubule-Associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus[END_REF][START_REF] Pawlica | Cytoplasmic Dynein Promotes HIV-1 Uncoating[END_REF] . During its intracytoplasmic transport, the capsid must avoid the host cell's restriction factors. In lentiviruses, this escape is mediated by the interaction of lentiviral p24 proteins with cellular cyclophilin A (CypA, UniProtKB: P62937) [START_REF] Goldstone | Structural and Functional Analysis of Prehistoric Lentiviruses Uncovers an Ancient Molecular Interface[END_REF] .

Capsid disassembly is then required for the efficient release of the viral dsDNA and its integration into the host cell's genome. While it is not completely clear whether this uncoating occurs in the nucleus or in the cytoplasm, recent experiments have revealed the presence of the capsid inside the nucleus [START_REF] Dharan | Nuclear Pore Blockade Reveals That HIV-1 Completes Reverse Transcription and Uncoating in the Nucleus[END_REF][START_REF] Selyutina | Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating[END_REF][START_REF] Arhel | Revisiting HIV-1 Uncoating[END_REF][START_REF] Burdick | HIV-1 Uncoats in the Nucleus near Sites of Integration[END_REF] . The HIV-1 p24 subunit is also involved in interactions with other cellular proteins, such as nucleoporins Nup153 and Nup358 and the cleavage and polyadenylation specificity factor subunit 6 (CPSF-6) [START_REF] Bhattacharya | Structural Basis of HIV-1 Capsid Recognition by PF74 and CPSF6[END_REF][START_REF] Xie | MxB Impedes the NUP358-Mediated HIV-1 Pre-Integration Complex Nuclear Import and Viral Replication Cooperatively with CPSF6[END_REF][START_REF] Matreyek | Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity[END_REF][START_REF] Price | CPSF6 Defines a Conserved Capsid Interface That Modulates HIV-1 Replication[END_REF][START_REF] Zhou | Transportin 3 Promotes a Nuclear Maturation Step Required for Efficient HIV-1 Integration[END_REF] (hNup153, hNup358, and hCPSF6: UniProtKB P49790, P49792, and Q16630, respectively). These cellular proteins are involved in both the transport of the capsid from the basal membrane to the nucleus and nuclear import. Therapeutic drugs targeting the capsid have been developed for their ability to impair the assembly process, prevent interactions with cellular partners required for HIV-1 infectivity, or prevent capsid uncoating [START_REF] Tang | Antiviral Inhibition of the HIV-1 Capsid Protein[END_REF][START_REF] Kortagere | Inhibiting Early-Stage Events in HIV-1 Replication by Small-Molecule Targeting of the HIV-1 Capsid[END_REF][START_REF] Zhang | A Cell-Penetrating Helical Peptide as a Potential HIV-1 Inhibitor[END_REF][START_REF] Sticht | A Peptide Inhibitor of HIV-1 Assembly in Vitro[END_REF][START_REF] Link | Clinical Targeting of HIV Capsid Protein with a Long-Acting Small Molecule[END_REF] .

In HIV-1, most mutations that affect the structure or stability of HIV-1 p24 assembly decrease infectivity [START_REF] Rihn | Extreme Genetic Fragility of the HIV-1 Capsid[END_REF] . Hence, drugs targeting the capsid seem promising as efficient treatments due to the limited adaptive capacities of p24. Therefore, compounds inhibiting the assembly, uncoating, and/or interactions between the capsid and the above-mentioned cellular partners are currently under study for their impact on HIV-1 infectivity, for example, PF74 and its derivatives, which target the nucleoporin-or CPSF-6-binding sites [START_REF] Zhou | Transportin 3 Promotes a Nuclear Maturation Step Required for Efficient HIV-1 Integration[END_REF][START_REF] Bhattacharya | Structural Basis of HIV-1 Capsid Recognition by PF74 and CPSF6[END_REF][START_REF] Link | Clinical Targeting of HIV Capsid Protein with a Long-Acting Small Molecule[END_REF][START_REF] Balasubramaniam | PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex[END_REF][START_REF] Mcarthur | HIV Capsid Inhibitors Beyond PF74[END_REF] .

Currently, there are no effective drugs to treat FIV 5 . Moreover, reference compounds targeting HIV-1 assembly, such as PF74, do not inhibit FIV replication in vitro [START_REF] Fricke | BI-2 Destabilizes HIV-1 Cores during Infection and Prevents Binding of CPSF6 to the HIV-1 Capsid[END_REF] . However, the identification of compounds inhibiting the function of FIV p24 is still anticipated. To identify molecules that interfere with FIV p24 capsid assembly, we performed in vitro assembly assays and screened approximately 400 compounds from our in-house chemical library, as previously described [START_REF] Sierra | Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus[END_REF] . This screening led to the identification of one promising lead compound, 696.

Microscale thermophoresis (MST) results revealed that 696 specifically binds p24, whose binding site was identified at the molecular level using liquid-state nuclear magnetic resonance (NMR) spectroscopy. To this purpose, we performed the first NMR resonance assignments of FIV p24 and then conducted chemical shift perturbation (CSP) experiments to characterize the binding site for 696 on p24. These experimental data were used to model the interaction between 696 and FIV p24 through molecular docking experiments. This model suggested a stabilizing role for 696 on FIV p24 assembly, which we were able to demonstrate in vitro. Taken together, this characterization of compound 696 paves the way for the identification of additional FIV capsid inhibitors that could ultimately lead to the development of an anti-FIV drug.

Materials and methods

Unlabeled protein production. Bacterial lysis, protein purification, and tag removal were performed using the full-length FIV p24 vector, as described earlier [START_REF] Serrière | Biophysical Characterization of the Feline Immunodeficiency Virus P24 Capsid Protein Conformation and In Vitro Capsid Assembly[END_REF] , except that the storage buffer was phosphate buffer (pH 6.8) containing 50 mM NaCl.

In vitro assembly/disassembly assay. The assembly of p24 was performed and monitored, as previously described [START_REF] Sierra | Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus[END_REF] . Assembly was triggered using a 1:1 (v/v) mix of 7 mg/mL purified protein in 50 mM NaPi buffer pH 7.4 containing 1M NaCl in a final volume of 50 µL. The optical density at 345 nm was measured every 40 sec using the Infinite M1000 multiplate reader (Tecan) at 38°C for 35 min. Compounds from our in-house library, which have been described elsewhere [START_REF] Sierra | Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus[END_REF] , were tested at a concentration of 200 µM [corresponding to a final concentration of 1% (v/v) dimethyl sulfoxide (DMSO)] by adding 0.5 µL of a 20 mM solution of compounds to the wells before starting the experiment. The control corresponded to 1% DMSO.

Compound 696 was synthesized, as described in the supporting information (supplementary scheme S1). End-point samples from the in vitro assembly assay in the presence of either compound 696 or 1% DMSO (control) were used for the disassembly assay. Briefly, these samples were centrifuged at 20,000 × g for 20 min at room temperature and resuspended in 400 µL of lowsalinity buffer (50 mM NaCl). Aliquots were sampled immediately after resuspension (t0) and at 8, 24, or 32 h thereafter. Aliquots were centrifuged immediately after sampling at 20,000 × g for 20 min at room temperature, and the pellets were collected and analyzed using 12% SDS-PAGE. Gels were stained using InstantBlue™ reagent (Expedeon). Gel images were acquired using a Fusion Fx camera (Vilber).

MST.

MST experiments were performed, as previously described [START_REF] Sierra | Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus[END_REF] . Briefly, we measured with a Monolith NT.115 instrument (NanoTemper Technologies) the diffusion of the fluorescentlabeled protein in capillaries when increasing the temperature of 1°C using an infrared light,. The diffusion properties of the fluorescent protein will be modified when binding 696, allowing the estimation of the affinity by titration with serial dilutions of the compound. To this purpose, Histagged proteins were labeled following the supplier's protocol (NanoTemper Technologies) using the Monolith His-Tag RED-tris-NTA Labeling kit (NanoTemper) [START_REF] Seidel | Microscale Thermophoresis Quantifies Biomolecular Interactions under Previously Challenging Conditions[END_REF] . The experiments were carried out at 40% MST power and 60% LED power at 37°C. The compound was tested using serial 2× dilutions, resulting in a concentration range between 1 mM and 31 nM. Kd estimation (data points averaging and curve fitting using the Kd model) was automatically calculated from the measured MST traces using the manufacturer's software (MO.Affinity Analysis 3).

Singly labeled 15 N protein. Escherichia coli bacteria (T7 Express Competent E. coli strain, New

England Biolabs) were transformed with pRSET-p24EΔCP-T, as previously described [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] . One colony was grown overnight in lysogenic broth (LB) media at 37°C. The inoculation was performed using 20 mL of this preculture per L of culture. The LB preculture volume used for inoculation was centrifuged for 5 min at 6,000 × g at room temperature to pellet the bacteria, the LB supernatant was removed, and bacteria were resuspended in M9 minimum media containing 4 g/L glucose, 1× MEM vitamins, 1 mM MgSO4, 100 µM CaCl2, 6 g/L Na2HPO4, 3 g/L KH2PO4, 5 g/L NaCl, 0.5 g/L ISOGRO-15 N Powder-Growth medium (all supplied by Sigma-Aldrich), and 1 g/L 15 NH4Cl (Euriso Top). Media were supplemented with ampicillin (Sigma-Aldrich) at a final concentration of 50 µg/L. After inoculation, bacteria were grown at 37°C until the OD600 reached 0.4; protein expression was induced using 1 mM isopropyl--D-thiogalactopyranoside (Sigma-Aldrich). Bacterial cells were then grown overnight at 25°C and centrifuged for 5 min at 6,000 × g at room temperature. Pellets were stored at -20°C until use.

Triple-labeled 15 N, [START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF] C, D protein. Bacteria were grown as described above for singly labeled protein but in M9 media prepared using D2O (Sigma-Aldrich), deuterated ISOGRO-13 C, [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N, D (Sigma-Aldrich), and 2 g/L D-glucose-13 C6,1,2,3,4,5,6,6-d7 (Euriso Top). Preculture and culture were performed, as described above, with inoculation in M9 media until the preculture reached a final OD600 of 0.1. Bacterial lysis. Bacteria were lysed by sonication (40% power, 2-min cycles) on ice at least three times until the medium was not viscous. Cell debris was then pelleted using centrifugation for 20 min at 11,000 × g at 4°C, and supernatants were purified using affinity chromatography.

Protein purification. We performed affinity chromatography for 6×His-tagged proteins using the Ni-TED resin (Macherey-Nagel) and removed the His tag using Factor Xa, as previously described [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] . The protein was then concentrated using a Vivaspin 20 centrifugal concentrator (10,000 MWCO; polyethersulfone, Sartorius Stedim Lab Ltd.) and stored in 50 mM phosphate buffer (pH 6.8) containing 50 mM NaCl and 2 mM DTT. Protein concentration was assessed using a Nanodrop instrument (Thermo Fisher Scientific). The protein solution was supplemented with D2O at a final concentration of 5%. We used samples at a final concentration of 2.8 mg/mL (120 µM) for the triple-labeled protein for NMR assignment and 2.2 mg/mL (92 µM) for CSP analysis.

NMR acquisition and data processing.

A 300-µL aliquot of labeled p24 sample (120 µM) was placed in 5-mm Shigemi tubes. All NMR spectra were recorded at 293 K using an AvanceNeo Bruker 900 MHz spectrometer equipped with a CPTCI cryoprobe. The sequence-specific backbone assignment was based on two-dimensional (2D) TROSY 1 H-15 N, and three-dimensional (3D) BEST-TROSY HNCO, BEST-TROSY-HN(CA)CO, BEST-TROSY-HNCACB, BEST-TROSY-HN(CO)CACB, BEST-TROSY-HNCA, BEST-TROSY-HNCOCA, and TROSY-HNCANNH pulse sequences. The proton chemical shifts were referenced using the sodium 3trimethylsillyl-[2,2,3,3-d4]propionate signal at 0 ppm. Data were transformed and processed using NMRPipe [START_REF] Delaglio | NMRPipe: A Multidimensional Spectral Processing System Based on UNIX Pipes[END_REF] and qMDD for acquisition with nonuniform sampling [START_REF] Mayzel | Time-Resolved Multidimensional NMR with Non-Uniform Sampling[END_REF] and analyzed using CCPN Analysis Suite software [START_REF] Vranken | The CCPN Data Model for NMR Spectroscopy: Development of a Software Pipeline[END_REF] .

For the determination of CSPs, we used [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N-labeled protein samples (2.2 mg/mL, 90 µM). We acquired HSQC spectra with samples containing compound 696 at a final concentration of 200 µM. Briefly, we added the required volume of compound stock solution diluted in deuterated-DMSO (DMSO-d6), or DMSO-d6 alone as a control, representing 3.3 µL in a protein sample of 330 µL (1% DMSO final).

Combined 1 H, [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N CSPs were calculated for each peak using the following formula: 𝐶𝑆𝑃 = √ (𝛿 1 𝐻) 2 + 𝜔 𝑁 𝜔 𝐻 (𝛿 15 𝑁) [START_REF] Brown | Feline Immunodeficiency Virus (FIV) in Wild Pallas' Cats[END_REF] with 𝛿 1 𝐻 and 𝛿 [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] 𝑁 as the chemical shift displacements with and without the tested compounds, respectively, and 𝜔 𝐻 and 𝜔 𝑁 as the spectral widths of 1 H and [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N, respectively, yielding 𝐶𝑆𝑃 = √(𝛿 1 𝐻) 2 + 0.237 (𝛿 15 𝑁) [START_REF] Brown | Feline Immunodeficiency Virus (FIV) in Wild Pallas' Cats[END_REF] .

Molecular docking and visualization. We used Chimera software (UCSF) to render the molecular structures [START_REF] Pettersen | UCSF Chimera--a Visualization System for Exploratory Research and Analysis[END_REF] , while molecular surfaces were plotted using the MSMS tool of the software [START_REF] Sanner | Reduced Surface: An Efficient Way to Compute Molecular Surfaces[END_REF] .

The monomeric crystal structure of full-length p24 (PDB entry 5NA2 [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] ) was used as the target in the docking experiment. Compound 696 was modeled in 3D using its smiles code produced by the ChemDraw software (PerkinElmer Informatics). Both p24 and 696 were then prepared using AutoDockTools (v1.5.6) [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility[END_REF] : The polar hydrogen atoms were added, the nonpolar hydrogens were merged, and the Gasteiger partial atomic charges were computed. Docking was carried out using the AutoDock Vina (v1.1.2) program [START_REF] Trott | AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading[END_REF] . Compound 696 was treated as fully flexible, while the target protein p24 was treated as rigid, with the exception of some residue side chains that were selected in an extended perimeter around the surface binding We finally used the LigPlot+ program (v2.2) [START_REF] Laskowski | LigPlot+: Multiple Ligand-Protein Interaction Diagrams for Drug Discovery[END_REF] to determine the residues involved in the predicted protein-compound complex and the nature of the interactions (i.e., hydrogen bonds and unbound contacts).

Results

Screening and identification of a specific compound interfering with FIV capsid assembly in vitro. The screening of 400 compounds from our in-house chemical library using a spectrophotometric in vitro assembly assay [START_REF] Sierra | Looking for Novel Capsid Protein Multimerization Inhibitors of Feline Immunodeficiency Virus[END_REF][START_REF] Bocanegra | Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity[END_REF] identified a compound, 696, belonging to the chemical series of benzimidazole compounds, which delayed capsid assembly (Figure 1). NMR assignment of FIV p24. Since the direct binding of 696 was confirmed by MST, we focused on the identification of its binding site on p24. To this end, we used liquid-state NMR spectroscopy. We used the truncated protein p24EΔCP-T for which the structure was previously determined using X-ray crystallography [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] . The p24 concentration was maintained at 2.8 mg/mL (120 µM) to avoid dimerization or oligomerization of the protein 29 during the NMR acquisition experiments. Using a 15 N, [START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF] C,D-triple-labeled FIV p24 sample, we obtained a goodquality 2D 1 H, [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N-HSQC spectrum using a 900-MHz NMR spectrometer (Figure 3). As no NMR data were available in the literature for FIV p24, we had to perform the NMR assignment. Thus, we collected the complete NMR dataset on 15 N, [START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF] C,D-triple-labeled FIV p24 samples from 3D 1 H, [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N, [START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF] C-HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, and HN(CA)NNH spectra during the sequential assignment process, which was performed using the CcpNmr software [START_REF] Vranken | The CCPN Data Model for NMR Spectroscopy: Development of a Software Pipeline[END_REF] (supplementary figure S1).

The NMR assignment of FIV p24 (Figure 3) was deposited in the Biological Magnetic Resonance Data Bank (entry 50794). This assignment covers 167 of the 201 non-proline residues (83% coverage) from the p24 sequence (Figure 4A andB). The missing assignments correspond to regions of the protein for which the signal:noise ratio was of low quality, including residues located in a proline-rich loop (residues Pro 84 to Phe 94) as well as part of helices α4 and α7 (Figure 4C). Using the experimental [START_REF] Meeker | Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration[END_REF] C NMR chemical shifts, we characterized the secondary structure of p24 in solution, which is in complete agreement with the crystal structure [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] , thereby confirming the quality of our NMR data (Figure 4D).

CSP analysis upon the binding of compound 696.

To identify the binding site for the compound in p24, we performed an NMR CSP analysis. For this purpose, we compared the 1 H, [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N-HSQC spectra of [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N-p24 samples in the absence (control with 1% DMSO-d6) and presence of compound 696 (200 µM, representing the final concentration of 1% DMSO-d6) and calculated the combined 1 H and [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N CSPs induced upon binding of the compound (Figure 5). CSPs were observed for a limited set of resonances in which most of the peaks were unaffected; these findings were compatible with a well-defined binding site in p24 (Figure 5). [START_REF] Schwartz | Antiviral Efficacy of Nine Nucleoside Reverse Transcriptase Inhibitors against Feline Immunodeficiency Virus in Feline Peripheral Blood Mononuclear Cells[END_REF] N-HSQC spectrum, corresponding to 21 p24 residues, displayed a CSP higher than a 2× standard deviation (SD), including 11 peaks with CSPs greater than 3× SD. Nine amino acids residues with the highest CSPs colocalized in a define area in the N-terminal domain (Figure 6A andB), which were mainly located in helices α3, α4, and α7 (Figure 6B). These amino acids define the binding site on the surface of the N-terminal domain of p24 (Figure 6C). Molecular modeling of the p24/696 interaction and experimental validation. Using these data, we performed a computer-aided molecular docking experiment to further characterize the binding mode of compound 696 to FIV p24. We used the monomeric structure of p24 (PDB entry 5NA2 [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] ) as a target. The docking search space was selected to encompass the surface area where we observed the NMR CSPs (Figure 6B andC). The peptide backbone was treated as rigid, while the residues side chains inside the above-mentioned area were treated as flexible. As a result, the best docking pose, with a ΔG binding energy estimated at -8.0 kcal/mol, was positioned in the putative binding site between helices α3 and α4 (Figure 7A). We used the LigPlot+ program [START_REF] Laskowski | LigPlot+: Multiple Ligand-Protein Interaction Diagrams for Drug Discovery[END_REF] to determine the predicted interactions between the p24 protein and compound 696 from this best docking solution. We identified nine amino acids involved in hydrogen bonding or hydrophobic interactions with the compound (Figure 7B). We also tested whether compound 696 could interfere with FIV p24 disassembly because this binding region corresponds, in HIV-1 p24, to a zone targeted by antiviral compounds that have been described to stabilize HIV-1 hexamers (e.g., PF74 51 ). Assembly products, obtained in the presence or absence of 696 at the end of our assembly experiments (Figure 1), were collected, centrifuged, and resuspended in a low-salinity buffer to promote disassembly. At different time points following the initiation of disassembly (t), samples were centrifuged, and the pellets were analyzed using SDS-PAGE (Figure 8). At t + 32 h, assembled p24 was still present in the presence of 696 but not in the untreated control, demonstrating the stabilizing role of 696 during p24 disassembly and re-enforcing our structural identification of the binding site of 696 by NMR and docking experiments.

Discussion

In this study, we identified one promising lead compound, named 696, which was shown to interfere with FIV capsid assembly/disassembly in vitro. The binding of this compound to FIV p24 was confirmed by both MST and NMR spectroscopy. The specific 696-binding site on p24, involving the α3, α4, and α7 helices, was identified by NMR experiments and refined using molecular modeling. This paper presents the first NMR assignment for the FIV p24 protein, which covers 83% of the total sequence (BRMB entry 50794). As observed on the NMR spectra, the quality of the signal is not uniform over the entire protein (supplementary figure S1). The requirement for perdeuteration, as well as disparities in the quality of the NMR signal, remain puzzling, considering the size of the protein (24 kDa), as larger proteins have been assigned previously without perdeuteration [START_REF] Kay | Solution NMR Spectroscopy beyond 25 KDa[END_REF][START_REF] Frueh | NMR Methods for Structural Studies of Large Monomeric and Multimeric Proteins[END_REF] . Indeed, initially acquired NMR spectra of p24 without perdeuteration showed a poor signal:noise ratio (spectra not shown). In addition, even though the deuterium labeling improved the overall signal:noise ratio, some resonances were still too broad with a very low signal:noise ratio. These observations may be linked to potential low-affinity p24/p24 interactions in the NMR samples, although the protein concentration was maintained at 120 µM to avoid multimerization [START_REF] Serrière | Biophysical Characterization of the Feline Immunodeficiency Virus P24 Capsid Protein Conformation and In Vitro Capsid Assembly[END_REF] . HIV-1 p24 subunits of the hexamer were shown to interact with each other through their N-terminal α1, α2, and α3 helices and their C-terminal α9 and α10 helices (PDB entries 3H47 and 3MGE, respectively) [START_REF] Pornillos | X-Ray Structures of the Hexameric Building Block of the HIV Capsid[END_REF] . Moreover, we cannot exclude that certain p24 residues exhibit conformational dynamics on the µs-to-ms scale (intermediate exchange) that could result in line broadening.

Although this assignment was incomplete, it was sufficient to identify the 696-binding site on FIV p24. As no known reference compound targeting FIV p24 has been described to date, our results represent a valuable tool for further studies of ligand-binding sites on FIV p24.

Interestingly, the relatively small number of NMR peaks that underwent CSPs upon 696 binding reveals the existence of a well-defined specific binding site on p24. Based on our NMR assignment, we were able to identify the p24 amino acid residues involved in binding. The combination of NMR spectroscopy and molecular docking allowed us to obtain a structural model of 696 bound to FIV p24. The results are consistent with a binding pocket delimited by the α3, α4, and α7 helices on the protein. We also observed that two residues located outside of the pocket, Gly 60 and Leu 104, underwent CSPs upon inhibitor binding, suggesting a slight conformational readjustment of the loops linking these helices. Because of the ability of p24 to undergo rearrangement during viral assembly and maturation, it is possible that these conformational changes after 696 binding could lead to the decrease in p24 assembly that we observed in vitro.

Compound 696 belongs to the benzimidazole family, one of the most common small molecule drug families listed by the U.S. Food and Drug Administration 68 . Some benzimidazoles have already been tested against HIV-1 p24. One (BM2) was shown to bind HIV-1 p24 but at a site delimited by the α1, α2, α3, and α4 helices 69 . These differences in binding sites can be explained by the low sequence homology (29%) between the HIV-1 and FIV p24 proteins [START_REF] Folio | Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline[END_REF] , which can impact the interactions between the compounds and proteins, or by differences between the chemical groups present on the benzimidazole rings of BM2 and 696. Nonetheless, interactions between 696 and HIV-1 p24 are currently being tested.

Interestingly, the 696 capsid pocket is structurally conserved in HIV-1, where it is targeted by inhibitors such as PF74 or one of its derivatives, GS-6207, which is currently in clinical trials against HIV [START_REF] Bhattacharya | Structural Basis of HIV-1 Capsid Recognition by PF74 and CPSF6[END_REF][START_REF] Link | Clinical Targeting of HIV Capsid Protein with a Long-Acting Small Molecule[END_REF] . These compounds do not inhibit FIV replication in vitro [START_REF] Fricke | BI-2 Destabilizes HIV-1 Cores during Infection and Prevents Binding of CPSF6 to the HIV-1 Capsid[END_REF] , but the molecular docking of 696 demonstrates that it interacts with FIV p24 similar to the interaction of PF74 with HIV-1 p24. It is interesting to note that these HIV-1 inhibitors have been demonstrated to target the p24:p24 interfaces in HIV-1 p24 capsomers [START_REF] Bhattacharya | Structural Basis of HIV-1 Capsid Recognition by PF74 and CPSF6[END_REF][START_REF] Link | Clinical Targeting of HIV Capsid Protein with a Long-Acting Small Molecule[END_REF][START_REF] Mcarthur | HIV Capsid Inhibitors Beyond PF74[END_REF] .

In HIV-1 p24, the conserved pocket also binds cellular partners such as human Nup153 (Figure 9A, PDB entry 4U0C) and CPSF6 (data not shown, PDB entry 4U0A [START_REF] Price | Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly[END_REF] ). Interestingly, the peptide fragment of human CPSF6, which is implicated in the binding of HIV-1 p24 in the crystal structure, is 100% identical to its counterpart in the feline CPSF6 protein (Felis catus sequence: UniProtKB A0A2I2UG80, residues 313-327). Both feline Nup153 (fNup153, UniProtKB M3WFC8) and human Nup153 have a high sequence identity of 89% for the region of hNup153 identified as interacting with HIV-1 p24. Therefore, we were able to model the binding of feline Nup153 on FIV p24 from the structure of human Nup153-HIV-1 p24 (Figure 9B).

We are currently investigating the molecular basis of the interactions between FIV p24 and feline Nup153 and CPSF6 to confirm this hypothesis. Anyhow, these structural similarities between HIV-1 and FIV p24 and the sequence conservation between human and feline Nup153 and CPSF6 suggest a conserved mechanism of interaction among lentiviruses with cellular partners, as previously described for CypA [START_REF] Goldstone | Structural and Functional Analysis of Prehistoric Lentiviruses Uncovers an Ancient Molecular Interface[END_REF] . We are currently seeking to solve the structure of the FIV p24 hexamer, which would confirm whether 696 indeed interacts with two adjacent monomers of capsomers. Such a finding would allow for structure-based improvements in compound 696 to target both sides of the p24:p24 interface in the hexamers. These observations are highly promising for the rational design of derivatives from 696 targeting FIV p24 that possess potent anti-FIV effects.

The affinity of 696 for monomeric p24 was estimated around 100 µM using MST, which is sufficient to consider 696 a promising lead scaffold for further optimization. Moreover, if 696 behaves in a similar fashion to PF74 and its derivatives [START_REF] Bhattacharya | Structural Basis of HIV-1 Capsid Recognition by PF74 and CPSF6[END_REF][START_REF] Link | Clinical Targeting of HIV Capsid Protein with a Long-Acting Small Molecule[END_REF][START_REF] Mcarthur | HIV Capsid Inhibitors Beyond PF74[END_REF] and indeed interacts with two adjacent monomers of FIV p24, the Kd value we obtained using monomeric FIV p24 might be underestimated compared to what it would be using FIV p24 hexamers. Moreover, the low solubility of 696 in water and the requirement for DMSO for its resuspension limit the end-point concentrations we could use in MST or NMR experiments. Consequently, we cannot exclude that the saturating values observed by MST are biased and that the real Kd value is lower than 100 µM. Solubility issues at high concentrations could also explain why the NMR CSP experiments did not reveal additional CSPs at 400 µM (data not shown). Efforts to develop 696-derived compounds that are more soluble in aqueous buffers will help clarify this point. Indeed, as 696 is now identified as the first compound to target FIV capsid assembly, derivatives of this reference molecule will be developed based on our NMR and molecular docking data and, when available, the structure of FIV p24 hexamers. These optimized compounds will first be characterized and selected using the strategy described here, and the more promising compounds will then be tested for their ability to inhibit FIV replication in cellulo. As the 696 compound is inexpensive to produce and thus compatible with the needs of veterinary medicine, this optimization strategy could eventually lead to the identification of drugs with better affinities for p24, low costs of synthesis, and antiviral efficacies, thus enabling their widespread use in veterinary practice.

Finally, it should be noted that the NMR assignment and molecular docking protocols we developed, and which were used to characterize the interaction between 696 and FIV p24, may not only be used to assess future 696 derivatives but can more generally be used to rationally design other inhibitory molecules targeting other regions of FIV p24, as described for HIV-1 [START_REF] Carnes | Inhibitors of the HIV-1 Capsid, A Target of Opportunity[END_REF] . Taken together, our work represents the first steps towards the development of potent, low-cost inhibitors targeting the FIV p24 protein as a treatment for FIV infection.

Associated content

Accession codes:

-HIV-1 Gag: UniprotKB P04591 -FIV Gag: UniprotKB P16087; subunit p24: BRMB 50794 -Human CypA: UniprotKB P62937 -hNup153: UniprotKB P49790 -hNup358: UniprotKB P49792 -hCPSF6: UniprotKB Q16630 -fCPSF6: UniprotKB A0A2I2UG80

  site identified by NMR-namely Leu 30, Phe 39, Thr 49, Met 51, Ile 55, Met 56, Cys 61, Asp 64, Lys 65, Glu 66, Ile 67, Leu 68, Asp 69, Glu 70, Leu 72, Lys 73, Thr 76, Asp 80, Met 100, Ile 102, Cys 121, Tyr 125, and Leu 129. The docking grid box was positioned to cover this area, and a large exhaustiveness value of 500 was used to maximize the thoroughness of the search. A visual examination of the best docking poses was performed using PyMOL (Schrödinger, LLC).

Figure 1 .

 1 Figure 1. FIV p24 assembly in the absence or presence of compound 696. The chemical structure

Figure 2 .

 2 Figure 2. Affinity of p24 for 696 measured by MST. Data points represent the mean of three

Figure 3 .

 3 Figure 3. The 1 H, 15 N-HSQC NMR spectrum of FIV p24. The NMR assignment was performed as

Figure 4 .

 4 Figure 4. NMR of FIV p24. A) Summary of the p24 NMR signal assignments according to atom

Figure 5 .

 5 Figure 5. 1 H, 15 N-HSQC NMR spectra of FIV p24. Overlay of 2D spectra of FIV p24 acquired in

Figure 6 .

 6 Figure 6. Interaction between p24 and 696. A) Combined 1 H, 15 N-NMR CSPs induced in p24 upon

Figure 7 .

 7 Figure 7. Molecular docking of compound 696 on the p24 surface using experimental NMR data.

Figure 8 .

 8 Figure 8. Effect of compound 696 on p24 disassembly analyzed by SDS-PAGE. The assembly of

Figure 9 .

 9 Figure 9. P24/Nup153 interactions. A) The experimental structure of the human Nup153 peptide

Acknowledgments

We thank M. Dujardin and L. Lecoq for their advices in NMR, and R. Monserret and C. Freton for their help with MST.

Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for

Funding sources

This work was supported by ECOS-Sud program U14S01 (Uruguay-France), French Ministry of Research, and by CSIC (Comisión Sectorial de Investigación Científica) program iniciación a la investigación 2017 number ID275. Financial support from the IR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged.

Author information

Corresponding authors

Notes

The authors declare no conflicts of interest.

Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.

Supporting Information

Supporting information content:


Mother-Tongue Instruction and Biliteracy Development in P'urhepecha (Mexico)

The dominant role of the P'urhepecha language at two primary schools in central Mexico provides an encouraging example of the promotion of an indigenous language in an educational context. P'urhepecha is a language isolate spoken by around 125,000 predominantly bilingual people in four regions of the central highlands of Michoacán. Intercultural Bilingual Education programs run in government schools throughout the state but offer only minimal indigenous language instruction. However, the two rural primary schools of San Isidro and Uringuitiro have developed a program and curriculum that emphasizes P'urhepecha language and culture, with instruction for all subjects provided in P'urhepecha from Grades 1 through 6. The schools are concerned with the maintenance of P'urhepecha language and culture as well as higher academic achievement and the transfer of P'urhepecha literacy skills to Spanish. Early literacy is introduced through P'urhepecha, and writing instruction is incorporated into the curriculum, with pupils given writing assignments across a range of school subjects. Native language literacy is crucial in facilitating cognitive development and the acquisition of competence in other domains, not just language. In this chapter we describe historical attempts to introduce P'urhepecha-based literacy, particularly the Tarascan Project, and a current initiative promoting biliteracy in P'urhepecha and Spanish through mother-tongue-based education. We then describe the instructional context for pupil writing at the two schools and analyse writing samples from four P'urhepecha-dominant fourth-grade pupils (aged 9-11) who were instructed to retell a story. We analyse these samples in terms of morphological complexity, narrative style, and orthographic accuracy, using the continua of biliteracy to frame the analysis. In so doing, we highlight the writing competence of the pupils in their contemporary version of P'urhepecha and illustrate the valued role of the language in the context of the school's native-language curriculum.

Introduction

P'urhepecha (previously known as Tarascan 1 ) is spoken by around 125,000 mostly bilingual people in four regions of the central highlands of Michoacán, Mexico (INEGI, 2010). This figure may suggest that P'urhepecha is quite vital, but the reality is that fewer and fewer children are learning the language at home (Chamoreau, 2000: 14); therefore it is potentially only a few generations away from grave endangerment. Economic circumstances have contributed to rapid language loss. In search of work, many P'urhepecha speakers have emigrated abroad, changing language use patterns and rupturing the transmission of the indigenous language from parent to child. Within this context of emerging language shift, our chapter presents positive examples of educational initiatives that have promoted literacy development in P'urhepecha and provides a preliminary analysis of writing samples by P'urhepecha pupils attending bilingual primary schools.

To survive under pressure from changing socio-economic circumstances and a dominant national language, it is important for the minority language to feature prominently in the education system [START_REF] Baker | Foundations of bilingual education and bilingualism[END_REF][START_REF] Cummins | Language, Power and Pedagogy[END_REF][START_REF] Fishman | Reversing Language Shift[END_REF]. In the case of P'urhepecha, children need to be schooled in their language as well as in Spanish, and to see the value in learning to read and write both languages. Despite the introduction of a compulsory eight-year programme of bilingual and bicultural primary education across Mexico in the 1970s, and the concomitant founding of the Dirección General de Educación Indígena (DGEI, Directorate General of Indigenous Education), Spanish remains the language of instruction at all levels. Only a few hours a week are devoted to the indigenous language at the primary school level and no provision is made for such lessons in secondary school. However, two rural primary schools in Michoacán have shifted to P'urhepecha-medium instruction in an encouraging but rare example of indigenous language promotion in an educational context [START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF][START_REF] Hamel | La noción de calidad desde las variables de equidad, diversidad y participación en la educación bilingüe intercultural[END_REF][START_REF] Hamel | The teaching of Spanish as a second language in an indigenous bilingual intercultural curriculum[END_REF].

As part of our ongoing analysis of biliteracy development through mother tongue instruction, we present here an evaluation of P'urhepecha writing samples penned by four P'urhepecha-dominant pupils at the end of fourth grade, who had been instructed to retell the 1 P'urhepecha can also be found spelled as Purepecha, Purépecha, P'urhépecha, and P'orhépecha, amongst others. The term 'Tarascan' was used to refer to the language in earlier studies but now is generally used only to refer to the precolonial population, as in the Tarascan State (see Section 3). P'urhepecha story Tukuru 'Owl'. We focus on how the texts are written in terms of narrative style, morphological complexity, and orthographic accuracy. Our analysis highlights how the P'urhepecha children can represent their own contemporary version of the language, including some (mostly lexical) elements from Spanish, in a grammatically and stylistically appropriate way. The following section provides the theoretical background to biliteracy development in indigenous education that motivates our analysis, focusing on the value of gaining initial literacy skills in one's own native language and on contesting established linguistic hierarchies in educational contexts.

Biliteracy development in indigenous education

Much has been said about the importance of educating children first in their native language, thus facilitating cognitive development and the acquisition of competence in other domains, not just language (Baker, 2000;Carlisle & Beeman, 2000;Dutcher & Tucker, 1996). Education in the native language or mother tongue is particularly important for linguistic minorities whose home language is undervalued, or in some cases not even recognised, by the dominant society around them. For indigenous communities attempting to preserve or revitalize their language, emphasis should be given to developing literacy skills in both the native, minority language, and the language of the dominant society. Previous research in the Mexican context, including at the schools featured in this chapter, has demonstrated that pupils who receive literacy instruction in their mother tongue or strongest language, whether that be an indigenous language or Spanish, achieve superior writing skills in both languages [START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF]. Such findings suggest a common underlying language proficiency in multilinguals that supports the transfer of literacy skills from one language to another [START_REF] Cummins | The construct of language proficiency in bilingual education[END_REF][START_REF] Cummins | Language, Power and Pedagogy[END_REF][START_REF] Hamel | The teaching of Spanish as a second language in an indigenous bilingual intercultural curriculum[END_REF].

In further exploring the connections between first and second language literacy, we make use of the continua of biliteracy framework, which brings together key research on literacy and multilingualism, emphasizing the interconnectedness among various aspects of biliteracy contexts, development, content, and media [START_REF] Hornberger | Continua of biliteracy[END_REF][START_REF] Hornberger | Revisiting the continua of biliteracy: International and critical perspectives[END_REF] see Figure 1). Biliteracy refers to "any and all instances in which communication occurs in two (or more) languages in or around writing" (Hornberger, 1990, 2). Of particular interest here is the interconnection among the various skills in the development of biliteracy, whether first language or second language, oral or written, receptive or productive. Although the opposite ends of the continua may appear to be dichotomous and finite, the notion of continua emphasizes a more nuanced and dynamic perspective. Indeed the framework draws attention to what these aspects of biliteracy have in common; e.g., the interrelatedness between first and second language skills. It also recommends the promotion of the traditionally undervalued aspects of biliteracy, giving attention, for example, to the first language of linguistic minorities, to oral skills as well as written, and to receptive as well as productive language skills. By providing a space for P'urhepecha literacy, the projects described in this chapter have contested established linguistic hierarchies, the historical context of which are described in the following section.

Context of biliteracy

Micro <-------------------> Macro Oral <-------------------> Literate Bi(multi)lingual <-------------------> Monolingual Development of biliteracy Reception <-------------------> Production Oral <-------------------> Written L1 <-------------------> L2

Content of biliteracy

Minority <-------------------> Majority Vernacular <-------------------> Literary Contextualized <-------------------> Decontextualized

Media of biliteracy

Simultaneous exposure <-------------------> Successive exposure Dissimilar structures <-------------------> Similar structures Divergent scripts <-------------------> Convergent scripts

History of the P'urhepecha

The beginning of a distinguishable P'urhepecha cultural tradition can be traced to the north and central zones of Michoacán in the Late Preclassic period (400 BCE -150 CE; Pollard, 2015: 93).

In the subsequent period (150 -900 CE), a major cultural transformation occurred under influence from central Mexico, leading to greater urbanisation. As well as regional interaction, facilitated along the Balsas-Tepelcatepec and Santiago-Lerma Rivers, there may also have been long-distance contact with Andean and coastal peoples of Peru and Ecuador, perhaps mediated by metallurgy [START_REF] Hosler | The Sounds and colors of power: The sacred metallurgical technology of ancient West Mexico[END_REF].

The formation of the Tarascan State began around 1000 CE as a number of competing small state societies emerged in Michoacán. Through warfare and strategic marriage alliances, the Wakúsecha "eagle warriors" emerged as the most dominant lineage in the region, fully consolidating their power under Lord Taríacuri (c. 1380-1420 CE), the first cazonci "chief" [START_REF] Roskamp | Tarascan (P'urhépecha) empire[END_REF], thereby founding the Tarascan State. Through a rapid process of cultural assimilation and political unification, the different groups in the region converged on a Tarascan ethnicity and socio-political system, which included use of the Tarascan language and centralised autocratic rule [START_REF] Gorenstein | The Tarascan civilization: A late prehispanic cultural system[END_REF]. By the mid-1400s, the Tarascans were the most formidable enemy of the Aztecs, being the only population to resist them militarily.

The Tarascan State was successfully invaded by the Spanish in 1523, with indigenous towns distributed among the colonisers as encomiendas. Spanish rule revolved around the exploitation of these encomiendas and other natural resources (e.g., mines) and the introduction of foreign socio-political structures [START_REF] Warren | The conquest of Michoacán: the Spanish domination of the Tarascan kingdom in western Mexico, 1521-1530[END_REF]. A continued struggle between the Tarascans and the Spanish ended abruptly on February 14, 1530, when the last cazonci was executed.

In the first two decades after the conquest, Michoacán, along with the rest of Mexico, saw a huge depopulation due to disease and forced resettlement. The Tarascan population was reduced by half in the first 30 years of Spanish occupation, with many survivors taking refuge deep in the Sierra (West, 1948: 12). Integrated into the Spanish colony, the former Tarascan State experienced profound socio-political and cultural transformations. One of the most obvious changes was linguistic; with the arrival of the Spaniards came the Spanish language, which quickly led to a situation of bilingualism and eventual shift away from P'urhepecha for many people.

The newly founded village structures were not delineated according to precolonial boundaries, leading to land disputes between some P'urhepecha communities. These disputes were compounded by a number of subsequent land reform acts, which persist to this day in some cases [START_REF] Roskamp | Visions of the past: The Tarascan kingdom and the late colonial primordial titles from Michoacán[END_REF]. Moreover, the rise of drug cartels in the region has compounded land disputes and increased violence across the state. For example, disillusioned by the lack of

6

governmental support to combat various local issues, the P'urhepecha town of Cherán declared independence from the state in 2010, setting up its own local council to govern according to the community's priorities [START_REF] Seneff | Ritual and public spheres in an ethnic celebration of communal sovereignty: The Purépecha transition[END_REF]. Indeed the past 40 years have seen the development of a clearer, more unified P'urhepecha identity, manifested in, for example, the inauguration of a flag representing the four regions, an anthem, and the motto juchari winapikwa, "our strength."

Language structure

A language isolate, P'urhepecha is characterised by its agglutinating structure and reliance on suffixation as a principal means of word formation. Four major dialect zones can be identified among the 110 P'urhepecha villages on the basis of linguistic, geographic, cultural, and social features: Meseta or Sierra, Lake Pátzcuaro Basin, Cañada de los 11 Pueblos, and Zacapu [START_REF] Chamoreau | Dialectología y dinámica: reflexiones a partir del purépecha[END_REF]. The variety represented in these samples is from the Meseta.

P'urhepecha's strongly agglutinative nature enables the formation of morphologically complex words entirely through suffixation. The core element of any word is the root, which can be either mono-or disyllabic. To this root can be added a sequence of suffixes, depending on the word class and meanings to be expressed. Most roots can also be reduplicated, yielding additional meanings of, for example, intensity, repetition, or multiple distribution in time and place (Friedrich, 1984: 66).

Two main types of nouns can be identified: fused and derived. Fused nouns comprise a root and suffix that are now inseparable (e.g., tsa=ki 'lizard'), while derived nouns add a nominalising suffix (usually -kwa) to a root (e.g., piré-kwa 'song,' from the root piré-'to sing').

Irrespective of their formation method, nouns are pluralised with the suffix -echa/-icha. There are seven cases in P'urhepecha: nominative, objective, genitive, instrumental, comitative, locative, and residential [START_REF] Chamoreau | Parlons Purepecha: Une langue du Mexique[END_REF]. The personal pronoun system is a standard six-way system, three singular and three plural, with the demonstratives ima and ts'ïma drafted in to function as third person singular and plural pronouns, respectively. P'urhepecha possesses a base 20 counting system, but it has largely been replaced by its Spanish counterpart. The numeral ma 'one' can also function as an indefinite article. There is no definite article.

Verbal morphology in P'urhepecha is remarkably elaborate, enabling the speaker to express combinations of location, direction, causative, voice/valency, mood, desiderative, adverbials, 3rd person plural object, aspect, tense, irrealis, mood, and subject/object person and number through the combination of suffixes, strictly in this order (Chamoreau, in press b).

Words can contain up to seven suffixes, but most are somewhat shorter (Friedrich, 1984: 65).

Members of the same category cannot co-occur, except for certain combinations of locative suffixes in the second slot. In the tense-aspect-mood domain, only mood is obligatory in a finite verb.

At the clausal level, word order is generally subject-verb-object (SVO), although historically it was more strictly SOV. The shift away from verb-final order is likely due to contact first with neighbouring Nahuatl (Uto-Aztecan) and Otomí (Otomanguean) and later with Spanish [START_REF] Chamoreau | [END_REF]. Coordinate clauses are linked with ka 'and'. In certain narrative styles, ka is ubiquitous, linking chain-medial clauses whose verbs take non-finite morphology as the subject has already been established [START_REF] Chamoreau | Non-finite chain-medial clauses on the continuum of finiteness in Purepecha[END_REF]; see Section 8).

Revitalisation efforts

P'urhepecha has been spoken in Michoacán since well before colonisation but was not a written language at that time. Franciscan missionaries encouraged literacy in P'urhepecha during the sixteenth century, but widespread literacy in the language was never established (Hamel 2008: 313). Colonial education policies focused primarily on forcibly assimilating indigenous peoples, both culturally and linguistically, through the direct imposition of Spanish in all grades in school (Hamel, 2013: 1). A second strategy comprised slow transition to Spanish and a very small number of language maintenance programs, but these were very much the exception (see Section 6).

Consequently, the Tarascan Project, initiated in 1939, represented a long-awaited shift to native language-medium education for P'urhepecha speakers. The project fostered literacy and language maintenance by teaching reading and writing in P'urhepecha, acting also as a bridge to Spanish literacy. Before launching the project, a combined team of Mexican and U.S. linguists and anthropologists devised a suitable, streamlined alphabet for P'urhepecha, as well as a set of primers for pedagogical purposes. P'urhepecha literacy classes were taught by 20 specially selected and trained native speakers. In Paracho, where the project was established, the project team produced additional materials, including instructional pamphlets regarding health and sanitation. Posters presenting the alphabet and contrasting segments were also displayed in village squares for consultation outside of class (Figure 2). The project ran for just over a year, from 1939-1941, and was reported as being immensely successful. Following its methodologically advanced, linguistic theory-based approach, previously illiterate individuals learned to read and write in 30 to 45 days (Barrera-Vásquez, 1953: 83). The project ended abruptly in 1941 due to the withdrawal of all funding. After the Tarascan Project ended, literacy in P'urhepecha advanced little, even with the introduction of bilingual and bicultural education in primary schools across Mexico in the 1970s and the later establishment of intercultural bilingual education (educación intercultural bilingüe, EIB) in the 1990s [START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF]. EIB is intended to integrate "content matters and competencies from indigenous funds of knowledge, as well as from national programs, [and] should be integrated in a culturally and pedagogically appropriate curriculum" [START_REF] Hamel | Multilingual education in Latin America[END_REF] 1-2). In contrast to earlier Spanish-centred programs, EIB should enable children to know and appropriate their own culture in their own language so that they can form sound competencies, values, and ethnic identity (see also [START_REF] López | Reaching the unreached: indigenous intercultural bilingual education in Latin America, Paper commissioned for the EFA Global Monitoring Report[END_REF].

Unfortunately, the reality of EIB is not as positive as its aims suggest. The vast majority of P'urhepecha-speaking children are not schooled in their native language first, or at all.

Instead, they continue to work through a system of "Castillanization," with Spanish as the vehicle for literacy and content instruction for all subjects. Government-run primary schools often provide a mere two hours a week of instruction in P'urhepecha, focusing only on language acquisition, albeit using P'urhepecha-medium materials in the form of workbooks and storybooks.

Revitalization efforts also exist in tertiary-level education institutions. The Instituto Tecnológico Superior P'urhepecha offers P'urhepecha language courses, while the Universidad Indígena Intercultural de Michoacán offers a number of Bachelor-level programs designed primarily for indigenous pupils, notably the Licenciatura in Language and Intercultural

Communication. The Universidad Michoacana in Morelia also offers P'urhepecha language classes. The first Diplomado in P'urhepecha language was run by the Centro de Investigaciones y Estudios Superiores en Antropología Social (CIESAS, Mexico City) in Santa Fe de la Laguna in July 2016, stimulating additional courses in legal translation and P'urhepecha syntax.

The main online community portal is hosted at http://www.purepecha.mx, which also includes a P'urhépecha-Spanish dictionary; Radio Xiranhua; and information regarding language, culture, and local initiatives and events (largely in Spanish). Local radio stations, such as Radio Juchári Uinápekua in Santa Fe de la Laguna, are also promoting the language to a wide audience. The revitalization efforts mentioned here, however, clearly do not constitute an exhaustive list. We focus in the following section on a specific effort to both promote mother tongue education and strengthen the language.

Language maintenance in a school context

A unique educational initiative has been developing in two government primary schools in the neighbouring towns of San Isidro and Uringuitiro, high in the Meseta P'urhepecha of western Michoacán. A group of committed P'urhepecha teachers were dissatisfied with the lack of attention to the P'urhepecha language under the national Intercultural Bilingual Education (EIB)

program and so began to develop a mother-tongue-based bilingual education program in the early 1990s. The national provisions for indigenous education mentioned above open up a space for the P'urhepecha language in government schools but do not provide for comprehensive literacy development in the mother tongue for those children who are primarily speakers of P'urhepecha. The new endeavour required awareness raising in the community, decisions about

10

an appropriate alphabet, and ongoing development of their own materials and curriculum [START_REF] Hamel | The teaching of Spanish as a second language in an indigenous bilingual intercultural curriculum[END_REF][START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF][START_REF] Hamel | La noción de calidad desde las variables de equidad, diversidad y participación en la educación bilingüe intercultural[END_REF].

The P'urhepecha educators have continued to improve the program over the last two decades, with support from researchers and experts through the Comunidad Indígena y Educación Intercultural Bilingüe (CIEIB) program, directed by R. Enrique Hamel. The curriculum that the schools are developing attempts to balance external educational demands with the maintenance of P'urhepecha language and culture, emphasizing P'urhepecha-medium instruction. In summarizing the schools' achievements, [START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF] writes that "[d]ifferent from most indigenous schools in Mexico, P'urhepecha has become the legitimate, unmarked language of all interaction at school, a sociolinguistic achievement still quite exceptional in indigenous education" (p. 320).

In San Isidro and Uringuitiro, P'urhepecha remains the primary home language.

Although in some cases Spanish is entering the home domain through marriages and returned migration, as well as through media and communication, the majority of the pupils start school with very little functional knowledge of Spanish. This is also true for many children in other P'urhepecha-dominant villages who must attend Spanish-medium public primary schools. Thus, the schools in San Isidro and Uringuitiro focus on three important objectives for the bilingual program: (i) supporting pupil content learning through the use of their mother tongue, (ii)

supporting pupils in learning Spanish as a second language, and (iii) developing and revitalizing P'urhepecha language and culture. Although often conceived of separately, in this instance the pedagogical and language learning objectives actually coincide with and mutually reinforce the preservation and revitalization objectives in these schools [START_REF] Hamel | The teaching of Spanish as a second language in an indigenous bilingual intercultural curriculum[END_REF].

Instructional writing practices

Writing in the P'urhepecha language forms an integral part of the educational program at these two primary schools. A number of alphabets still exist for P'urhepecha, leading to difficulties in carrying out unified literacy initiatives. The educators in San Isidro and Uringuitiro have developed a pedagogical alphabet appropriate for literacy instruction and employ a contrastive method to introduce letters in Spanish that do not exist in P'urhepecha [START_REF] Hamel | La lecto-escritura en la lengua propia: Educación indígena bilingüe en la región P'urhepecha de México[END_REF], a method reminiscent of the Tarascan Project. As the principal medium of instruction and the "legitimate, unmarked language of all interaction" at the schools [START_REF] Hamel | Bilingual education for indigenous communities in Mexico[END_REF], P'urhepecha is naturally used for note taking, learning exercises, and writing assignments throughout the primary school years.

According to the overall plan for bilingual instruction at the two schools, Grades 1 and 2 emphasize literacy development in P'urhepecha, with introduction to oral Spanish. By Grade 3, pupils should be able to read and write in P'urhepecha, after which they learn to read and write in Spanish. The teaching of Spanish follows the principles of content and language integrated learning (CLIL), with selected content instruction in Spanish forming the basis for teaching Spanish as a second language (see [START_REF] Coyle | Content and language integrated learning: Towards a connected research agenda for CLIL pedagogies[END_REF][START_REF] Haley | Content-based second language teaching and learning[END_REF]Richard & Rodgers, 2001). Classroom observations by the second author of this chapter, including in Grade 4 classes, confirmed that P'urhepecha was the primary language for both speaking and writing. Although the program plans for one hour of Spanish-medium instruction per day, P'urhepecha is by far the most frequently used language for communication in the classroom, even when Spanish textbooks are being used. Lessons are usually followed by writing assignments, whether pupils are copying notes, providing example sentences, describing the results of an experiment, or retelling stories from home. When pupils finish an assignment, they bring their notebooks to the teacher for verification and feedback.

Building from the requirements of the Mexican education system, the schools have developed a curriculum that is divided into the five school terms and that integrates concepts and vocabulary from the L1 (P'urhepecha) and the L2 (Spanish). Thus, the development of language skills, including writing, is incorporated into the content learning. By Grade 5, writing is listed as an important learning goal in the various subject areas, and by Grade 6, pupils are expected to be able to write opinions and arguments, as well as informative and descriptive texts in both languages.

Observers conducting research at the schools through CIEIB were impressed by the writing abilities of pupils in both languages by the end of Grade 3 (age 8-10). Also significant in the assessment of language skills at the two schools is the clearly parallel development of reading and writing skills in the two languages [START_REF] Hamel | La noción de calidad desde las variables de equidad, diversidad y participación en la educación bilingüe intercultural[END_REF]. One of the primary research goals of CIEIB in the current phase of the project is to explore the process of acquiring and developing P'urhepecha and Spanish literacy skills in the context of primary education. The P'urhepecha writing we analyse in the following section formed part of an assessment administered to all 12 pupils at the two schools in 2013, including components for both Spanish and P'urhepecha written expression.

Development of pupils' writing

The four writing samples under analysis were written by four pupils completing fourth grade, whose first language is P'urhepecha and whose Spanish proficiency was quite low at the time of assessment. The pupils were selected based on their representative performance in the assessment and on the availability of later writing samples, which form part of our ongoing analysis of biliteracy development. They were asked to re-write a story that had been read to them entitled Tukuru 'Owl,' with emphasis placed on their ability to articulate the key points in the story. The global scores given by teachers on the selected essays ranged from 3 to 6 out of 10, indicating that there were fragments of narrative-capturing elements of the story, though the stories lack a full story line. The same assessment was administered to pupils at all grade levels, so a sixth-grade pupil would be expected to score higher, for example, than a fourth-grade pupil.

Thus, the mid-range scores achieved by the fourth-grade pupils were not unexpected. An example of one of these samples is presented in Figure 3. The writing samples range from 15 to 40 lines of text on the test paper, with the original story comprising 12 sentences (around 620 words). The writing samples were analysed with attention to morphological complexity, narrative style, and orthographic accuracy. Our goal in this initial analysis was to identify features of pupil writing that suggest biliteracy development and that could be compared across time and across languages.

One of the salient features in all four essays was the frequent use of a very typical P'urhepecha discourse structure. This narrative style relies heavily on the coordinator ka 'and', as well as non-finite verbs ending in -ni, usually after the main actor of a sentence has been introduced [START_REF] Chamoreau | Non-finite chain-medial clauses on the continuum of finiteness in Purepecha[END_REF]. See example (1), where these features are underlined.

(1) ka tukuru no uxe-ni uanda-ni ka and owl NEG can-NF speak-NF and 'and the owl could not speak, and…' Pupils also use Spanish connectors such as komu (como) 'like', pari/para 'for, in order to', and porka (porque) 'because' as in example (2). This is a common feature of spoken P'urhepecha [START_REF] Chamoreau | [END_REF] and is also found in other indigenous languages that are in contact with Spanish, such as Otomí, also spoken in Mexico, as the coordinators sit in prominent positions outside of the clause and are therefore easily replaced with loanwords (Bakker & Hekking, 2012).

(2) ka porka tukuru no sesi jasï uka-s-p-ti and because owl NEG well type do-AOR-PST-3S.ASS 'And because the owl did it harm' (lit. did not do it well) P'urhepecha verbs can be fairly complex (see Section 4), but the pupils have clearly mastered them by Grade 4. The verbs in the writing samples generally include up to three suffixes, including aspect (usually in the form of the aorist), tense (past, as this is the only one that is marked), and mood/person (3).

(3) arhi-s-p-ti say-AOR-PST-3.ASS 's/he said'

Despite being immersed in their native language, with low proficiency in Spanish, the impact of the national language is still observable in the writing samples. While no Spanish loanwords were used in the original essay that was read to the pupils, several Spanish loanwords appear in the pupil essays, fully integrated into P'urhepecha morphology. Various orthographic inconsistencies can be identified in the writing samples, such as in the name of the story itself, which is spelled as tukuru, tukurhu and tuk'urhu, but this is to be expected for various reasons. First, the pupils are still quite young; therefore, one should not expect complete consistency. A low emphasis on orthographic conventions is a currently accepted pedagogical strategy and is not to be censured. Second, given the relatively recent development of written materials in the language, standards are still being developed, and consistency among materials from different sources is yet to be achieved. Third, it can be difficult to distinguish between the aspirated and non-aspirated stops, and also between the tap and retroflex rhotic (/rh/) in everyday speech. However, one does see a certain amount of consistency in the way the language is represented orthographically. The use of /b d g/ for /p t k/ after a nasal phoneme reflects the voicing that occurs in speech, and /l/ for /ɽ/ reflects a regional pronunciation of this phoneme.

In sum, these examples show that the children can represent their own contemporary version of the language, which includes some elements from Spanish, in a grammatically appropriate way. More importantly, these essays represent the opportunity that pupils have to demonstrate their skills in written P'urhepecha in a legitimized educational context. Rather than pinpointing a specific method for writing instruction, we emphasize here the legitimate, unmarked use of a minority language in all aspects of these pupils' primary education. This extensive space for P'urhepecha and for a P'urhepecha discourse style reflects the broader valuing of P'urhepecha cultural norms evident in the two schools, where their language is given as much, if not more, value than the dominant national language.

Like the other chapters in this volume, we focus at a micro level on pupil writing samples within a classroom and school environment that supports minority language development, even if the more macro level (i.e. national, government) context is unsupportive. In such contexts of biliteracy, the traditionally less powerful orality and bilingualism [START_REF] Hornberger | Revisiting the continua of biliteracy: International and critical perspectives[END_REF]; see Figure 1) are given value. The content of biliteracy in the case of the San Isidro and Uringuitiro schools is consciously minority and contextualized. Although the school remains tied to decontextualized, majority curriculum requirements, the development of a P'urhepecha curriculum based on traditional P'urhepecha values and discourse styles has been an ongoing priority. The media of biliteracy in this case emphasizes a gradual introduction of Spanish, which is structurally rather dissimilar from P'urhepecha and has a somewhat divergent orthography. In terms of the development of biliteracy, we expect that the receptive and productive skills of listening, speaking, reading, and writing will be interconnected, especially in a context where these skills are nurtured in both the L1 and the L2. This preliminary analysis shows features of an oral narrative style incorporated into a formal writing assignment and L2

features incorporated into well-structured ("grammatically correct") L1 sentences.

Promising directions for future revitalization efforts

Much remains to be learned regarding the interrelated development of literacy skills in multiple languages. The analysis presented here forms part of an ongoing exploration of biliteracy development, including analysis of written and oral samples, in both P'urhepecha and Spanish across time and grade levels. Such investigations will also shed light on the language acquisition process and can help to feed back into refining teaching methods. Clearly the transfer of literacy skills is also relevant to contexts in which pupils are (re-)learning an indigenous language and have stronger skills in the dominant national language, which is the case for some P'urhepecha children.

In some parts of rural Michoacán, P'urhepecha is still fortunate to be in a language maintenance situation, with its associated intergenerational transmission of the language.

However, the risk of language shift in the coming generations is great. The use of P'urhepecha as medium of instruction in primary schools is a much-needed, longer-term solution to the current low levels of P'urhepecha literacy transmission across the region. The schools in San Isidro and Uringuitiro provide an example of best practice in mother-tongue instruction that can be emulated by other schools in Michoacán as well as further afield . These schools provide for the educational needs of P'urhepecha-dominant pupils and also promote P'urhepecha language and culture in a valued educational context. However, for children to develop full biliteracy skills, the less valued aspects of biliteracy require promotion. This entails continuous support for and development of proficiency in minority languages such as P'urhepecha in the spaces where they have been undervalued, including onward into secondary and higher education.

Figure

  Figure 1: The Continua of Biliteracy (from Hornberger & Skilton-Sylvester, 2000) Traditionally less powerful Traditionally more powerful

Figure 2 :

 2 Figure 2: Example of Tarascan Project teaching material: a mural newspaper bearing the title kerénda ȼiȼʌki 'crag flower'. A younger man, probably a teacher, stands by as members of the community read local and national news. Photo by Frances L. (Swadesh) Quintana, 1939/1940.

Figure 3 :

 3 Figure 3: Extract of a writing sample from the P'urhepecha-medium Miguel Hidalgo primary school, San Isidro, Michoacán

  Bellamy, Kate & Cynthia Groff. 2019. Mother-Tongue Instruction and Biliteracy Development in P'urhepecha. In: Ari Sherris & Joy Peyton (Eds.), Early Writing in Indigenous Languages, London: Routledge, pp. 202-217.

  Bellamy, Kate & Cynthia Groff. 2019. Mother-Tongue Instruction and Biliteracy Development in P'urhepecha. In: Ari Sherris & Joy Peyton (Eds.), Early Writing in Indigenous Languages, London: Routledge, pp. 202-217.

  The noun pajarito 'little bird' and the verb buscar 'to look for' are evident in examples (4a-b).

	'And that he is looking for (them) the birds'
	(4a) kwiripu-echa	ka	ima=ksi	pajaritu-cha mantini=ksi
	person-PL	and	DEM=3PL	bird.DIM-PL by.one=3PL
	'The people and those birds one by one…'
	(4b) y	que	busqi-ti=ksɨ	pajaritu-cha
	and	that look.for-3.ASS=3PL bird.DIM-PL