WO2008062309A2 - Molécules anti-coronavirus et leur utilisation dans des compositions et procédés pour traiter et/ou prévenir une infection provoquée par un coronavirus - Google Patents

Molécules anti-coronavirus et leur utilisation dans des compositions et procédés pour traiter et/ou prévenir une infection provoquée par un coronavirus Download PDF

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WO2008062309A2
WO2008062309A2 PCT/IB2007/004174 IB2007004174W WO2008062309A2 WO 2008062309 A2 WO2008062309 A2 WO 2008062309A2 IB 2007004174 W IB2007004174 W IB 2007004174W WO 2008062309 A2 WO2008062309 A2 WO 2008062309A2
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protein
coronavirus
cellular
sars
fragment
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WO2008062309A3 (fr
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Béatrice Thérèse Marie NAL-ROGIER
Ralf Marius Altmeyer
François KIEN
Jean Millet
Kim Tat Teoh
Yu Lam Siu
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Hong-Kong Pasteur Research Centre
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to the field of coronavirus and more particularly to anti-coronavirus molecules and their use in compositions and methods for treating and/or preventing infection caused by a coronavirus such as the one responsible for
  • the present invention also relates to a method for screening new anti- coronavirus molecules.
  • Coronaviruses are found in a wide variety of animals and can cause disorders of diverse severity. Considering the number of recently discovered human and animal coronaviruses, including the highly pathogenic virus responsible of the SARS syndrome (SARS-CoV), research on novel antiviral strategies against coronaviruses is a priority (Peiris et al., 2003; Rota et al., 2003; Woo et al., 2005; Woo et al., 2006). Understanding mechanisms elaborated by viruses to hijack or exploit cellular pathways is a clue to identify novel cellular targets (Marsh and Helenius, 2006).
  • SARS-CoV highly pathogenic virus responsible of the SARS syndrome
  • coronavirus infection There is currently no vaccine or efficient anti-viral treatment available against human coronavirus infection. Coronaviruses, including SARS-CoV-like viruses, are present in animal reservoirs, particularly in bats. As happened in 2002 with the emergence of SARS, one cannot exclude that these bat precursor viruses will readapt to human transmission at some unpredictable future date. The causes for the severity of illness in SARS-CoV infected patients are not yet clearly understood.
  • the SARS-CoV mainly targets the epithelial cells, the respiratory tract being the primary site of infection.
  • DAD diffuse alveolar damage
  • One of the major pathological features of SARS-CoV infection is diffuse alveolar damage (DAD) of the human lung, more prominent in the terminal stage, with sometimes, abrupt deterioration of the lung epithelium.
  • DAD diffuse alveolar damage
  • the present invention provides a method of screening for anti-coronavirus molecules comprising the steps of:
  • the present invention also relates to an anti-coronavirus composition
  • an anti-coronavirus composition comprising at least one purified cellular interacting protein or a fragment thereof and/or an antiviral molecule obtained by the above-mentioned screening method.
  • the present invention further relates to a composition for the treatment and/or prevention of infection caused by a coronavirus, the composition comprising:
  • the present invention is also concerned with the use of at least one coronavirus protein and/or at least one cellular interacting protein or cellular interacting protein contemplated by the present invention, to reduce or inhibit an infection of human cells caused by a coronavirus.
  • the present invention is further concerned with the use of at least one coronavirus protein and/or at least one cellular interacting protein or cellular interacting protein contemplated by the present invention, for the treatment of infection caused by a coronavirus.
  • the present invention is also concerned with the use of at least one coronavirus protein and/or at least one cellular interacting protein or cellular interacting protein contemplated by the present invention, for the preparation of a composition in the treatment of of infection caused by a coronavirus.
  • the present invention also provided a method for treating an infection caused by a coronavirus, comprising the step of administrating an anti-coronavirus composition as defined above and/or a composition as defined above.
  • the present invention further provides a method for identifying inhibitors of early stages of SARS-CoV replication cycle by using lentiviral Spike pseudothyped particles.
  • Figure 1 shows an amino acid sequence (SEQ ID NO: 1) of an example of a contemplated coronavirus protein, namely the SARS-CoV Spike protein, and more particularly, its Spike cytosolic tail.
  • Figure 2 shows an amino acid sequence (SEQ ID NO: 2) of an example of a contemplated cellular interacting protein, namely a protein of the ERM family: ezrin.
  • Figure 3 shows an amino acid sequence (SEQ ID NO: 3) of an example of a contemplated cellular interacting protein, namely the E-codherive.
  • Figure 4 shows an amino acid sequence (SEQ ID NO: 4) of an example of a contemplated cellular interacting protein, namely the alpha 5 integrin.
  • Figure 5 shows an amino acid sequence (SEQ ID NO: 5) of an example of a contemplated coronavirus protein, namely the SARS-CoV E protein, and more particularly, its E cytosolic tail.
  • Figure 6 shows an amino acid sequence (SEQ ID NO: 6) of an example of a contemplated cellular interacting protein, namely a PDZ domain containing cellular interacting protein: PALSI (MPPS).
  • Figure 7 shows an amino acid sequence (SEQ ID NO: 7) of an example of a contemplated coronavirus protein, namely the SARS-CoV M protein, and more particularly, its M cytosolic tail.
  • Figure 8 shows an amino acid sequence (SEQ ID NO: 8) of an example of a contemplated cellular interacting protein, namely the cellular DIP/SPIN90 protein.
  • Figure 9 shows the Spike-ezrin interaction in vitro by GST-PuII Down co- precipitation.
  • the GST-S cterm fusion protein co-precipitates ezrin from HeLa and Vero-E6 cell lysates.
  • FIG. 11 Ezrin is relocalized to SARS-CoV budding compartment where S acumulates. Confocal immunofluorescence microscopy experiment on SARS-CoV infected cells.
  • Figure 12 shows a decrease in intracellular ezrin levels dramatically increases entry of SARS-Spike pseudotyped particles.
  • Figure 13 shows a decrease in ezrin expression levels enhances entry of SARS- Spike pseudotyped particles, a decrease in radixin expression levels restricts entry.
  • FIG. 14A PALS1 PDZ domain is sufficient to precipitate SARS-CoV E protein from cell lysates.
  • GST pull down assay Cell lysate from SARS-CoV E-transfected VeroE ⁇ cells were incubated with glutathione-sepharose beads pre-bound to glutathione serine-transferase (GST)-PALSI fusion proteins. Beads only and beads bound to GST were used as negative controls. 0.5 and 1 ⁇ g of each GST-PASL1 fusion proteins have been used for the pull down assay and are illustrated by a triangle.
  • Figure 14B Schematic representation of PALS1 sequences and domains.
  • FIG. 15 PALS1 is recruited to E-rich sub-cellular areas in E-transfected and SARS-CoV infected VeroE ⁇ cells.
  • Panel A VeroE ⁇ cells were transfected with pCDNA-E and pCDNA-PALS1-EYFP (gift from Dr Margolis) constructs and fixed at 20 hours posttransfection.
  • Panels B to D SARS-CoV infected VeroE ⁇ cells were fixed at 24 hours postinfection.
  • E SARS-CoV E protein
  • ERGIC Endoplasmic reticulum-Golgi intermediary compartment
  • S:SARS-CoV Spike protein Shows aberrant localization of PALS1 at a perinuclear site that colocalises with SARS-CoV structural viral proteins; shows normal PALS1 distribution profile at cell-cell junctions.
  • FIG. 16 GFP-Vpr is incorporated into SCoV Spike pseudotyped HIV particles (SARS pp).
  • FIG. 17 GFP-Vpr is incorporated into HIV particles. Supernatant was collected after transfection of 293T cells with GFP-Vpr, optimized Spike and pNL E- R- Luc plasmids, filtered through a 0.45 ⁇ m membrane and applied to a 20% sucrose cushion for concentration. Pseudotyped paricles 1Ox concentrated were exposed to clean coverslips in the presence of 8 ⁇ g/mL Polybrene. Coverslips were rinsed, fixed and immunostained with a mAb specific to p24. Middle panel is the merged images so that overlapping red and green signals appear yellow. Left and right panels show the individual fluorescent images. White arrows denote double labelled particle; the pink arrows show single labeled (GFP-negative) particles.
  • Figure 18 GFP-Vpr is incorporated into SARSpp.
  • White arrows denote double labelled particle; the pink arrows show single labeled (GFP-negative but Spike- positive, SARSpp Luc) particles, the green arrows show single labeled (GFP-positive but Spike negative, ⁇ envpp GFP-Vpr Luc) particles.
  • FIG. 19 Anti-Spike serum neutralize entry of SARSpp GFP-Vpr Luc into VeroE ⁇ cells.
  • Supernatant was collected after transfection of 293T cells with GFP-Vpr, optimized Spike and pNL E- R- Luc plasmids, filtered through a 0.45 ⁇ m membrane and applied to a 20% sucrose cushion for concentration. Pseudotyped particles 10x concentrated were incubated with anti-Spike mouse serum (1/1000) for 1 hr at 37 0 C then applied on VeroE6 targeting cells. Transduction was measured as SARSpp-expressed luciferase activity 64 hr post-infection. All experiments were performed in triplicates and data are presented as average ⁇ standard deviation.
  • FIG. 20 After SCoV Spike-mediated entry, GFP-Vpr accumulates in the perinuclear region, often at the MTOC. Supernatant was collected after transfection of 293T cells with GFP-Vpr, optimized Spike and pNL E- R- Luc plasmids, filtered through a 0.45 ⁇ m membrane and applied to a 20% sucrose cushion for concentration. Pseudotyped paricles 1Ox concentrated were applied on coverslips seeded with VeroE ⁇ targeting cells for 30 (left panel) or 150 min (right panel) at 37 0 C, rinsed and fixed in PFA then stained with DAPI. GFP fluoresecence is shown in green. BRIEF DESCRIPTION OF THE INVENTION
  • the present invention relates to the identification and/or use of anti- coronavirus molecules or agents.
  • the invention relates to the identification and/or use of anti-coronavirus molecules that inhibit or disrupt viral protein- cellular protein interactions that are important or essential in the viral life cycle of a coronavirus.
  • coronavirus protein refers to any protein, peptide or polypeptide of coronavirus origin that binds to an eukaryotic cells in such a way that the binding interaction is important or essential in the viral life cycle of a coronavirus.
  • peptide and “polypeptide” are used herein interchangeably. They refer to sequences of more than three amino acids. Preferably, peptides are sequences of less than about 250 amino acids, particularly, less than 100, 75, 50, and 30 amino acids. For instance, contemplated peptides of the invention contain in the range of about 5-50, 7-30, or 15-25 amino acids.
  • amino acid refers to a monomeric unit of a protein.
  • amino acids found in naturally occurring proteins, all of which are L-isomers.
  • amino acid may also include analogs of the amino acids, D- isomers of the protein amino acids and their analogs.
  • isolated when applied to interacting and specificity peptides of the present invention means a peptide or a portion thereof, which, by virtue of its origin or manipulation, (a) is present in a host cell as the expression product of a portion of an expression vector; (b) is linked to a protein or chemical moiety other than that to which it is linked in nature; (c) does not occur in nature, or (d) is such that its manufacture or production involved the hand of man.
  • isolated it is, alternatively or additionally, meant that the peptide of interest is chemically synthesized; or expressed in a host cell and purified away from at least some other proteins.
  • the peptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) which are used to purify it.
  • inhibitor and “inhibitory agent” are used herein interchangeably.
  • inhibitory agents of the invention inhibit the life cycle of a coronavirus responsible of SARS.
  • small molecule refers to any natural or synthetic organic compound or factor with a molecular weight, for instance, less than about 600-700 Daltons.
  • coronavirus disease refers to any kind of infection or disorder caused by a coronavirus.
  • the term includes symptoms and side effects of any coronavirus infection, including latent, persistent and sub-clinical infections, whether or not the infection is clinically apparent.
  • the term "treatment” refers to administration of an anti-viral agent to a patient (who is either the host of a coronavirus infection or may be at risk of being infected by a coronavirus). If it is administered prior to exposure to the virus, the treatment is preventive or prophylactic (i.e., it protects the patient against infection), whereas if the administration is performed after infection or initiation of the disease, the treatment is therapeutic (i.e., it combats the existing infection).
  • composition as used herein, is defined as comprising an effective amount of the active element, such as the antiviral molecule obtained by the method of the present invention, or a physiologically tolerable salt thereof, and at least one pharmaceutically acceptable carrier.
  • the term "effective amount” refers to any amount of an inhibitory molecule, or pharmaceutical composition thereof, that is sufficient to fulfill its intended purpose(s).
  • the purpose(s) may be: to protect against infection by a coronavirus; to combat a coronavirus; to prevent the onset of a disease caused by the coronavirus; to slow down or stop the progression, aggravation, or deterioration of the symptoms of a coronavirus disease; to bring about amelioration of the symptoms of the disease; or to cure the disease.
  • cellular interacting protein refers to a human or an animal protein or a fragment of a said protein wich interacts with at least a SARS- CoV viral protein among as S, M or E protein C-terminal domains.
  • the interaction can be obtained by using as an example the Yeast Two-Hybrid (Y2H) assay.
  • a method of screening for anti-coronavirus molecules comprising the steps of:
  • any kind of compounds, small molecules or factors can be tested using the inventive screening method.
  • the method of the invention may also be used for screening collections or libraries. Any collection or library of molecules can be screened using the method provided herein.
  • selection refers to any set of compounds, molecules, or agents, while the term “library” refers to any set of structural analogs of compounds, molecules or agents.
  • binding interaction between the coronavirus protein and said cellular interacting protein concerns a binding interaction that is important in the life cycle of a coronavirus, such as a coronavirus responsible of Severe Acute Respiratory Syndrome Coronavirus (SARS-Co-V).
  • a coronavirus responsible of Severe Acute Respiratory Syndrome Coronavirus (SARS-Co-V).
  • the coronavirus protein may be the SARS-CoV Spike protein and a fragment of the coronavirus protein, which the latter may be the Spike cytosolic tail.
  • the Spike cytosolic tail may have for instance the amino acid sequence of SEQ ID NO: 1.
  • amino acid sequences of the Spike cytosolic tail that can mediate binding to cellular interacting proteins have been further defined by GST pull down experiments using truncated forms of the Spike cytosolic tail fused to GST (see Example 2).
  • the contemplated cellular interacting protein or a fragment thereof by the present invention may be obtained for instance from a cell lysate.
  • the contemplated cellular interacting protein or a fragment thereof may be a purified cellular interacting protein or a fragment thereof.
  • the coronavirus protein is the SARS-CoV Spike protein or a fragment thereof
  • the cellular interacting protein contemplated by the present invention may be of the ERM family, such as ezrin, radixine and moesin.
  • such cellular interacting protein may comprise a FERM domain, such as a sub-domain selected from the group consisting of F1 , F2 and F3 sub- domains.
  • a contemplated cellular interacting protein may be an ezrin protein that has for instance the amino acid sequence of SEQ ID NO: 2. This amino acid sequence of ezrin corresponds to ezrin F1 sub-domain of the FERM domain.
  • the cellular interacting protein may alternatively be selected from the group consisting of E-cadherine and alpha 5 integrin.
  • the E-cadherin has for instance the amino acid sequence of SEQ ID NO: 3
  • the alpha ⁇ integrin has for instance the amino acid sequence of SEQ ID NO: 4.
  • coronavirus protein contemplated by the present invention may be the SARS-CoV E protein or a fragment thereof.
  • the fragment of the coronavirus may be, for instance, the E cytosolic tail (E CT) which may have the amino acid sequence of SEQ ID NO: 5.
  • E CT E cytosolic tail
  • the cellular interacting protein may consist of a PD2 domain containing cellular interacting protein.
  • the PDZ domain containing cellular interacting protein may be the PALS1 (MPP5) protein or fragment thereof.
  • the PALS1 protein may, for instance, comprises the amino acid sequence of SEQ ID NO: 6.
  • coronavirus protein may be the SARS-CoV M protein or a fragment thereof. More specifically, the fragment of the coronavirus may be the M cytosolic tail (M CT) which may have the amino acid sequence of SEQ ID NO: 7.
  • M CT M cytosolic tail
  • cellular interacting protein of the invention may be the cellular DIP/SPIN90 protein or fragment thereof which may have the amino acid sequence of SEQ ID NO: 8.
  • the present invention provides a method for identifying inhibitors of early stages of SARS-CoV replication cycle by using lentiviral Spike pseudotyped particles. This method may be used following the identification of
  • SARS-CoV Spike binding inhibitors with their corresponding cellular interacting proteins as exemplified in Example 1.
  • Another aspect of the invention concerns an anti-coronavirus composition
  • an anti-coronavirus composition comprising at least one purified cellular protein or a fragment thereof and/or an antiviral molecule obtained by the screening method of the invention.
  • a further aspect of the invention concerns a composition for the treatment of an infection caused by a coronavirus.
  • the composition comprises at least one coronavirus protein or fragments thereof, and at least one cellular interacting protein or fragments thereof as defined previously.
  • contemplated coronavirus proteins and/or cellular interacting proteins as defined above are useful to reduce or inhibit an infection of human cells caused by a coronavirus. Furthermore, the contemplated coronavirus proteins and/or cellular interacting proteins as defined above are usefull for the treatment of infection caused by a coronavirus.
  • coronavirus proteins and/or cellular interacting proteins are particularly useful in the preparation of a composition in the treatment of an infection caused by a coronavirus.
  • a further aspect of the invention is to provide a method for treating an infection caused by a coronavirus.
  • the method comprises the step of administering to an individual an effective amount of one of the compositions of the invention.
  • compositions contemplated by the present invention may further comprise the compositions of the invention may be given to an individual through various routes of administration.
  • the compositions may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub- cutaneously by injection, by infusion or per os.
  • Suitable dosages will vary, depending upon factors such as the amount of each of the components in the compositions, the desired effect (short or long term), the route of administration, the age and the weight of the subject to be treated. Any other methods well known in the art may be used for administering the composition of the invention.
  • yeast two-hybrid screening strategy (Hybrigenics company, Paris)
  • the inventors have identified cellular factors that interact with SARS-CoV Spike, E and M envelope protein CT.
  • the inventors identified ezrin (ERM protein) and PALS1 (PDZ domain-containing protein) proteins with significantly high confidence scores for interaction with the CT domains of Spike and E 1 respectively.
  • Both ezrin and PALS1 i/ belong to families of scaffolding proteins specialized in the functional organization of specific plasma-membrane domains by providing a link between filamentous actin and membrane proteins; ii/ are involved in signal transduction and iii/ have been found functionally linked to the regulation of polarized epithelial secretion in gastric parietal cells (Cao et al., 2005; Gautreau et al., 2000; Roumier et al., 2001 ; Straight et al., 2006).
  • ERM actin-binding proteins involvement of ERM actin-binding proteins in microbial infections has been occasionally reported during the entry, trafficking and spreading processes of bacteria (Pust et al., 2005; Tran Van Nhieu et al., 2000).
  • the cell cytoskeleton is implicated at several stages of the viral life cycle and cytoskeletal proteins including ERM proteins have been isolated from viral particles, which support the hypothesis that interactions between cell actin cytoskeleton components and viral proteins are implicated in viral assembly and budding (Dohner and Sodeik, 2005; Matarrese and Malomi, 2005; Ott et al., 1996).
  • the inventors focused on the characterisation of Spike CT (aa 1216-1255) interaction with ezrin.
  • the inventors determined that the subdomain F1 of the ezrin N-terminal FERM region is the binding site for SARS-Spike CT.
  • the ezrin-Spike interaction was confirmed by pull-down experiments.
  • the inventors have confirmed the interaction between SARS-Spike cytosolic tail and ezrin using GST pull-down assays. Incubation in presence of a rabbit serum that recognizes the Spike cytosolic tail domain efficiently decreased ezrin binding to Spike cytosolic tail.
  • the inventors have shown that the interaction is diminished by a factor 3 when the Spike cytosolic tail (1216-1255) is replaced by the Spike cytosolic tail truncated forms 1216-1247 ( ⁇ 8) and 1216-1236 ( ⁇ 19).
  • This result shows that the C-terminal 8 amino-acids (1248-1255: KGVKLHYT) are important for optimal interaction between SARS-Spike cytosolic tail and ezrin in vitro.
  • the membrane proximal cysteihe-rich domain 1216-1236 CCMTSCCSCLKGACSCGSCC
  • CCMTSCCSCLKGACSCGSCC membrane proximal cysteihe-rich domain
  • ezrin subcellular distribution is drastically affected in VeroE ⁇ cells infected by SARS- CoV.
  • ezrin is enriched in subcortical areas of the cell in normal conditions, it is recruited to ERGIC/Golgi apparatus compartments where Spike concentrates in infected cells. These compartments are the sites of coronavirus assembly and budding, which suggests a potential role of ezrin in these events.
  • the inventors have found that the FERM domain of ezrin cosediments with SARS-CoV virus-like particles (VLP) ultracentrifuged in a sucrose gradient.
  • VLP SARS-CoV virus-like particles
  • SARSpp Spike-pseudotyped lentiviral particles
  • siRNA small interfering RNAs
  • Tight junctions the most apical structures of cell-cell junctions, are crucial for the formation and maintenance of cell polarity, and thus of epithelium structure. Tight junctions define the boundary between the apical and baso-lateral domains of epithelial cells and form a barrier against free diffusion of fluids, electrolytes and macromolecules as well as luminal micro-organisms and their secreted products.
  • Several microorganisms have evolved strategies to either generate signals that disrupt tight junctions or directly target tight junction components, leading to epithelial barrier dysfunction and thus contributing to disease. Of particular relevance, the causes for the severity of illness in SARS-CoV infected patients are still not clearly understood.
  • the SARS-CoV mainly targets the epithelial cells, the respiratory tract being the primary site of infection.
  • DAD diffuse alveolar damage
  • the SARS-CoV E protein could be a major factor leading to the lung epithelium damages observed in SARS-CoV infected patients.
  • E CT aa 34-76
  • PALS1 PALS1
  • PALS1 that contains the PDZ domain and a portion of the SH3 domain.
  • the 4 last amino acids of the E CT domain share strong similarities with the 4 C-terminal amino-acids forming the PDZ-binding motif of the natural partner of PALS1 PDZ domain, the membrane protein CRB3.
  • CT domain share strong similarities with the consensual PDZ binding motif and 2 other clones coding for other PDZ containing proteins were also isolated from the screen. Interestingly, it was reported recently that the PDZ ligand motif at the C- terminus end of the H5N1 highly pathogenic avian influenza NS1 protein is a potential virulence determinant (Obenauer et al., Science 2006, vol 311 , pp 1576).
  • the following cell lines are used in the experiments: human fetal epithelial kidney cells HEK-293T, african green monkey epithelial kidney cells Vero-E6, epithelial cells derived from human cervix carcinoma HeLa cells stably transfected to express either the SARS-CoV receptor ACE2 (Hel_a-F5, Wang et al. 2004) or the HIV receptor (CD4) and co-receptor CXCR4 (Hel_a-P4, Clavel et al. 1994).
  • the cells are cultured in DMEM medium containing Glutamax I and Sodium Pyruvate (Gibco BRL), supplemented with 5% Fetal Calf Serum (FCS) and in presence of 100 U/mL of penicillin and 100 ⁇ L/mL of streptomycin.
  • the cells are incubated at 37°C in humid atmosphere with CO 2 adjusted at 5%.
  • the pCDNA-opt S-Flag, pCDNA-opt E, pCDNA-opt N-Flag, plRES-M-E plasmids contain codon optimized sequences of the Spike (S), the envelop (E), the membrane (M) and the nucleocapsid (N) proteins of SARS-CoV.
  • the cDNA encoding the M2 Flag peptide (DYKDDDDK) is introduced in frame at the 3' end of both cDNA encoding S and N.
  • the pGEX-GST-S CT and pG EX-GST-PALS 1 plasmids are derived from the pGEX-4T1 plasmid (Pharmacia Biotech).
  • the cDNA encoding the C-terminal tail (AA 1216 through 1255) of the SARS-CoV S protein has been amplified by polymerase chain reaction (PCR) from the pcDNA-opt S-Flag plasmid.
  • PCR polymerase chain reaction
  • BamHI and Xhol restriction sites were introduced at the 5' and 3' ends of the S CT cDNA respectively (sense oligonucleotide: GTTAGGATCCTGTTGCATGACCTCC; antisense oligonucleotide containing a STOP codon: GTAGCTCGAGTCATGTGTAATGCAGCTT).
  • These restriction sites enable to clone the S CT cDNA in frame with the Glutathione S-Transferase (GST) cDNA.
  • the pGEX-GST-S CT ⁇ 8 and ⁇ 19 truncated forms were subcloned using similar strategy using the same sense oligonucleotide and different antisense oligonucleotides (G TAG CTC GAG TCA CAG CAC TGG CTC AGA GTC and G TAG CTC GAG TCA GCA GCA AGA TCC ACA AGA, respectively).
  • the pG EX-GST FERM encodes the FERM domain of ezrin (AA 1 through 309) fused to the GST.
  • the pCB6-EzrinNter-VSV encodes the FERM domain of ezrin fused to a VSV peptide tag.
  • PALS1 SH3 were constructed by inserting PCR amplified sub-domains between the BamHI and Xhol sites of the pGEX-4T1 vector from Pharmacia Biotech. Oligonucleotides used for sub-clonings of PALS1 domains into the pGEX4T1 vector are summarized in the following table :
  • the PALS 1 -EYFP plasmid construct is a gift from Benjamin Margolis.
  • the pNL4.3.Luc R- E- lentiviral vector that encodes the sequences of a defective proviral sequence of HIV ⁇ env ⁇ nef and the luciferase reporter gene was a generous gift from N. R. Landau (Aaron Diamond AIDS Research Centre, Rockefeller University, New York, USA).
  • the GFP-Vpr plasmid was a generous gift from P. Bieniasz (Aaron Diamond AIDS Research Centre, Rockefeller University, New York, USA).
  • the following antibodies are used in the experiments: Serum from rabbits immunized with a peptide derived from ezrin (Monique Arpin). lmmunopurified serum from rabbits immunized with a peptide derived from ezrin (Monique Arpin).
  • the serum from mice immunized with the S protein was provided by J. Kam (HKU-PRC).
  • the serum from rabbits immunized with the S Cterm was purchased from ProScience.
  • Serum from rabbits immunized with the E endodomain (E CT) was provided by Nicolas Escriou (Institut Pasteur, Paris).
  • the goat monoclonal antibodies targeting radixin (IgG, C-15) were provided by Santa-Cruz Biotechnologies.
  • the mouse monoclonal IgG antibodies targeting ERGIC-53 were a generous gift from H. P. Hauri.
  • the mouse monoclonal IgG antibodies targeting Golgi-58K were provided by AbCam.
  • the mouse monoclonal IgG antibodies targeting the GAPDH (Glyceraldehyde 3 Phosphate Deshydrogenase) protein were provided by AbCam.
  • the rabbit anti-VSV-G tag (anti peptide 497-511 of the VSV-G protein) was from Sigma, rabbit anti-PALS1 antibodies were from SantaCruz, mouse anti-p24 was from Abeam, anti-GFP antiserum was from Invitrogen.
  • BL21-Gold(DE3)pLysS electrocompetent E.coli bacteria were transformed by electroporation with the following plasmids: pGEX-GST S CT, pGEX-GST-S CT ⁇ 8, pGEX-GST-S CT ⁇ 19, pGEX-GST FERM, pGEX-GST- PALS1 , pGEX-GST-PALS1 L27, pGEX-GST-PALS1 L27,PDZ,SH3, pGEX-GST- PALS1 L27.PDZ, pGEX-GST-PALS1 PDZ, pG EX-GST-PALS 1 SH3.
  • Transformed bacterial clones were subsequently grown on liquid LB broth in presence of Ampicillin.
  • GST-fusion protein production was induced by adding 1mM IPTG (Isopropyi-Thio- ⁇ -D-Galactopyranoside) in the culture medium.
  • IPTG Isopropyi-Thio- ⁇ -D-Galactopyranoside
  • the bacteria were incubated 2 hours at 37°C with shaking.
  • the bacteria were then centrifuged at 4°C for 5 min at 7000 g.
  • the pellet was resuspended in 1 mL of lysis buffer (400 mM NaCI, 50 mM Tris, 1 mM DTT, 0.3% Triton X-100, PMSF 0.6 mM).
  • Glutathione sepharose 4B beads (Amersham Biosciences) are sedimented at 500 g for 5 min. They are subsequently rinsed three times with 1 ml_ of cold lysis buffer (150 mM NaCI, 50 mM Tris, 0.1% Triton X-100, PMSF 1 mM, BSA 50 ⁇ g/mL). The beads are resuspended in 100 ⁇ l_ of lysis buffer.
  • the bacterial lysis supernatant containing GST fusion proteins are incubated with 10 ⁇ L of Glutathione sepharose beads.
  • the coupled beads are then rinsed three times with a buffer containing: 150 mM NaCI, 50 mM Tris, 0.2% Triton X-100, PMSF 1 mM and BSA 50 ⁇ g/mL.
  • Lysis is performed on ice for 15 min on 70% confluent cells that were plated a day beforehand.
  • the lysis buffer used contains: 0.5% Triton X-100, 150 mM NaCI, 20 mM Tris, 1 mM EDTA, 50 mM NaF, 1X Protease Inhibitor Cocktail (Roche), 1mM PEFABIoc (Roche).
  • the lysate is centrifuged at 10000 g for 10 min at 4°C and the pellet is discarded. 500 ⁇ L of lysates are added to the Glutathione sepharose beads coupled with the GST fusion proteins. The mix is incubated 3 hours at 4 0 C on a spinning wheel.
  • the beads are then washed 5 times with 400 ⁇ L of the buffer used for mammalian cell lysis.
  • the washed beads are mixed with NuPage Loading Buffer (Invitrogen) in the presence of 10 mM DTT (dithiolthreitol).
  • the samples are analyzed by Western Blot as described in the paragraph "Western Blot protein quantifications".
  • VeroE6 cells were grown on glass coverslips and co-transfected with both pCDNA-opt E and pCDNA-PALS1-EYFP using the FuGene ⁇ transfection reagent. Cells were permeabilized and undergo blocking treatment by incubation for 30 min in a PBS + 0.2% Tween 20 solution (PBST 0.2%) supplemented with 10% Normal Goat Serum (NGS) or Fetal Calf Serum (FCS).
  • NGS Normal Goat Serum
  • FCS Fetal Calf Serum
  • the labeling with primary antibodies was performed by incubating the slides for 45 min with different combinations of the following antibodies: mouse serum against the S protein (1/100), rabbit anti-S antibodies (1/100), rabbit anti-ezrin antibodies (1/1000), mouse anti-Golgi 58K antibodies (1/50), goat anti-radixin antibodies, mouse anti-ERGIC 53 antibodies (1/1000), rabbit anti-E antibodies (1/250) and rabbit anti-PALS1 antibodies (1/300).
  • the slides were then washed in a PBST 0.2% solution with shaking for 5 min.
  • the slides were then incubated with the following secondary antibodies: goat anti-Rabbit IgG antibodies coupled with FITC (ImmunoResearch, 1/100), goat anti-mouse IgG antibodies coupled with Texas Red (Oncogene, 1/100), Rabbit anti-Goat IgG antibodies coupled with FITC (ImmunoResearch, 1/100).
  • the dilution of antibodies were all performed with a PBST 0.2% solution supplemented with 5% NGS or FCS (for radixin labeling).
  • the slides were washed for 5 min with the PBST 0.2% solution. They were then incubated in a solution containing DAPI staining marker (4',6-Diamidino-2- phenylindole).
  • HEK-293T cells 2.5 10 6 HEK-293T cells were seeded onto 10 cm plates. After 24 hours incubation in 37 0 C humid atmosphere with CO 2 adjusted at 5%, the culture medium was replaced with fresh medium. The cells were subsequently transfected using the CalPhos Mammalian transfection Kit (Clontech). SARS-CoV S pseudotyped lentiviral particles were obtained by using a 2-plasmid system. The two plasmid used were pNL4.3.Luc R- E- and pcDNA-opt S Flag and were transfected at a 10:10 ratio (10 ⁇ g of each plasmid are simultaneously used to transfect the cells).
  • a third plasmid coding for the GFP tagged vpr HIV accessory protein was co- transfected to produce fluorescent pseudotyped lentiviral particles. 36-40 hours after transfection, the culture medium was collected, centrifuged at 2000 g for 10 min at 4 0 C and filtered through 0.45 ⁇ m membranes. The pseudotyped particle solution were aliquoted at stored at -80 0 C. VSV G, HIV gp120/gp41 and Influenza HA glycoprotein pseudotyped lentiviral particles were obtained by F. Kien, JM Garcia and I. Nelfkens respectively using similar strategy (HKU-PRC).
  • Hel_a-F5 or Hel_a-P4 cells were transfected with pools of 4 oligonucleotides (Dharmacon) that specifically target either ezrin mRNAs (NCBI accession # of human ezrin cDNA upon which the design of siRNAs was based: NM_003379.
  • Duplex 1 Sense GCUCAAAGAUAAUGCUAUGUU Antisense
  • Duplex 4 Sense GAUCAGGUGGUAAAGACUAUU Antisense 5'PUAGUCUUUACCACCUGAUCUU) or radixin mRNAs (NCBI accession # of human radixin cDNA upon which the design of siRNAs was based: NM_002906.
  • Duplex 1 Sense GAAGGAAGCCUGAUACUAUUU Antisense ⁇ 'PAUAGUAUCAGGCUUCCUUCUU.
  • Duplex 2 Sense CACAAGAUCUAGAAAUGUAUU Antisense 5'PUACAUUUCUAGAUCUUGUGUU.
  • Duplex 3 Sense
  • the pseudotyped particles were diluted: 1/2 for SARS S pseudotyped particles (SARSpp), 1/100 for VSV G pseudotyped particles (VSVGpp) and Influenza
  • HApp HA pseudotyped particles
  • HAVpp HIV gp120/gp41 pseudotyped particles
  • 50 ⁇ l_ of pseudotyped particles were added for each well. Each condition was performed in quadruplicates. 12 hours after pseudotyped particles were added, the medium was refreshed with DMEM containing 5% FCS and antibiotics. 48 hours after medium change 100 ⁇ l_ of BrightGlo (Promega) reagent was added to each well. After 5 min of incubation with the reagent, the 96-well plate was analyzed by a MicroBeta luminescence counter (PerkinElmer). Western Blot protein quantifications
  • the lysates of each type of quadruplicates were pooled. 5 ⁇ l_ of NuPage loading buffer and 2 ⁇ l_ of DTT (200 mM) were added to 13 ⁇ L of each of the pooled samples. The mixes were then heated at 95°C for 5 min and then loaded onto a precast NuPage Novex Bis-Tris 4-12% gel (Invitrogen). The loaded samples undergo electrophoresis at 200 V and 110 mA for 1 hour using the MOPS SDS-PAGE running buffer (Invitrogen).
  • the proteins contained inside the gel were transfered on a PVDF membrane (Amersham Biosciences) that was dehydrated with EtOH and rehydrated with (i) distilled water and (ii) Nupage transfer buffer (Invitrogen).
  • the transfer was performed under the following conditions: 2 hours at 30V and 170-110 mA using Nupage transfer buffer.
  • the membrane was washed three times for 10 min using PBST 0.1 %.
  • the membrane was blocked with a PBST solution supplemented with 5% powdered milk.
  • the membrane was incubated with primary antibodies for 2 hours at room temperature using anti-ezrin (1/1000) or anti-radixin (1/100) antibodies diluted with PBST- 5% Milk solution.
  • the membrane was washed three times as described previously.
  • the membrane was then incubated for one hour with secondary antibodies that target either rabbit or goat IgGs and coupled with Horse- Radish Peroxidase (HRP, Zymed).
  • HRP Horse- Radish Peroxidase
  • the membrane was washed three times as described previously.
  • the membrane revelation was performed using the ECL kit from Amersham BioSciences.
  • the membranes were placed in a revelation cassette with a film (Kodak) for 5 sec or 1 min.
  • the film was then developed using FPM 100A film processing unit (FujiFilm).
  • a second protein revelation was performed on top of the first one by using antibodies targeting house-keeping genes that were not affected by the siRNA treatments: GAPDH and actin.
  • Example 1 Method for identifying binding inhibitors that inhibit the binding of the C-terminal domains of the S, M, and E viral proteins of the SARS-CoV virus to their identified cellular partners.
  • Electrocompetent bacteria BL21- Gold (DE3) pLysS (Stratagene) are transformed by electroporation with the PGEX GST-S CT, PGEX GST-M CT, PGEX
  • IPTG Isopropyl-Thio- ⁇ -D-Galactopyranoside
  • the samples undergo a rapid shock through freezing/defreezing and are sonicated with 5 pulses of a maximum amplitude of 15 s on ice.
  • the lysate is centrifuged at 14 000 g for 20 min at 4°C to eliminate bacterial debris and insoluble proteins. The supemantant that contains the soluble proteins is kept.
  • the sepharose-glutathione beads (Glutathione SepharoseTM 4B, Amersham Biosciences) settle at 500 g for 5 min at the bottom of the wells of the 96-well plates.
  • Tris 0.1 % Triton X-100, PMSF 1 mM, BSA 50 ⁇ g/mL.
  • the beads are resuspended in 100 ⁇ L of this lysis buffer. Coupling of fusion proteins to the sepharose-glutathione beads
  • the bacterial supernatants are incubated with the sepharose-glutathione beads for 2 hours at 4°C in order to immobilize the fusion proteins solubilized onto the beads in the 96-well plate. Finally, the beads are rinsed 3 times with the following lysis buffer: 150 mM NaCI, 50 mM Tris, 0.2% Triton X-100, PMSF 1 mM, BSA 50 ⁇ g/mL.
  • cell lysates from cells expressing cellular partners Preparation of cell lysates from cells expressing cellular partners.
  • cell lysates from cells transfected with plasmid construct allowing expression of viral proteins S, M or E are prepared. Lysis is performed on ice during 15 min with the following lysis buffer: 0.5% Triton-X100, 150 mM NaCI, 20 mM Tris, 1 mM EDTA, 50 mM NaF, PIC 1X, 1mM PEFABIoc on cells that were inoculated the previous day and that have a confluence of 70%. The lysate is centrifuged at 10 000 g for 10 min at 4 0 C and the pellet is eliminated.
  • the compounds to be screened are added to the 96-well plates containing the fusion proteins GST-S CT, PGEX GST-M CT, PGEX GST-E CT and PGEX GST coupled to the beads (negative controls: well containing only beads and well without beads and without fusion proteins).
  • the plates are incubated with agitation for 30 minutes.
  • the lysates are added to the sepharose-glutathione beads coupled to the fusion proteins which are pre-incubated with the potential inhibitor compounds. This mixture is incubated for 3 hours at 4°C. The beads are then rinsed 5 times.
  • the cellular protein or the viral protein of interest is revealed by using a specific primary antibody and a secondary antibody coupled to alkaline phosphatase. The revealing of the protein requires a substrate of the alkaline phosphatase and an ELISA plate reader. The signals are quantified and reflect the binding efficiency of the cellular partner to the C-terminal domain of the S, M and E viral proteins.
  • Example 2 Identification of proteins that interact with Sars CoV-S c-terminal domain( HK1) by yeast two-hybrid (Y2H) screening.
  • Bait cloning
  • HK1 was PCR-amplified and cloned in a Y2H vector optimized by Hybrigenics, S.A., Paris, France.
  • the bait construct was checked by sequencing the entire insert, and was subsequently transformed in the L40 GAL4 yeast strain (Fromont-Racine et al. 1997).
  • the screen was first performed on a small scale to adapt the selective pressure to the intrinsic property of the bait. The bait was found to auto-activate the Y2H system, and 2mM 3-aminotriazole was found to be the optimal concentration to reduce background colonies. Then, the full-scale screen was performed in conditions ensuring a minimum of 50 million interactions tested, in order to cover five times the primary complexity of the yeasttransformed cDNA library (Rain et al. 2001).
  • the PBS relies on two different levels of analysis: firstly a local score takes into account the redundancy and independency of prey fragments, as well as the distributions of reading frames and stop codons in overlapping fragments. Secondly, a global score takes into account the interactions found in all the screens was performed using the same library. In addition, potential false-positives are flagged by a specific "E" PBS score. This is done by discriminating prey proteins containing "highly connected" domains, previously found several times in screens performed on libraries derived from the same organism. The PBS scores have been shown to positively correlate with the biological significance of interactions (Rain et al. 2001 ; Wojcik et al. 2002).
  • VIL2 mRNA] - E-cadherin: hCDHl; [gi
  • Ezrin (6-93: containing the F1 subdomain of ezrin FERM N-terminal domain): NVRVTTMDAELEFAIQPNTTGKQLFDQWKTIGLREVWYFGLHYVDNKGFPTWLKLDK KVSAQEVRKENPLQFKFRAKFYPEDVAEEL E-cadherin interaction domain with S CT: E-Cadherin preproprotein (5-72):
  • lntegrin alpha 5 interaction domain with S CT lntegrin alpha 5 preprotein (24-334):
  • the cytosolic tail of SARS CoV Spike glycoprotein interacts with Ezrin N- terminal domain
  • the inventors adopted a yeast-two-hybrid screening strategy to identify cellular proteins which interact with the cytosolic carboxy-terminal tail (CT) of SARS- Spike glycoprotein (aa 1216-1255).
  • CT carboxy-terminal tail
  • the screening of a human placenta random- primed cDNA library was performed by Hybrigenics company (Hybrigenics SA 1 Paris, France).
  • 233 clones came out from 147 millions of interactions tested. Sequencing of positive prey clones revealed that 35% of the clones (82/233 clones) correspond to human ezrin cDNA fragments.
  • GST pull-down assays To confirm the interaction between SARS-Spike CT and ezrin F1/SBD, the inventors performed GST pull-down assays. First, a GST-Spike CT fusion protein was tested for its capacity of binding to ezrin protein. Briefly, GST-Spike CT protein bound on gluthatione-sepharose beads was incubated with cell extracts from human epithelial HeLa cells or green monkey epithelial VeroE ⁇ ceils. In parallel gluthatione- sepharose beads alone or bound to GST were also incubated with cells extracts. Only gluthatione-sepharose beads bound to GST-Spike CT protein was able to pull down ezrin protein from cell extracts in a concentration dependant manner.
  • the inventors have constructed GST-Spike CT truncated forms S CT 1216-1247 ( ⁇ 8) [1216 CCMTSCCSCLKGACSCGSCCKFDEDDSEPVL 1247] and and S CT 1216-1236 ( ⁇ 19) [1216 CCMTSCCSCLKGACSCGSCC 1236].
  • the GST-Spike CT ⁇ 8 and ⁇ 19 were used to precipitate ezrin and radixin ERM proteins from total cell lysates of VeroE6 and HeLa cells.
  • Ezrin is recruited to S-enriched sites in SARS-CoV infected cells
  • inactivated ezrin is present within the cytosol and activated form is associated to cortical areas where it interacts with both filamentous actin and membrane receptors.
  • ezrin is efficiently recruited to the ERGIC- Golgi apparatus region in VeroE ⁇ infected cells where it colocalises with SARS-S- rich sites. This subcellular region is the site of SARS-S maturation as well as the site of SARS-CoV assembly and budding. This result suggests a role of ezrin during the assembly/budding steps of SARS-CoV.
  • Ezrin restricts SARS CoV entry into permissive cells
  • the inventors have set up an anti-ezrin siRNA strategy which can shut down ezrin expression up to 90% in human cell lines.
  • the inventors have demonstrated that ezrin shut down lead to a strong enhancement of SARS-CoV pseudotype viruses entry into permissive cells (5 to 12 times depending on experiments), showing that ezrin is a restricting factor of SARS CoV infection.
  • the shut down of the radixin protein (which belongs to the same ERM protein family than ezrin, and which has also been identified by the Y2H screen with a B score) inhibits the infection of cells by SARS-CoV pseudotype viruses with a factor of ⁇ 2.5. Moreover, simultaneous shut down of both ezrin and radixin ERM proteins leads to increase of infection by a factor ⁇ 3. Altogether, these results demonstrate a regulatory role of ERM proteins on early stages of SARS-CoV infection mediated by the specific interaction with SARS-S CT domain and an antagonist effect of both ezrin and radixin proteins.
  • Fluorescent SARSpp are infection-competent as revealed by measurement of luciferase activity and observation of GFP-Vpr intracellular localization in infected HeLa-F5 cells. Fluorescent SARSpp constitute a powerful tool for the analysis of role of cellular partners, including ERM proteins, during entry steps of SARS-CoV. They could also be used in later studies for monitoring the effect of anti-coronavirus molecules on early stages of SARS-CoV infection, as shown in Example 5.
  • Example 3 Identification of proteins that interact with SARS-CoVM C-terminal domain (HK3) by yeast two-hybrid (Y2H) screening.
  • Bait cloning
  • HK3 was PCR-amplified and cloned in a Y2H vector optimized by Hybrigenics, S.A., Paris, France.
  • the bait construct was checked by sequencing the entire insert, and was subsequently transformed in the L40 GAL4 yeast strain (Fromont-Racine et al. 1997). Y2H Screening.
  • the screen was first performed on a small scale to adapt the selective pressure to the intrinsic property of the bait. Neither toxicity nor autoactivation of the bait was observed. Then, the full-scale screen was performed in conditions ensuring a minimum of 50 million interactions tested, in order to cover five times the primary complexity of the yeasttransformed cDNA library (Rain et al. 2001). 152 millions of interactions were actually tested with HK3.
  • the PBS relies on two different levels of analysis: firstly a local score takes into account the redundancy and independency of prey fragments, as well as the distributions of reading frames and stop codons in overlapping fragments. Secondly, a global score takes into account the interactions found in all the screens was performed using the same library. In addition, potential false-positives are flagged by a specific "E" PBS score. This is done by discriminating prey proteins containing "highly connected" domains, previously found several times in screens performed on libraries derived from the same organism. The PBS scores have been shown to positively correlate with the biological significance of interactions (Rain et al. 2001 ; Wojcik et al. 2002). Results:
  • SARS coronavirus Membrane (M) protein C-terminal sequence SARS coronavirus Membrane (M) protein C-terminal sequence:
  • SPIN90/DIP interaction domain with M CT SPIN90/DIP (310-478):
  • Example 4 Identification of proteins that interact with SARS CoV E C-terminal domain (HK4) by yeast two-hybrid (Y2H) screening.
  • Bait cloning
  • HK4 was PCR-amplified and cloned in a Y2H vector optimized by Hybrigenics, S.A., Paris, France.
  • the bait construct was checked by sequencing the entire insert, and was subsequently transformed in the L40 GAL4 yeast strain (Fromont-Racine et al. 1997).
  • the screen was first performed on a small scale to adapt the selective pressure to the intrinsic property of the bait. Neither toxicity nor autoactivation of the bait was observed. Then, the full-scale screen was performed in conditions ensuring a minimum of 50 million interactions tested, in order to cover five times the primary complexity of the yeasttransformed cDNA library (Rain et al. 2001). 140 millions of interactions were actually tested with HK4.
  • the PBS relies on two different levels of analysis: firstly a local score takes into account the redundancy and independency of prey fragments, as well global score takes into account the interactions found in all the screens performed at Hybrigenics using the same library. In addition, potential false- positives are flagged by a specific "E" PBS score. This is done by discriminating prey proteins containing "highly connected" domains, previously found several times in screens performed on libraries derived from the same organism. The PBS scores have been shown to positively correlate with the biological significance of interactions (Rain et al. 2001 ; Wojcik et al. 2002).
  • the inventors have found that a smaller region of Spike CT can still pull down ezrin from total cell lysate.
  • CT cytosolic tail
  • endogenous PALS1 or exogenous PALS1- EYFP show aberrant sub-cellular distribution in SARS-CoV-infected and E- transfected cells 9 (Fig. 15). Indeed, in these cells PALS1 localizes at the peri- nuclear area of the cell, at close proximity of the ERGIC, where viral structural proteins, including E, accumulate for virion assembly.
  • the inventors work on the characterization of a novel mechanism by which the SARS-CoV hijacks cellular machineries of the lung epithelium and may delineate a new important determinant of the pathogenesis of SARS-CoV infection.
  • SARS-CoV leads to a disseminate infection in humans.
  • organs are affected, in particular the gastro-intestinal tract.
  • One cannot exclude that similar mechanism is involved in dysfunction of infected epithelia others than the airway epithelium. Delineation of such pathogenic mechanisms with SARS-CoV may be also relevant to other infectious diseases.
  • Yeast Two Hybrid Screening using SARS-CoV E CT sequence as bait Identification of clones coding for 3 different PDZ domain-containing proteins: HK4v1_pB27A-131 (1 out of 28 clones) hMPP5; [gi
  • HK4v1_pB27A-122 hMPDZ [gi
  • E SARS coronavirus Envelope (E) protein C-terminal sequence: E CT (34-76):
  • golgi reassembly stacking protein 2 (PDZ containing protein)
  • the inventors have defined the common sequence shared by all 28 PaIsI cDNA clones identified by yeast-two-hybrid as the minimal binding domain of PaIsI to SARS CoV E protein C-terminal domain.
  • the minimal binding site consists in PALS1 PDZ domain and truncated SH3 domain.
  • PALS1 PDZ domain is crucial for binding with cellular factors, such as Crumb 1 (CRB1) and 3 (CRB3), via its PDZ domain motif
  • the PDZ-binding motif on CRB3 consists in the 4 C-terminal amino-acids
  • Example 5 Method for Identifying inhibitors of early stages of SARS-CoV replication cycle by using lentiviral Spike pseudotyped particles
  • HEK-293T cells 2.5 10 6 HEK-293T cells are seeded onto 10 cm plates (alternatively, larger production scales are made). After 24 hours incubation in 37 0 C humid atmosphere with CO 2 adjusted at 5%, the culture medium is replaced with fresh medium. The cells are subsequently transfected using the CalPhos Mammalian transfection Kit (Clontech). SARS-CoV S pseudotyped lentiviral particles are obtained by using a 2- plasmid system. The two plasmid used are pNL4.3.Luc R- E- and pcDNA-opt S Flag and are transfected at a 10:10 ratio (10 ⁇ g of each plasmid are simultaneously used to transfect the cells).
  • a third plasmid coding for the GFP tagged Vpr HIV accessory protein (GFP-Vpr) is co-transfected to produce fluorescent pseudotyped lentiviral particles. 36-40 hours after transfection, the culture medium is collected, centrifuged at 2000 g for 10 min at 4 0 C and filtered through 0.45 ⁇ m membranes. The pseudotyped particles are aliquoted at stored at -8O 0 C. Cell plating and incubation with binding inhibitors*
  • Susceptible cells for example VeroE ⁇ or Hel_a-F5 are plated in 96-well plates and incubated at 37 0 C, 5% CO2, in appropriate culture medium. 16 hours after seeding, inhibitors of Spike-cellular partner binding are added to the cells. Cells are either incubated at 37 0 C or kept on ice with serial dilutions of binding inhibitors.
  • Endocytosis is then stopped by placing cells back on ice. Cells are washed 3 times to eliminate non-internalized SARSpp with cold PBS1X and detached with EDTA
  • Inhibitors of SARS-CoV binding and entry should inhibit SARSpp binding or internalization and therefore lead to a lower signal for GFP.
  • Endocytosis is then stopped by placing cells back on ice.
  • Cells are washed 3 times to eliminate non-internalized SARSpp with cold PBS1X.
  • Cells are fixed in PBS 1X, 4% paraformaldehyde, washed in PBS 1X and analyzed by fluorescence microscopy (Confocal microscopy, Large throughput screening and quantification modules).
  • Fluorescence microscopy Confocal microscopy, Large throughput screening and quantification modules.
  • Subcellular localization of the GFP signal is determined (cell surface, endosomal compartment) and GFP fluorescence is quantified.
  • Inhibitors of SARS-CoV binding and entry should inhibit SARSpp binding or internalization and therefore lead to either a change of localization of fluorescent SARSpp or a diminution of intensities of GFP signals, or both.
  • cells are infected with fluorescent or non-fluorescent SARSpp. 12 hours after addition of pseudotyped particles, the medium is refreshed with DMEM containing 5% FCS and antibiotics. 48 hours after medium change 100 DL of BrightGlo (Promega) reagent is added to each well. After 5 min of incubation with the reagent, the 96-well plate is analyzed by a MicroBeta luminescence counter (PerkinElmer). Luciferase activity is quantified and reflect efficacy of cell transduction by SARSpp.
  • SARSpp are pre-incubated with various concentration of binding inhibitors before addition to susceptible cells.
  • HIV envelope protein replaced by another envelope protein of a heterologous virus
  • the heterologous envelope protein determines the entry mechanism of the particle - Reporter gene (luciferase): quantification of transduction by pseudotyped particles -
  • a GFP-vpr protein is incorporated to produce fluorescent SARS-Spp particles (analysis of SARS-CoV entry by fluorescence microscopy.
  • Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature. 446:801- 805 (2007).
  • Formstecher E Aresta S, Collura V, Hamburger A, Meil A, Trehin A, Reverdy C, Betin V, Maire S, Brun C 1 Jacq B, Arpin M, Bellaiche Y, Bellusci S, Benaroch P, Bornens M, Chanet R, Chavrier P, Delattre O, Doye V, Fehon R, Faye G, GaIIi T, Girault JA, Goud B, de Gunzburg J, Johannes L, Junier MP,

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Abstract

La présente invention porte sur le domaine des coronavirus et, plus particulièrement, sur des molécules anti-coronavirus et sur leur utilisation dans des compositions, et sur des procédés pour traiter et/ou prévenir une infection provoquée par un coronavirus, tel que celui responsable du syndrome respiratoire aigu grave (SARS). La présente invention porte également sur un procédé pour cribler de nouvelles molécules anti-coronavirus.
PCT/IB2007/004174 2006-11-20 2007-11-20 Molécules anti-coronavirus et leur utilisation dans des compositions et procédés pour traiter et/ou prévenir une infection provoquée par un coronavirus WO2008062309A2 (fr)

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CA002569080A CA2569080A1 (fr) 2006-11-20 2006-11-20 Molecules anti-coronavirus et leur utilisation dans les compositions, et methodes de traitement et/ou prevention de l'infection causee par un coronavirus
CA2,569,080 2006-11-20

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WO2022024108A1 (fr) * 2020-07-28 2022-02-03 Rapo Yerape B.H. Ltd. Peptide d'ezrine (hep-1) destiné à être utilisé dans le traitement d'une maladie à coronavirus

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