METHODS FOR SCREENING INHIBITORS AGAINST CHIKUNGUNYA VIRUS AND FOR DETERMINING WHETHER SUBJECTS ARE PREDISPOSED TO INFECTION BY SAID VIRUS

Abstract

Chikungunya virus (CHIKV) has caused recent outbreaks associated with severe morbidity. Currently no vaccine or treatment exists to protect humans from CHIKV infection. Treatment is therefore purely symptomatic and is based on non-steroidal anti-inflammatory drugs. Accordingly, there is a high medical need exists to have new methods of screening of compounds which could inhibit chikungunya virus. Further to a CRISPR-Cas9 genetic screen the inventors now identify the four and a half LIM domains protein 1 (FHL1) has an essential host factor for CHIKV infection. In particular, they show that primary myoblast and fibroblast from FHL1 deficient patient are resistant to CHIKV infection. They also demonstrate that depletion of FHL1 prevents CHIKV replication. Finally, they show that CHIKV non-structural protein 3 interacts specifically with FHL1A through its hypervariable domain. Thus compounds that are capable of inhibiting the interaction between the non-structural protein 3 and FHL1 would be suitable for inhibiting the replication capacity of the virus. Determining the expression level of FHL1 and/or identifying some genetic variant would also be suitable for determining whether some subjects are predisposed to CHIKV infection.

Claims

1. A method for identifying a substance useful for inhibiting the replication capacity of chikungunya virus (CHIKV) comprising the steps of (a) contacting a polypeptide (P1) containing an amino acid sequence of the human FHL1 protein with a polypeptide (P2) having an amino acid sequence of the CHIKV NSP3 protein, under conditions and for a time sufficient to permit binding and the formation of a complex between the two polypeptides (P1) and (P2), in the presence of a test substance, and (b) detecting the formation of the complex, in which the ability of the test substance to inhibit the interaction between the two polypeptides (P1) and (P2) is indicated by a decrease in complex formation as compared to the amount of complex formed in the absence of the test substance and (c) selecting the substance that inhibits the interaction.

2. The method of claim 1 wherein the polypeptide (P1) comprises an amino acid sequences having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

3. The method of claim 1 wherein the polypeptide (P2) comprises an amino acid sequence having at least 90% of identity with the amino acid sequence ranging from the amino acid residue at position R326 to amino acid residue at position L524 in SEQ ID NO:4.

4. The method of claim 1 wherein the polypeptide (P1) and/or (P2) is labelled with a detectable molecule.

5. The method of claim 1 wherein step (b) comprises generating physical values which illustrate or not the ability of said test substance to inhibit the interaction between the polypeptides (P1) and (P2) and comparing said physical values with standard physical values obtained in the same assay performed in the absence of the test substance, and wherein the physical values encompass light absorbance values, radioactive signals and intensity value of fluorescence signal.

6. The method of claim 5 wherein if after the comparison of the physical values with the standard physical values, it is determined that the said test substance inhibits the binding between polypeptides (P1) and (P2), then the candidate is positively selected at step (c).

7. The method of claim 1 wherein step (b) involves an assay selected from the group consisting of a two-hybrid assay, a gel migration assay, an assay that includes the use of an optical biosensor, an assay that includes the use of affinity chromatography, and an assay that involves detection of a fluorescence signal.

8. The method of claim 1 which further comprises a step (d) of determining whether the substance selected at step (c) inhibits the replication of CHIKV in a host cell and a step (e) of positively selecting the test substance capable of inhibiting the replication of said CHIKV in said host cell.

9. The method of claim 8 further comprising the steps of i) infecting said host cell with said CHIKV and ii) culturing an infected host cell in presence of the test substance, iii) comparing the replicating capacity of the virus in the host cell with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the virus.

10. A method of treating a subject who is predisposed for a CHIKV infection comprising the steps of i) measuring the expression level of FHL1 in a sample obtained from the subject and ii) treating the subject for CHIKV infection when a differential between the measured expression level and the predetermined reference value is detected.

11. A method of treating a subject thought to have or to be predisposed to having a CHIKV infection, comprising analysing a sample of interest obtained from said subject to detect the presence of a genetic variant in the gene encoding for FHL1 protein, and treating the subject when the genetic variant is detected.

12. The method of claim 11 that comprises detecting one or more single nucleotide polymorphisms (SNP).

13. A method of treating a subject thought to have or be predisposed to having a CHIKV infection, comprising analysing a sample of interest obtained from said subject to detect post-translational modifications of FHL1 protein, and treating the subject when at least one post-translational modification is detected.

Description

FIGURES

[0087] FIG. 1. CRISPR-Cas9 genetic screen identifies FHL1 has an essential host factor for CHIKV and ONNV infection.

[0088] (A) Results of the CHIKV 21 strains screen analyzed by MAGeCK. Each circle represents individual gene. Y-axis represents the significance of sgRNA enrichment of genes in the selected population compared to the non-selected control population. X-axis represents a random distribution of the genes. (B-D) HAP1 cells were edited with a control or two different FHL1 sgRNA. (B) Immunoblotting of FHL1 in control and FHL1.sup.KO clones. (C) Viability of control and FHL1.sup.KO HAP1 cells over a 72 hours period using the Cell-Titer Glo assay. Data shown are representative of two experiments. (D) Control or FHL1.sup.KO cells were exposed to CHIKV 21 strains (HAP1: MOI of 10; 293T: MOI of 2) and stained for E2 protein. Data shown are mean+/−SD from three experiments (n=6, one-way ANOVA with Dunnett's test). (E) Control, FHL1.sup.KO or FHL1.sup.KO HAP1 cells transduced with the three FHL1 isoforms were inoculated with CHIKV 21 strains (MOI of 10) and E2 expression was analyzed. Data shown are mean+/−SD from two experiments (n=4, one-way ANOVA with Dunnett's test). (F) Control or FHL1.sup.KO cells were inoculated with CHIKV (MOI of 10), MAYV (MOI of 10) and ONNV (MOI of 10), and E2 expression was analyzed. Data shown are mean+/−SD from two experiments (n=4, one-way ANOVA with Dunnett's test). (G) Control or FHL1.sup.KO HAP1 cells were inoculated with TCID50 of the indicated alphaviruses, and infection was assessed by qRT-PCR. Data shown are mean+/−SD from one representative experiment. (H) Control or FHL1.sup.KO HAP1 cells were inoculated with DENV (MOI of 10) and ZIKV (MOI of 20), and E expression was analyzed. Data shown are mean+/−SD from three experiments (n=6, one-way ANOVA with Dunnett's test) * P<0.05; **** P<0.0001; ns not significant.

[0089] FIG. 2. Primary myoblast and fibroblast from FHL1 deficient patient are resistant to CHIKV infection.

[0090] (A) Schematic representation of FHL1 protein in control (C1 and C2) or patient carrying a mutation (P1 to P3), and genomic organization of FHL1 gene carrying a LINE1 insertion in exon 4 (P4). (B) Immunoblotting of FHL1 in the lysate from control and patient primary myoblast. (C) Control and patient primary myoblast were inoculated with CHIKV (MOI of 2), and E2 expression was analyzed. Data shown are mean+/−SD from two experiments (n=4, one-way ANOVA with multi-comparison test). (D) Quantification of viral particles released by infected primary myoblast at 24, 48 and 72 hours post-infection. FIU, flow cytometry infectious particles. Data shown are mean+/−SD from one representative of two experiments (n=2). (E) Primary fibroblasts were inoculated with CHIKV (MOI of 0.4), MAYV (MOI of 2) and DENV (MOI of 20), and analyzed for E2 or E protein expression. Data shown are mean+/−SD from two experiments (n=4, one-way ANOVA with multi-comparison test). (F) Quantification of viral particles released by infected primary fibroblast at 48 hours post-infection. FIU, flow cytometry infectious particles. Data shown are mean+/−SD from one representative of two experiments (n=2). **** P<0.0001; ns not significant.

[0091] FIG. 3. Depletion of FHL1 prevents CHIKV replication.

[0092] (A) Transfection of CHIKV replicon RNA expressing luciferase into control and FHL1.sup.KO HAP1 cells. Luciferase activity was monitored at indicated time point. RLU, relative light units. Data shown are mean+/−SD from three experiments (n=12). (B) Control 293T cells were transfected with the indicated CHIKV capped in vitro transcribed RNA expressing renilla luciferase (Rluc). Rluc activity was monitored at indicated time points. RLU, relative light units. Data shown are mean+/−SEM (n=2 independent experiments in quadruplicate; Two-way ANOVA with Tukey's multiple comparisons test). (C) Negative-stranded viral RNA quantification by RT-qPCR from samples (h.p.i., hours post-infection; NI, not infected). Data are mean±s.d. n=2 independent experiments in quadruplicate. One-way ANOVA with a Tukey's multiple comparison test. Dashed line represents the experimental background threshold.

[0093] FIG. 4. CHIKV non-structural protein 3 interacts specifically with FHL1A through its hypervariable domain.

[0094] (A) Immunoassay of the interaction between viral replication complex and endogenous FHL1 in 293T cells infected with CHIKV expressing nsp3-mCherry, assessed by immunoprecipitation with anti-RFP antibody followed by immunoblot analysis with anti-mCherry and anti-FHL1. (B) Immunoassay of the interaction of endogenous FHL1 with CHIKV nsP proteins in 293T cells transfected with plasmids encoding Flag-tagged individual nsP, assessed by co-immunoprocipitation with anti-FLAG and immunoblot analysis with anti-FHL1 and anti-FLAG. (C) Top panel shows full-length CHIKV nsP3 and constructs of nsP3 containing various combination of MD, AUD and HVD. Bottom panel shows immunoassay in 293T cells transfected with the plasmids encoding FLAG-tagged nsP3 constructs. Cellular lysates were subject to immunoprecipitation with anti-FLAG followed by immunoblot analysis with anti-FLAG and anti-FHL1. (D) Immunoassay of the interaction between CHIKV nsP3 and FHL1 isoform in 293Tcells transfected with FLAG-tagged nsP3 and empty vector or HA-tagged plasmids encoding the three FHL1 isoforms (top panel). Cellular lysates were subject to immunoprecipitation with anti-HA followed by immunoblot analysis with anti-FLAG and anti-HA. (E) Immunoassay of the interaction between FHL1 and nsP3 protein from various alphaviruses in 293T cells transfected with plasmid encoding FLAG-tagged CHIKV, Sindbis (SINV) and Semliki forest virus (SFV) nsP3. Cellular lysates were subject to immunoprecipitation with anti-FLAG followed by immunoblot analysis with anti-FLAG and anti-FHL1. (A-E) Data are representative of two experiments with similar results.

[0095] FIG. 5. FHL1 is a factor of susceptibility to CHIKV infection in mice. Viral titres in tissues of 9-day-old mice. Wild-type (WT) male littermates (n=5) and Fhl1−/y mice (n=7) were inoculated with 105 plaque-forming units of CHIKV by intradermal injection and euthanized 7 days after infection. The amount of infectious virus in tissues was quantified as the TCID50. The dashed line indicates the detection threshold. Data are mean±s.e.m. Two-tailed t-test.

EXAMPLE 1

[0096] Chikungunya virus (CHIKV) has caused recent outbreaks associated with severe morbidity. Currently no vaccine or treatment exists to protect humans from CHIKV infection. Treatment is therefore purely symptomatic and is based on non-steroidal anti-inflammatory drugs. Accordingly, there is a high medical need exists to have new methods of screening of compounds which could inhibit chikungunya virus. Further to a CRISPR-Cas9 genetic screen the inventors now identify the four and a half LIM domains protein 1 (FHL1) has an essential host factor for CHIKV infection (FIG. 1A-H). In particular, they show that primary myoblast and fibroblast from FHL1 deficient patient are resistant to CHIKV infection (FIG. 2A-F). They also show that transfection of CHIKV(GAA) RNA in ΔFHL1 or control cells resulted in similar Rluc activities (Data not shown), indicating that FHL1 is dispensable for viral RNA translation. When similar experiments were performed with wild-type CHIKV RNA, a large increase in Rluc activity was observed in control—but not ΔFHL1—cells 24 h after infection, demonstrating that FHL1 is essential for viral RNA replication. Quantitative reverse-transcription PCR (RT-qPCR) experiments showed that ablation of FHL1 resulted in severely reduced synthesis of CHIKV negative-strand RNA. They demonstrate that depletion of FHL1 prevents CHIKV replication (FIG. 3A-C).

[0097] Finally, they show that CHIKV non-structural protein 3 interacts specifically with FHL1A through its hypervariable domain (FIG. 4A-E). Thus compounds that are capable of inhibiting the interaction between the non-structural protein 3 and FHL1 would be suitable for inhibiting the replication capacity of the virus. Determining the expression level of FHL1 and/or identifying some genetic variant would also be suitable for determining whether some subjects are predisposed to CHIKV infection.

EXAMPLE 2

[0098] Methods:

[0099] Cell culture. HAP1 cells (Horizon Discovery), which are derived from near-haploid chronic myeloid leukemia KBM7 cells, were cultured in IMDM supplemented with 10% FBS, 1% penicillin-streptomycin (P/S) and GlutaMAX (Thermo Fisher Scientific). 293FT (Thermo Fisher Scientific), HEK-293T (ATCC), Vero E6 (ATCC), HepG2 (kind gift of Olivier Schwartz, Institut Pasteur, Paris, France), primary myoblasts and primary fibroblasts were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX and 25 mM Hepes. Human placenta choriocarcinoma Bewo cells were cultured in in DMEM supplemented with 5% FBS, 1% penicillin-streptomycin, 1% GlutaMAX and 25 mM Hepes. AP61 mosquito (Aedes pseudoscutellaris) cells (gift from Philippe Despres, Institut Pasteur, Paris, France) were cultured at 28° C. in Leibovitz medium supplemented with 10% FCS, 1% P/S, 1% glutamine, 1× non-essential amino acid, 1× Tryptose phosphate and 10 mM Hepes. All cell lines were cultured at 37° C. in presence of 5% C02 with the exception of AP61 that were maintained at 28° C. with no C02.

[0100] Virus strains and culture. CHIKV21 (strain 06-21), ZIKV (HD78788) (both are kind gift from Philippe Despres, Institut Pasteur, Paris, France), CHIKV West Africa (strain 37997, accession nb AY726732.1) and dengue virus serotype 2 DENV (16681) viruses were propagated in mosquito AP61 cell monolayers with limited cell passages. CHIKV-Brazza-MRS1 2011, CHIKV-Ross, CHIKV-St Martin H20235 2013-Asian, RRV (strain 528v), MAYV (strain TC 625), ONNV (strain Dakar 234), SINV (strain Egypt 339), EEEV (strain H178/99), VEEV (strain TV83 vaccine), WEEV (strain 47A), SFV (strain 1745) were obtained from the European Virus Archive (EVA) collection and propagated with limited passage on Vero E6 cells.

[0101] pCHIKV-M-Gluc (see plasmid sections) and pCHIKV-mCherry molecular clones were derivate of pCHIKV-M constructed from a CHIKV (strain BNI-CHIKV_899) isolated from a patient during Mauritius outbreak in 2006. To generate infectious virus from CHIKV molecular clones, capped viral RNAs were generated from the NotI-linearized CHIKV plasmids using a mMESSAGE mMACHINE SP6 or T7 Transcription Kit (Thermo Fischer Scientific) according to manufacturer's instructions. Resulting RNAs were purified by phenol:chloroform extraction and isopropanol precipitation, resuspended in water, aliquoted and stored at −80° C. until use. Thirty μg of purified RNAs were transfected in BHK21 with lipofectamine 3000 reagent and supernatants harvested 72 hours later were used for viral propagation on Vero E6 cells.

[0102] For all the viral stock used in flow cytometry analysis experiments, viruses were purified through a 20% sucrose cushion by ultracentrifugation at 80,000×g for 2 hours at 4° C. Pellets were resuspended in HNE1X pH7.4 (Hepes 5 mM, NaCl 150 mM, EDTA 0.1 mM), aliquoted and stored at −80° C. Viral stock titers were determined on Vero E6 cell by plaque assay and are expressed as PFU per ml. Virus stocks were also determined by flow cytometry as previously described.sup.38,39 Briefly, Vero E6 cells were incubated for 1 h with 100 μl of 10-fold serial dilutions of viral stocks. The inoculum was then replaced with 500 μl of culture medium and the percent of E2 expressing cells was quantified by flow cytometry at 8 hpi. Virus titers were calculated using the following formula and expressed as FACS Infectious Units (FIU) per ml. [Titer (FIU/ml)=(average % of infection)×(number of cells in well)×(dilution factor)/(ml of inoculum added to cells)].

[0103] Reagents. The following antibodies were used: anti-FHL1 mAb (ref MAB5938, R & D Systems), anti-FHL1 rabbit Ab (ref NBP1-88745, Novus Biologicals), anti-vimentin antibody (ab24525, abcam), anti-GAPDH mAb (ref SC-47724, Santa Cruz Biotechnology), polyclonal rabbit anti-HA (ref 3724, Cell Signaling Technology), anti-FLAG M2 mAb (ref F1804, SIGMA), anti-RFP (ref 6G6, Chromotek), anti-CHIKV E2 mAb (3E4 and 3E4 conjugated-CY3), anti-alphavirus E2 mAb (CHIK-265 was a kind gift from Michael Diamonds, University school of medicine, St Louis, USA), anti-EEEV E1 mAb (ref MAB8754, Sigma), anti-pan-flavivirus E protein mAb (4G2), anti-dsRNA J2 mAb (Scicons), Alexa Fluorm 488-conjugated goat anti-rabbit IgG (A11034, Invitrogen), Alexa Fluori-647-conjugated goat anti-chicken IgG (ab150175, abcam), Alexa Fluor™ 488-conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch), Alexa Fluorm 647-conjugated goat anti-mouse IgG (115-606-062, Jackson ImmunoResearch), peroxydase-conjugated donkey anti-rabbit IgG (711-035-152, Jackson ImmunoResearch), and anti-mouse/HRP (P0260, Dako Cytomotion). FLAG magnetic beads (ref M8823, SIGMA), HA-magnetic beads (ref 88837, Thermo Fisher Scientific) and anti-RFP coupled to magnetic agarose beads (RFP-Trap MA, Chromotek) were used for immunoprecipitation experiments.

[0104] CRISPR genetic screen. The GeCKO v2 human CRISPR pooled libraries (A and B) encompassing 123,411 different sgRNA targeting 19,050 genes (cloned in the plentiCRISPR v2) were purchased from GenScript. Lentiviral production was prepared independently for each half-library in 293FT cells by co-transfecting sgRNA plasmids with psPAX2 (Kind gift from Nicolas Manel, Institut Curie, Paris, France) and pCMV-VSV-G at a ratio of 4:3:1 with lipofectamine 3000 (Thermo Fisher Scientific). Supernatants were harvested 48 h after transfection, cleared by centrifugation (750×g for 10 min), filtered using a 0.45 μM filter and purified through a 20% sucrose cushion by ultracentrifugation (80,000×g for 2 hours at 4° C.). Pellets were resuspended in HNE1X pH7.4, aliquoted and stored at −80° C. HAP1 cells were transduced by spinoculation (750×g for 2 hours at 32° C.) with each CRISPR-sgRNA lentiviral libraries at a multiplicity of infection (MOI) of 0.3 and a coverage of 500 times the sgRNA representation. Cells were selected with puromycin for 8 days and expanded. Sixty million cells from each library were pooled and infected with CHIKV21 using a MOI of 1. Simultaneously forty million of non-infected pooled cells were pelleted and kept at −80° C. to serve as a reference of the library representation at time of infection. Approximately 5 days after infection, cytopathic effect was detectable and surviving cells were collected 2 weeks later. Genomic DNA was extracted from selected cells or non-infected pooled cells using QIAamp DNA column (Qiagen), and inserted gRNA sequences were amplified and subject to next generation sequencing on an Illumina MiSeq (Plateforme MGX, Institut Génomique Fonctionelle, Montpellier, France). gRNA sequences were analyzed using the MAGeCK software. Additionally, gRNA sequences were analyzed using the RIGER software following previously published recommendation.sup.40.

[0105] FHL1 editing. FHL1 was validated using two independent sgRNA targeting the exon 3 and exon 4, which are common to all FHL1 isoforms. sgRNAs were cloned into the plasmid lentiCRISPR v2 according to Zhang lab's recommendation. HAP1 and 293FT cells were transiently transfected with the plasmid expressing individual sgRNA and selected with puromycin until all mock-transfected cells died (approximately 72 hours). Transfected cells were used to ascertain gRNA-driven resistance to CHIKV cytopathic effect, and clonal cell lines were isolated by limiting dilution and assessed by immunoblot for FHL1 expression.

[0106] Infection assay. For infection quantification by flow cytometry analysis, cells were plated in 24-well plates. Cells were infected for 24 (293T) or 48 hours (HAP1), trypsinized and fixed with 2% (v/v) paraformaldehyde (PFA) diluted in PBS for 15 min at room temperature. Cells were incubated for 30 min at 4° C. with 1 μg/ml of either the 3E4 anti-E2 mAb for CHIKV strains and ONNV) or the CHIKV 265 anti-E2 mAb for MAYV or the anti-E1 mAb for EEEV or anti-pan-flavivirus E 4G2 for DENV and ZIKV. Ab were diluted in permeabilization flow cytometry buffer (PBS supplemented with 5% FBS, 0.5% (w/v) saponin, 0.1% Sodium azide). After washing, cells were incubated with 1 μg/ml of Alexa Fluor 488 or 647-conjugated goat anti-mouse IgG diluted in permeabilization flow cytometry buffer for 30 min at 4° C. Acquisition was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analysis was done by using FlowJo software (Tree Star). To assess infectious viral particles release during infection, cells were inoculated for 3 hours with viruses, washed once and then maintained in culture medium over a 72-hour period. At indicated time points supernatants were collected and kept at −80° C. Vero E6 cells were incubated with 10-fold serial dilution of supernatant for 24 hours and E2 expression was quantified by flow cytometry as described above.

[0107] For detection of infected cells by immunofluorescence, control and ΔFHL1 HAP1 cells were plated on Lab-Tek II CC2 glass slide 8 wells (Nunc). Cells were inoculated with CHIKV21 strain (MOI of 20) or CHIKV-nsP3-mCherry (MOI of 20) for 48 hours, then washed thrice with cold PBS and fixed with 4% (v/v) PFA diluted in PBS for 20 min at room temperature. CHIKV E2 protein was stained with the 3E4 mAb at 5 μg/ml, followed by a secondary staining with 1 μg/ml of Alexa 488-conjugated goat anti-mouse IgG. Both antibodies were diluted in PBS supplemented with 3% (w/v) BSA and 0.1% saponin. Slides were mounted with ProLong Gold antifade reagent containing 4,6-diamidino-2-phenylindole (DAPI) for nuclei staining (Thermo Fisher Scientific).

[0108] For colocalization experiments, cells infected with CHIKV-nsP3-mCherry (MOI of 20) were stained with 10 μg/ml of the anti-FHL1 mAb, followed by a secondary staining with 1 μg/ml of Alexa 488-conjugated goat anti-mouse IgG.

[0109] For detection of dsRNA foci, control and ΔFHL1 293T cells were plated on Lab-Tek II CC2 glass slide 8 wells (Nunc) and infected with CHIKV21 strain (MOI of 50) for 4 or 6 hours. After fixation with 4% (v/v) PFA diluted in PBS, cells were stained with 5 μg/ml of the anti-dsRNA mAb, followed by a secondary staining with 1 μg/ml of Alexa 488-conjugated goat anti-mouse IgG. Both antibodies were diluted in PBS supplemented with 3% (w/v) BSA and 0.1% Triton 100×. Of note, no dsRNA foci were detectable at 4 hpi.

[0110] Fluorescence microscopy images were acquired using a LSM 800 confocal microscope (Zeiss).

[0111] Plasmid constructions. To generate the C-terminal HA-tagged FHL1 isoforms, the cDNAs of FHL1A (NM_001449.4), FHL1B (XM_006724746.2) and FHL1C (NM_001159703.1) were purchased from Genscript. Coding sequence (CDS) were amplified and cloned into pLVX-IRES-ZsGreenl vector (Takara). Using the same approach, coding sequence of murine FHL1 (NM_001077362.2) and cloned into pLVX-IRES-ZsGreenl vector. C-terminal HA-tagged FHL2 coding sequence was synthesized by Genscript and subcloned into pLVX-IRES-ZsGreenl vector. The plasmids pCI-neo-3×FLAG plasmids expressing the CHIKV nsP3 and nsP4, the Sindbis virus (SINV) and Semliki Forest virus (SFV) nsP3 proteins were previously described.sup.41. The CHIKV nsP3 ΔHVD, ΔR1 to ΔR4 were generated by site-directed mutagenesis (QuickChange XL Site-Directed Mutagenesis Kit, Agilent)

[0112] The plasmids expressing the chimeric nsP3 CHIKV-HVD SINV and nsP3 SINV-HVD CHIKV were obtained as follows. First, the DNA sequence coding for the N-terminal parts of the CHIKV or SINV nsP3 (MD-AUD region) are obtained by PCR using the pCI-neo-3×FLAG expression plasmids as templates and the following sets of primers: 3×FLAG_NotI-F and Overlap-CHIKV-SINV-R, or 3×FLAG_NotI-F and Overlap-SINV-CHIKV-R for CHIKV and SINV constructs, respectively. HVD coding sequences were also generated by PCR using the following primers: Overlap-CHIKV-SINV-F and nsP3-SINV_BamHI-R for SINV HVD, and Overlap-SINV-CHIKV-F and nsP3-CHIKV_BamHI-R for CHIKV HVD. Next, the CHIKV-HVD-SINV and SINV-HVD-CHIKV PCR-fragments were obtained by overlap extension PCR using the previously obtained PCR-products and the following sets of primers: 3×FLAG_NotI-F and nsP3-SINV_BamHI-R or nsP3-CHIKV_BamHI-R. Finally, the chimeric PCR fragments were cloned into a NotI-BamHI digested pLVX-IRES-ZsGreenl vector (Takara).

[0113] The plasmid expressing FHL1A-R4 and FHL1A-R4* fusion proteins were obtained by overlap extension PCR approach as well. First, the FHL1A part which is common to both constructs was amplified from a cDNA template (Genscript, NM_001449.4). Second, nsP3-R4 and -R4* portions were obtained by PCR using either the pCI-neo-3×FLAG-nsP3 expression plasmid or the pCHIKV-SG45-R4* plasmid (containing the randomized R4 region) as templates. Next, the FHL1A-R4 and FHL1A-R4* PCR-fragments were obtained by PCR using the previously obtained PCR-products and the outer sets of primers: FHL1A Fwd and FHL1-fusion-Rev or FHL1-fusion-Rand-Rev. Amplification fragments were cloned into a NotI-EcoRI digested pLVX-IRES-ZsGreenl vector (Takara).

[0114] To obtain pCHIKV-M-Gluc a viral sequence encompassing the CHIKV 26S promoter and a part of the capsid protein sequence was amplified from pCHIKV-M, cut with PmeI and BssHII and assembled together with an AgeI-PmeI fragment from pCHIKVRepl-Gluc.sup.42 into an AgeI-BssHII cut vector. From the resulting plasmid the AgeI-BssHII fragment was released and ligated together with a BssHII-SfiI fragment from pCHIKV-M.sup.43 into pCHIKV-M cut with AgeI and Sfi.

[0115] To establish pCHKV-Rluc-GAA two PCR fragments were amplified from pCHIKV-WT using primers CHIKV 5590 F and Bo422 or Bo421 and CHIKV 8512 R, respectively. The obtained fragments were fused via PCR amplification using the outer primers CHIKV 5590 F and CHIKV 8512 R. The resulting fragment was cut with AgeI and BglI and inserted into pCHIKV-Rluc cut with the same restriction enzymes.

[0116] For generation of CHIKV-Rluc-ΔR4 and CHIKV-Rluc-R4* first PCR fragments encompassing the desired changes were amplified and assembled as follows: 1) CHIKV-Rluc-ΔR4: two fragments amplified from CHIKV-Rluc using Bo408 and Bo1259 or Bo1258 and Bo409, respectively, were fused together using the outer primers Bo408 and Bo409. 2) CHIKV-Rluc-R4*: the randomized sequence cassette was obtained sequentially from three successive PCRs: First PCR fragment was generated using primers Bo1260 and. Then, it was fused at the 5′ end with a PCR fragment amplified from CHIKV-Rluc with Bo408 and Bo1262. Next, the resulting fragment is further fused at the 3′ end with a PCR fragment amplified from CHIKV-Rluc with Bo1263 and Bo409, using the outer primers Bo408 and Bo409. Finally, the PCR fragments containing the ΔR4 and R4* mutations were cut with Sac and AgeI and fused in each case with a NgoMIV-SacII fragment derived from CHIKV-Rluc (SG45) and were cloned into a NgoMIV-AgeI digested SG45 plasmid.

[0117] Trans-complementation and over expression experiments. The lentiviral plasmids containing FHL1 isoforms were packaged as described above (see ‘CRISPR genetic screen’ section). Cells of interest were stably transduced by spinoculation (750×g for 2 hours at 32° C.) with these lentiviruses and, when necessary, sorted for GFP-positive cells by flow cytometry. For trans-complementation assays cells were inoculated with CHIKV21 for 48 hours. Cells were then collected and processed for E2 expression by flow cytometry. For ectopic expression, cells were plated on 24-well plates (5×10.sup.4) and incubated with CHIKV-M-GLuc and CHIKV21, and either processed for E2 expression by flow cytometry or infectious virus yield quantification on Vero E6 cells.

[0118] Kinetic of infection by qPCR assay. Control and ΔFHL1 HAP1 cells were plated on 60 mm dishes (400,000 cells) and inoculated with CHIKV21 (MOI of 5). At indicated time point cells were washed thrice with PBS, incubated with trypsin 0.25% for 5 min at 37° C. to remove cells surface bound particles, and total RNA was extracted using the RNeasy plus mini kit (Qiagen) according to manufacturer's instruction. cDNAs were generated from 500 ng total RNA by using the Maxima First Strand Synthesis Kit following manufacturer's instruction (Thermo Fisher Scientific). Amplification products were incubated with 1 Unit of RNAse H for 20 min at 37° C., followed by 10 min at 72° C. for enzyme inactivation, and diluted 10-fold in DNAse/RNAse free water. Real time quantitative PCR was performed using a Power Syber green PCR master Mix (Fisher Thermo Scientific) on a Light Cycler 480 (Roche). The primers used for qPCR were: E1-C21_F, E1-C21_R for viral RNA quantification, and Quantitect primers for GAPDH were purchased from Qiagen. The relative expression quantification was performed based on the comparative threshold cycle (C.sub.T) method, using GAPDH as endogenous reference control. CHIKV negative strand RNA was quantified as previously described”. Briefly, cDNA were generated from 1 μg total RNA using a primer containing a 5′ tag sequence CHIKV(−)Tag and the SuperScript II reverse transcriptase following the manufacturer's instruction (Thermo Fisher Scientific). Amplifications products were diluted 10-fold and used for real time quantitative PCR with the following primers CHIKV(−)fwd and CHIKV(−)rev. The 133 bp sequence corresponding to the amplified cDNA was synthesized by Genescript and serially diluted (650 to 6.5×10.sup.9 genes copies/μl) to generate standard curves.

[0119] Genomic viral RNA transfection and kinetic of viral amplification. To assess CHIKV RNA replication within the cells, we transfected control and ΔFHL1 cells with capped genomic viral RNA generated from pCHIKV-M-Gluc (see ‘Virus strains and culture’ section). Cells were plated on 48 well plate (3×10.sup.4 cells) and transfected with 100 ng of purified RNA using the Lipofectamine MessengerMax reagent according to the manufacturer's instruction (Thermo Fisher Science), and cells were cultured in absence or presence of 15 mM NH.sub.4Cl to prevent subsequent viral propagation. At specific times, cells were washed once with PBS and lyzed with Gaussia lysis buffer. Lysates were kept at −20° C. until all samples were collected. Luciferase activity was measured by using the Pierce Gaussia Luciferase Glow assay kit on a TriStar2 LB 942 with 20 μl of cell lysate, 20 μl of substrate and 2 s integration time.

[0120] The same experimental approach was used to monitor luciferase activity from capped genomic viral RNA generated from pCHIKV-Rluc WT (SG45), pCHIKV-Rluc-GAA, pCHIKV-Rluc-ΔR4 and pCHIKV-Rluc-R4* mutants. Luciferase activity was measured using the Renilla Luciferase assay system (Promega) on a TriStar2 LB 942 with 20 μl of cell lysate, 20 μl of substrate and 2.5 s integration time.

[0121] Immunoblot. Cell pellet were lysed in Pierce™ IP Lysis Buffer (Thermo Fisher Scientific) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fischer Scientific) for 30 min at 4° C. Equal amount of protein, determined by DC™Protein Assay (BioRad), were prepared in LDS Sample Buffer 4× (Pierce™) containing 25 mM dithiothreitol (DTT) and heated at 95° C. for 5 min. Samples were separated on Bolt™ 4-12% Bis-Tris gels in Bolt® MOPS SDS Running Buffer (Thermo Scientific), and proteins were transferred onto a PVDF membrane (BioRad) using the Power Blotter system (Thermo Fischer Scientific).

[0122] Membranes were blocked with PBS containing 0.1% Tween-20 and 5% non-fat dry milk and incubated overnight at 4° C. with primary antibody. Staining was revealed with corresponding horseradish peroxidase (HRP)-coupled secondary antibodies and developed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) following manufacturer's instructions. The signals were acquired through Fusion Fx camera (VILBERT Lourmat).

[0123] Co-immunoprecipitation assay. HEK-293T cells were plated in 10 cm dishes (5.Math.10.sup.6 cells/dish). Twenty-four hours later, the cells were transfected with a total of 15 μg of DNA expression plasmids (7.5 μg of each plasmid in co-transfection assays). Twenty-four hours post-transfection the cells washed once with PBS and collected with a cell scrapper. After 5 min centrifugation (400×g for 5 min), cells pellets were lysed for 30 min in cold IP lysis buffer supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail, and then cleared by centrifugation for 15 min at 6,000×g. Supernatants were incubated overnight at 4° C., with either anti-FLAG magnetic beads or HA magnetic beads (see ‘reagent’ section above). Beads were washed three times with B015 buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.5 mM EDTA, 0.05% Triton, 0.1% Tween-20). The retained complexes were eluted twice with either 3×FLAG-peptide (200 μg/ml; SIGMA F4799-4MG) or HA peptide (400 μg/ml; Roche #11666975001) for 30 min at room temperature. Samples were prepared and subjected to immunoblot as described above. For input, 1% of whole cell lysate were loaded on the gel.

[0124] Bacterial expression, purification and GST pull down assay. To express nsP3, nsP3ΔHVD as glutathione S-transferase fusion proteins, their respective open reading frame (orf) were subcloned into pGEX-4T-1. Similarly, FHL1A cDNA was subcloned into the pET47b (+) and expressed as a 6×His fusion protein. The following oligonucleotides were used to amplify nsP3 and nsP3ΔHVD cDNAs (sense: 5′-ccccggaattcATGgcaccgtcgtaccgggtaa-3′; antisense: 5′-ccgctcgagTCAtaactcgtcgtccgtgtctg-3′) and FHL1A (sense: 5′-ccggaattccATGgcggagaagtttgactgcc-3′; antisense: 5′-ccgctcgagTTAcagctttttggcacagtc-3′). E. Coli strain BL21 Star (Invitrogen) was transformed with recombinant expression vectors encoding GST-nsP3, GST-nsP3ΔHVD or 6×His-FHL1A recombinant proteins. Transformed bacteria were induced with isopropylthio-β-Dgalactoside (IPTG) for 3 hours at 37° C. Cells were collected by centrifugation and the pellets were resuspended in lysis buffer containing lysozyme (1 mg/mL), incubated 30 min at 4° C. followed by three subsequent freeze-thawed cycles and sonication. The bacterial lysates were centrifuged at 13,000 r.p.m for 20 min and the supernatants were incubated with glutathione-Sepharose beads for GST-nsP3 and GST-nsP3ΔHVD, or Ni-NTA column (Qiagen) for 6×His-FHL1A. Column washing and recombinant protein elution were performed according to the manufacturer's instructions. Five μL of eluted GST fusion proteins and 3 μL of Ni-NTA eluted 6×His-FHL1A were analyzed by SDS-PAGE and proteins were visualized by Coomassie staining. For pull-down assay, GST, GST-nsP3 or GST-nsP3ΔHVD bound beads were incubated with 6×His-FHL1A for 1 hour at 4° C. in presence of 100 μM ZnSO4. The resin was washed extensively with a buffer containing 500 mM KCL. The beads were then resuspended in Laemmli buffer, resolved on SDS-PAGE and the presence of 6×His-FHL1A was assessed by western blot using anti-FHL1 antibody.

[0125] Genetic analysis, fibroblasts and myoblasts from Emery-Dreifuss muscular dystrophy patients. Dermal fibroblasts and myoblasts were taken from 4 patients carrying FHL1 gene mutations. FHL1 gene was analyzed as previously reported.sup.6 as they had, among other symptoms, features reminiscent of Emery-Dreifuss muscular dystrophy. Patients P1, P2 and P3 were previously reported.sup.6 with detailed clinical description (respectively as patient F321-3, F997-8 and F1328-4) while patient P4 was not yet published. Briefly, patient P4 had myopathy with joint contractures, hypertrophic cardiomyopathy, vocal cords palsy, short stature, alopecia, skin abnormalities and facial dysmorphism. In this patient, FHL1 analysis revealed an insertion of a full-length LINE-1 retrotransposon sequence together with poly A tail of unknown length (i.e., ? thereafter) after 27 bp of the start of exon 4 (c.183_184ins [LINE1;?; 171_183]) that results at mRNA level in altered splicing with retention of 108 bp of the inserted LINE sequence leading to predicted premature termination codon and shorter FHL1A (Extended Data FIG. 7b).

[0126] Ethics statement. All materials (skin and/or muscle biopsies) from patients and controls included in this study were taken with the informed consent of the donors and with approval of the local ethical boards. All the procedures were followed alongside the usual molecular diagnostic procedure during patient follow-up, and in accordance with the ethical standards of the responsible national committee on human experimentation.

[0127] In vivo studies. Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (EC Directive 2010/63, French Law 2013-118, Feb. 6, 2013). All experiments were approved by the Ethics Committee #89 (and registered under the reference APAFIS #6954-2016091410257906 v2). Male mice either deficient for FHL1 (FHL1-null) or not (WT littermates) were obtained by crossing heterozygous females for FHL1.sup.4 with WT male Black Swiss mice. Nine day-old male littermates, both FHL1-null and WT mice, were injected with CHIKV21 (10.sup.5 PFU/20 μl) by intradermal route and viral load was determined in tissues by day 7 post infection. Virus titers in tissue samples were determined on Vero E6 cells by tissue cytopathic infectious dose 50 (TCID50/g). For histology experiments, muscles were snap frozen in isopentane cooled by liquid nitrogen for cryo-sectioning then processed for histological staining (hematoxylin and eosin) or immunolabelling.

[0128] Transmission electron microscopy. Cells were scrapped and fixed for 24 h in 1% glutaraldehyde, 4% paraformaldehyde, (Sigma, St-Louis, Mo.) in 0.1 M phosphate buffer (pH 7.2). Samples were then washed in phosphate-buffered saline (PBS) and post-fixed for 1 h by incubation with 2% osmium tetroxide (Agar Scientific, Stansted, UK). Cells were then fully dehydrated in a graded series of ethanol solutions and propylene oxide. Impregnation step was performed with a mixture of (1:1) propylene oxide/Epon resin (Sigma) and then left overnight in pure resin. Samples were then embedded in Epon resin (Sigma), which was allowed to polymerize for 48 hours at 60° C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Sections were stained with 2% uranyl acetate (Agar Scientific), 5% lead citrate (Sigma) and observations were made with a transmission electron microscope (JEOL 1011, Tokyo, Japan).

[0129] Cell viability assay. Cell viability and proliferation were assessed using the CellTiter-Glo 2.0 Assay (Promega) according to the manufacturer's protocol. In brief, cells were plated in 48-well plates (3×10.sup.4). At specific times, 100 μl of CellTiter-Glo reagent were added to each well. After 10 min incubation, 200 μl from each well were transferred to an opaque 96-well plate (Cellstar, Greiner bio-one) and luminescence was measured on a TriStar2 LB 942 (Berthold) with 0.1 second integration time.

[0130] Statistical analysis. Graphical representation and statistical analyses were performed using Prism7 software (GraphPad Software). Unless otherwise stated, results are shown as means+/−standard deviation (SD) from at least 2 independent experiments in duplicates. Differences were tested for statistical significance using the unpaired two-tailed t test, One-way or Two-way Anova with multiple comparison post-test.

[0131] Results:

[0132] Several host factors implicated in CHIKV infection have been identified, however none of them accounts for CHIKV tropism for joint and muscle tissues.sup.7-10. To identify key host factors dictating CHIKV cell permissiveness, we performed a genome-wide CRISPR-Cas9 screen in the HAP1 haploid cell line (data not shown). HAP1 cells expressing the human GeCKO v2 single guide RNA libraries A and B, which contains each 3 unique sgRNAs targeting 19,050 genes.sup.11, were inoculated with CHIKV21, a strain isolated from a patient infected during the 2005-2006 CHIKV outbreak in La Reunion Island.sup.12. Genomic DNA from lentivirus-transduced cells that survived to CHIKV infection was isolated, amplified and the corresponding integrated sgRNA sequenced. Gene enrichment was assessed using the MAGeCK software.sup.13 (data not shown). The top hit of our screen was the gene encoding the Four-and-a-Half LIM protein 1 (FHL1) (data not shown), the founding member of the FHL protein family.sup.14. FHL1 is characterized by the presence of four and a half highly conserved LIM domains with two zinc fingers arranged in tandem.sup.14. FHL1 is strongly expressed in skeletal muscles and heart.sup.414. In human, there are three FHL1 splice variants: FHL1A, FHL1B and FHLIC.sup.4,15,16. FHL1A is the most abundantly expressed, primarily detected in striated muscles.sup.4 and fibroblasts.sup.17. The two other variants FHL1B and C are expressed in muscles, brain and testis.sup.15,16. We functionally validated the requirement of FHL1 in CHIKV21 infection by using two distinct gRNAs targeting all three FHL1 isoforms (data not shown). We generated HAP1 and 293T knockout FHL1 clones (ΔFHL1) and confirmed gene editing by sequencing and western blot analysis (data not shown). FHL1 knockout did not alter cell proliferation and viability as determined by CellTiter-Glo assay (data not shown). CHIKV infection and release of infectious particles was drastically inhibited in ΔFHL1 cells (data not shown). Trans-complementation of ΔFHL1 cells with a human cDNA encoding FHL1A, but not FHL1B or C, restored both susceptibility to CHIKV21 infection and virus release (data not shown), indicating that FHL1A is a critical factor for CHIKV21 infection. Expression of FHL2, a member of the FHL family predominantly expressed in heart.sup.18, restored CHIKV infection in ΔFHL1 cells, albeit to a lower efficiency than FHL1 (data not shown). We then assessed FHL1 dependency of CHIKV strains from distinct genotypes. FHL1 is important for infection by strains belonging to the Asian (strain St Martin H20235 2013), the ECSA (East, Central, and South African) strains Ross and Brazza (MRS1 2011) and the Indian Ocean (IOL) (strain M-899) lineages (data not shown). Of note, the requirement for FHL1 was less pronounced with CHIKV 37997, a strain from the West African genotype (data not shown). We next tested the requirement of FHL1 for infection by other alphaviruses. Interestingly, O'nyong-nyong virus (ONNV), an Old World alphavirus that is phylogenetically very close to CHIKV.sup.1, showed a dramatically reduced infection level in ΔFHL1 cells (data not shown). In sharp contrast, other Old World alphaviruses such as Mayaro virus (MAYV), Sindbis virus (SINV), Semliki Forest Virus (SFV) and Ross River virus (RRV), and New World encephalitic viruses such as Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV) or Venezuelan equine encephalitis virus (VEEV) infected HAP1 cells in a FHL1-independent manner (data not shown). No effect of FHL1 was observed for infection by Dengue virus (DENV) or Zika virus (ZIKV), two members of the Flavivirus genus (data not shown). Consistent with the requirement of FHL1 for CHIKV infection, BeWo or HepG2 cells which are poorly susceptible to CHIKV infection.sup.20,21 and do not express endogenous FHL1 (data not shown) became permissive to the virus upon FHL1A expression (data not shown). This highlights the major role played by FHL1A in human cell permissiveness to CHIKV.

[0133] To determine which step in CHIKV life cycle requires FHL1, we challenged parental and ΔFHL1 cells with CHIKV particles and quantified the viral RNA at different time points (data not shown). We did not observe any major difference in CHIKV RNA levels in FHL1-deficient cells compared to WT cells at 2 h post-infection (data not shown). In contrast, a massive reduction of CHIKV RNA was observed in ΔFHL1 cells as early as 6 h post-infection (data not shown) which was even greater 24 h post-infection, suggesting that FHL1 expression is involved in an early post-entry step of the CHIKV life cycle. We therefore bypassed virus entry and uncoating by transfecting CHIKV RNA into controls or ΔFHL1 cells in the presence of NH.sub.4Cl to inhibit further rounds of infection.sup.9. Upon CHIKV RNA transfection, viral replication was drastically impaired in ΔFHL1 cells compared to WT cells (data not shown). To evaluate the contribution of FHL1 in incoming genome translation versus RNA replication, we generated a replication-deficient CHIKV molecular clone (with the GDD motif of the viral polymerase nsP4 mutated to GAA) encoding a Renilla luciferase (Rluc) fused to the nsP3 protein as described.sup.22. Transfection of CHIKV GAA RNA in ΔFHL1 or control cells resulted in a similar Rluc activity (data not shown), indicating that FHL1 is dispensable for CHIKV incoming RNA translation. When similar experiments were performed with the WT CHIKV RNA, a massive increase in Rluc activity was observed in control cells but not ΔFHL1 24 hpi (data not shown), demonstrating that FHL1 is essential for viral RNA replication. Furthermore, qRT-PCR experiments showed that ablation of FHL1 resulted in a severely reduced synthesis of CHIKV negative strand RNA (data not shown). We then investigated the impact of FHL1 in the production of dsRNA intermediates which are a marker of viral replication complex (vRC) assembly.sup.23. At 6 h post-infection, a massive reduction of dsRNA-containing complexes was observed in ΔFHL1 cells stained with anti-dsRNA mAb when compared to parental cells (data not shown). Consistent with this observation, transmission electron microscopy showed that the formation of plasma membrane-associated spherules and cytoplasmic vacuolar membrane structures, which are alphavirus-induced platforms required for viral RNA synthesis.sup.24, are absent in ΔFHL1 cells (data not shown). Altogether, these data show that FHL1 is critical for CHIKV RNA replication and vRC formation in infected cells.

[0134] We next investigated FHL1 location during infection. Confocal microscopy studies showed that FHL1 displays a diffuse cytoplasmic distribution in uninfected human fibroblasts. In cells infected for 6 h, FHL1-containing foci appeared and colocalized with nsP3 (data not shown), a CHIKV non-structural protein orchestrating viral replication in the cytoplasm.sup.25,26. Indeed, CHIKV nsP3 contains a large C-terminal hypervariable domain (HVD).sup.26 known to mediate assembly of protein complexes and regulate RNA amplification.sup.2526. Interestingly, FHL1 and FHL2 have been reported as putative nsP3 HVD binding partners in mass spectrometry analyses.sup.26,27. We experimentally validated FHL1-nsP3 interaction (data not shown) and found that endogenous FHL1 co-immunoprecipitates with nsP3 from CHIKV-infected cells (data not shown). Consistent with infection studies, both FHL1A and FHL2 co-precipitated with CHIKV nsP3 (data not shown). FHL1A-nsP3 interaction is specific for CHIKV as it was not observed with other alphaviruses such as SINV or SFV, which do not depend on FHL1 for infection (data not shown). Of note, in ΔFHL1 cells, nsP3 retained its ability to bind G3BP1 and 2, two components of the stress granules implicated in CHIKV replication.sup.22,26 (data not shown). We next generated chimeric proteins where the HVD region of CHIKV nsP3 is swapped with the corresponding domain of SINV nsP3 and vice versa. Whereas CHIKV-SINV(HVD) chimeric protein lost its ability to bind FHL1, the HVD of CHIKV in the context of SINV nsP3 protein conferred binding to FHL1 (data not shown). Pull-down experiments with purified proteins showed that FHL1A directly binds to WT nsP3 but not to the HVD-deficient variant (data not shown). We then mapped the binding region within CHIKV nsP3HVD responsible for FHL1A interaction (data not shown). The FHL1 binding domain, referred as HVD-R4, is found in all CHIKV and ONNV strains and is located upstream of the short repeating peptide corresponding to G3BP1/2 binding sites.sup.6 (data not shown). Deletion of the HVD-R4 region strongly impaired FHL1 interaction with nsP3, without affecting G3BP1/2 binding to the viral protein (data not shown). To investigate whether FHL1 interaction with the HVD region of nsP3 is required for FHL1 proviral role, we generated two chimeric FHL1A protein either fused to the HVD-R4 peptide (FHL1A-R4) or to a randomized peptide sequence of HVD-R4 (FHL1A-R4*) as a positive control (data not shown) and assessed their ability to interact with nsP3. Whereas FHL1A-R4 failed to bind nsP3 (data not shown), FHL1A-R4* interacted with nsP3 as efficiently as WT FHL1A protein (data not shown). These results indicate that the fused HVD-R4 peptide likely hides the binding site of FHL1A to nsP3, inhibiting their interaction. Furthermore, trans-complementation of ΔFHL1 cells with a cDNA encoding FHL1A-R4 did not restore CHIKV21 infection when compared to FHL1A-R4* or WT FHL1A (data not shown). Consistent with this, in vitro transcribed RNA from CHIKV molecular clone mutated in FHL1 binding site (ΔR4 or R4*) showed a strong defect in replication after transfection in 293T cells (data not shown). Together these data strongly suggest that the interaction between the HVD region of nsP3 with FHL1 is critical for FHL1 proviral function.

[0135] Mutations in the FHL1 gene have been associated with X-linked myopathies.sup.5,28, including the Emery-Dreifuss muscular dystrophy (EDMD).sup.6, a rare genetic disease characterized by early joint contractures, muscular wasting and adult-onset cardiac disease.sup.29. We studied the permissiveness to CHIKV of dermal fibroblasts and myoblasts from four EDMD male patients carrying FHL1 gene mutations as well as from two healthy donors (data not shown). A detailed clinical description of P1, P2 and P3 has been reported.sup.6, and patient P4 presented with EDMD and additional clinical abnormalities (see methods). Analysis of P4 FHL1 gene revealed the insertion of a full-length LINE-1 retrotransposon sequence in exon 4 (data not shown). FHL1 expression is severely reduced in primary cells from all four EDMD patients as established by immunoblot analysis (data not shown). Infection studies showed that fibroblasts and myoblasts from those EDMD patients are resistant to CHIKV21 and M-899 Mauritian strains (data not shown), and exhibit a massive defect in the release of infectious particles (data not shown), in contrast to healthy donor cells. Similar results were obtained with the CHIKV strains Brazza, Ross and H20235 (data not shown). FHL1-null myoblasts and fibroblasts remained highly susceptible to MAYV, which does not rely on FHL1 for replication (data not shown). Trans-complementation of EDMD fibroblasts by a lentivirus encoding WT FHL1A restored CHIKV viral antigen synthesis (data not shown) and infectious particle release (data not shown).

[0136] To directly assess the role of FHL1 in chikungunya pathogenesis, we conducted in vivo experiments in mice expressing or not FHL1. Human and mouse FHL1 orthologues are highly conserved (data not shown). Murine FHL1 interacts with CHIKV nsP3 and enhances viral infection, albeit less efficiently that its human orthologue (data not shown). Moreover, CHIKV infection was strongly impaired in the murine muscle cell C2C12 deleted for the fhl1 gene (data not shown). Susceptibility to CHIKV infection of young mice deficient or not for FHL1 was then tested. CHIKV actively replicated in tissues of WT littermates, as previously reported.sup.20, but virtually no infectious particles were detected in tissues of FHL1-null mice (FIG. 5). Moreover, necrotizing myositis with massive infiltrates and necrosis of the muscle fibers were observed in skeletal muscle of WT littermates, while FHL-null mouse muscle showed no detectable pathology (data not shown). Immunolabelling with Ab against CHIKV E2 protein, FHL1 and vimentin in muscle revealed that in young WT mice, CHIKV mainly targets muscle fiber expressing FHL1, whereas muscle cells of FHL1-null mice show no label for CHIKV nor for FHL1 (data not shown). These experiments demonstrate that FHL1 knock out mice are resistant to CHIKV infection.

[0137] In summary, this study shows that FHL1 is a critical CHIKV host dependency factor for infection and pathogenesis. In vivo, FHL1 expression pattern, which accounts for the clinical presentation of EDMD, also reflects CHIKV tissue tropism for skeletal muscles and joints. This suggests that the hijacking of FHL1 by CHIKV during infection may, on top of allowing viral replication, lead to cellular dysfunctions contributing to muscular and joint pains that are the hallmark of chikungunya disease.sup.12. Mechanistically, FHL1 interacts with the HVD domain of nsP3 to enable viral RNA synthesis and viral replication complex formation. The alphavirus nsP3 HVD domain is an intrinsically disordered region that binds distinct sets of cellular proteins.sup.23,26,30 such as the G3BP1 and G3PB2, two key components and markers of stress granules that are important for the replication of CHIKV and other alphaviruses.sup.22,26. G3BP1/2 nsP3 interactions are thought drive a common alphavirus-specific mechanism that is important for assembly of the replication complex and stabilization of viral G RNA.sup.22,23,26. FHL1 interacts with a nsP3 HVD region which is located away from G3BP1/2 binding sites. Therefore, FHL1 and G3BP proteins likely play distinct roles during CHIKV replication. In contrast to G3BPs, FHL1 is selectively used by CHIKV, suggesting that it may accomplish a specific and essential function in CHIKV RNA amplification. Upon interaction with FHL1, CHIKV nsP3 HVD may adopt a unique conformation that is critical for the initiation of viral replication. Interestingly, intrinsically disordered domains (IDD) such as the nsP3 HVD have also been shown to induce liquid-liquid phase separations.sup.31 and negative-stranded RNA viruses use proteins displaying IDDs to form liquid organelles for their replication.sup.32. Indeed, in CHIK-infected cells, nsP3 forms intracellular granules reminiscent of these virus-induced inclusions.sup.33,34 FHL1 may regulate the formation and/or the dynamic of such granules to create an optimal environment for efficient CHIKV RNA amplification. FHL1 contains four LIM domains arranged in tandem known to function as a modular protein binding interface regulating diverse cellular pathways.sup.35. FHL1 has been shown to scaffold MAPK components (Raf-1/MEK2/ERK2) to the stretch sensor Titin N2B to transmit MAPK signals that regulate muscle compliance and cardiac hypertrophy.sup.36,37. One may speculate that, during CHIKV infection, FHL1 may be hijacked from its physiological function in sarcomere extensibility and intracellular signaling to act as scaffolding protein promoting CHIKV RNA amplification.

[0138] In conclusion, this study provides major insights into the understanding of CHIKV interactions with its target host cell. Although other host-factors have been identified as required for CHIKV infection, none of them fully account for the specific joint and muscular pathology which is the hallmark of CHIKV and gave its name to its associated disease, chikungunya, which means “that which bends up” in Makonde, to describe the posture of patient with muscle and joint pain. The hijacking of FHL1 by nsP3 during CHIKV infection is unique and constitutes a critical clue that paves the way to fully decipher the pathogenesis of chikungunya disease. Targeting FHL1A-nsP3 interactions now stands as an attractive therapeutic approach to combat CHIKV pathogenesis.

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