PEPTIDES INCLUDING A BINDING DOMAIN OF THE VIRAL PHOSPHOPROTEIN (P) SUBUNIT TO THE VIRAL RNA FREE NUCLEOPROTEIN (N0)
20170158741 ยท 2017-06-08
Inventors
- Viktor VOLCHKOV (Lyon Cedex 07, FR)
- Marc Jamin (Grenoble, FR)
- Philip LAWRENCE (Lyon Cedex 07, FR)
- Filip Yabukarski (Stanford, CA)
Cpc classification
C12N2760/18433
CHEMISTRY; METALLURGY
C12N2760/18222
CHEMISTRY; METALLURGY
C12N2760/18233
CHEMISTRY; METALLURGY
International classification
Abstract
The invention related to isolated peptides including a binding domain of the viral phosphoprotein (P) subunit to the viral RNA free nucleoprotein (N.sup.0) which has the property to inhibit the replication of viruses from the subfamily Paramyxovirinae (like Henipavirus, Rubulavirus or Morbillivirus). These isolated peptides may be used for the prevention or the treatment of Paramyxovirinae infection.
Claims
1. An isolated peptide of at most 100 amino-acids comprising an amino acid sequence of formula (I):
Valine-Xaa1-Xaa2-Glycine-Leucine-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8, wherein Xaa1 is glutamine (Q), serine (S), asparagine (N), lysine (K) or an equivalent polar amino acid; Xaa2 is glutamic acid (E), aspartic acid (D) asparagine (N), lysine (K), or an equivalent negatively charged (or acid) amino acid; Xaa3 is glutamic acid (E), aspartic acid (D), lysine (K) or glutamine (Q), serine (S) or asparagine (N); Xaa4 is cysteine (C) or isoleucine (I); Xaa5 is isoleucine (I), leucine (L) or valine (V) or an equivalent apolar aliphatic amino acid; Xaa6 is glutamine (Q), lysine (K), arginine (R) or aspartic acid (D); Xaa7 is alanine (A), or phenylalanine (F) or an equivalent apolar amino acid; and Xaa8 is isoleucine (I), leucine (L) or valine (V) or an equivalent apolar aliphatic amino acid.
2. The isolated peptide of claim 1 wherein Xaa1 is asparagine (N) or glutamine (Q); Xaa2 is aspartic acid (D) or glutamic acid (E); Xaa3 is asparagine (N) or glutamic acid (E); Xaa4 is isoleucine (I) or cysteine (C); Xaa5 is isoleucine (I); Xaa6 is aspartic acid (D) or glutamine (Q); Xaa7 is phenylalanine (F) or alanine (A); and Xaa8 is isoleucine (I).
3-9. (canceled)
10. The isolated peptide according to claim 1, wherein the peptide is selected from the group consisting of i) an amino acid sequence ranging from the valine residue at position 7 to the isoleucine residue at position 17 in SEQ ID NO:1, ii) an amino acid sequence ranging from the valine residue at position 9 to the leucine residue at position 19 in SEQ ID NO:2, and iii) an amino acid sequence substantially homologous to the sequence of (i) or (ii).
11. The isolated peptide according to any claim 1 comprising the amino acid sequence of formula (II):
Yaa1-Yaa2-Yaa3-Yaa4-Yaa5-Valine-Xaa1-Xaa2-Glycine-Leucine-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Yaa6-Yaa7-Yaa8, wherein Xaa1-Xaa2 Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8 are as defined in claim 1, Yaa1 is aspartic acid (D), glutamic acid (E), or an equivalent acidic amino acid, Yaa2 is glutamine (Q) or lysine (K), Yaa3 is alanine (A), leucine (L) or tyrosine (Y), Yaa4 is glutamic acid (E), tyrosine (Y) or arginine (R), Yaa5 is asparagine (N), histidine (H) or leucine (L), Yaa6 is glutamine (Q), lysine (K), or arginine (R), Yaa7 is lysine (K), alanine (A) or glutamic acid (E), and Yaa8 is asparagine (N), glutamic acid (E) or serine (S).
12-19. (canceled)
20. The isolated peptide according to claim 11, wherein the peptide is selected from the group consisting of i) the amino acids sequence consisting of MDKLELVNDGLNIIDFIQKNQKEIQKTYGRSSIQQPSIKD (SEQ ID NO: 1); ii) an amino acid sequence ranging from the leucine residue at position 6 to the aspartic acid residue at position 40 in SEQ ID NO:1; iii) an amino acid sequence ranging from the leucine residue at position 11 to the aspartic acid residue at position 40 in SEQ ID NO:1; iv) an amino acid sequence ranging from the methionine residue at position 1 to the asparagine residue at position 20 in SEQ ID NO:1; v) an amino acid sequence ranging from asparagine residue at position 20 to the glutamine residue at position 35 in SEQ ID NO:1; vi) the amino acids sequence consisting of MAEEQAYHVSKGLECLKALRENPPDIEEIQEVSSLRDQTC (SEQ ID NO: 2); vii) an amino acid sequence ranging from the methionine residue at position 1 to the asparagine residue at position 22 in SEQ ID NO:2; viii) the amino acids sequence consisting of DQAENVQEGLECIQAIQKN (SEQ ID NO: 3); ix) the amino acids sequence consisting of MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEIS (SEQ ID NO: 4); x) an amino acid sequence ranging from the valine residue at position 9 to the leucine residue at position 19 in SEQ ID NO:4; and xi) an amino acid sequence substantially homologous to the sequence of one of (i) to (x).
21. The isolated peptide of claim 1, wherein said isolated peptide is linked to at least one cell-penetrating peptide.
22. (canceled)
23. A polynucleotide comprising or consisting of a nucleic acid encoding a peptide as defined in claim 1.
24. A method for producing a peptide as defined in claim 1, wherein said method comprises the steps of: a) culturing a recombinant cell comprising a recombinant vector comprising a polynucleotide comprising or consisting of a nucleic acid encoding a peptide as defined in claim 1 in conditions allowing the expression of the peptide; b) optionally, purifying the peptide obtained at step a).
25-29. (canceled)
30. The isolated peptide according to claim 10, wherein the amino acid sequence of (iii) is an amino acid sequence at least 80% identical to the sequence of (i) or (ii).
31. The isolated peptide according to claim 20, wherein the amino acid sequence of (xi) is an amino acid sequence at least 80% identical to the sequence of one of (i) to (x).
32. A method for preventing and/or treating a Paramyxovirinae infection comprising a step of administering at least one isolated peptide as defined in claim 1.
33. The method for preventing and/or treating a Paramyxovirinae infection as defined in claim 32; wherein said Paramyxovirinae infection is selected from the group consisting of Rubulavirus infection, Avulavirus infection, Henipavirus infection, Henipavirus-like infection, Morbillivirus infection, Morbillivirus-like (TPMV-like viruses) infection, Respirovirus infection and Ferlavirus infection.
34. The method for preventing and/or treating a Paramyxovirinae infection as defined in claim 33, wherein said Avulavirus infection is an infection with the Newcastle disease virus; said Henipavirus infection is an infection with the Nipah virus (NiV) or with the Hendra virus (HeV); said Morbillivirus infection is an infection with the Measles virus (MeV), the Rinderpest virus, the Canine distemper virus, the phocine distemper virus or the Ovine rinderpest virus; said TPMV-like virus infection is an infection with the Tupaia paramyxovirus, the Mossman virus, the Nariva virus or the Salem virus; said Ferlavirus infection is an infection with the Fer-de-Lance virus; said Rubulavirus infection is an infection with the Mumps virus, the parainfluenza type 2, 4 viruses, the Achimota virus 1 and 2, the Simian parainfluenza virus 5, the Menangle virus, the Tioman virus, or the Tuhokovirus 1, 2 and 3; and said Respirovirus infection is an infection with the Sendai virus or the human parainfluenza viruses 1 and 3.
35. A pharmaceutical composition comprising a peptide according to claim 1, and one or more pharmaceutically acceptable excipients.
36. A method for preventing and/or treating a Paramyxovirinae infection comprising a step of administering at least one isolated peptide derived from a N-terminal phosphoprotein (P) of a Paramyxovirinae virus, having the capacity to inhibit an interaction between the phosphoprotein (P) and the nucleoprotein (N.sup.0) of said Paramyxovirinae virus.
37. The method as defined in claim 36, wherein said isolated peptide comprises at least 9 and at most 100 amino acids.
38. The method as defined in claim 36, wherein said isolated peptide is linked to at least one cell-penetrating peptide.
39. The isolated peptide as defined in claim 1, wherein said peptide is a modified peptide.
40. The method as defined in claim 36, wherein said isolated peptide is a modified peptide.
Description
FIGURES
[0179]
[0180]
[0181]
[0182]
[0183]
EXAMPLES
Example 1
[0184] Material & Methods
[0185] Reconstitution of the N.sup.0-P Core Complex.
[0186] Constructs comprising residues 1-50 (P50) of P and residues 32-383 (N.sub.32-383) or 32-402 of N (N.sub.32-402) from the Malaysian isolate UMMC1 of Nipah virus (Uniprot numbers Q9IK91 and Q9IK92) were cloned in pETM40 vector in fusion with an N-terminal maltose binding protein (MBP) tag. All proteins were expressed in E. coli BL21 (DE3) Rosetta cells. Cells were grown at 37 C. in LB medium until O.D. reached 0.6, and protein expression was induced overnight at 20 C. by addition of isopropyl--D-thiogalactoside (IPTG) to a final concentration of 1 mM. Cells were harvested and the pellet was suspended in buffer A for P construct (20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl, 50 mM arginine, 50 mM glutamate and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)) and in buffer B for N constructs (Tris-HCl buffer at pH 7.5 containing 150 mM NaCl). All buffers were supplemented with Complete protease inhibitor cocktail (Roche). Cells were disrupted by sonication, and the crude extract was cleared by centrifugation at 45,000 g at 4 C. for 20 min. The supernatant was loaded onto an amylose resin column (New England Biolabs) equilibrated in buffer A or B. The column was washed with 10 volumes of buffer A or B containing 500 mM NaCl and the protein was eluted with 50 mM maltose (Sigma) in buffer A or B.
[0187] The P-MBP fusion protein was cleaved with TEV protease to remove the MBP tag. The protease was added at an approximate weight ratio of 100:1 (fusion protein:TEV) and digest was performed in buffer A overnight at 4 C. After concentration using Vivaspin concentrators (GE Healthcare) with a 3 kDa cut-off, the protein solution was loaded onto a S75 Superdex (GE Healthcare) column equilibrated in buffer A at 4 C. The purified P peptide was mixed with purified N-MBP, and the mixture was incubated overnight at 4 C. After concentration, the solution was loaded onto a S75 Superdex column equilibrated in buffer A. The fractions containing the N.sup.0-MBP-P complex were pooled, and the MBP tag was cleaved by incubation overnight at 4 C. in the presence of TEV protease at a weight ratio of 100:1. The solution was concentrated and loaded onto a S75 Superdex (GE Healthcare) column coupled to a short amylose resin (NEB) column equilibrated in buffer B to completely remove cleaved MBP. The fractions containing the N.sup.0-P complex were pooled and concentrated using Amicon concentrators (Millipore) with a 10 kDa cut-off. During the purification process, protein purity was checked by SDS-PAGE.
[0188] A construct comprising residues 1-100 (P.sub.100) of P was cloned in pET28 vector with a C-terminal His-tag and expressed in E. coli BL21 (DE3) Rosetta cells. To produce unlabeled P.sub.100, cells were grown at 37 C. in LB medium until O.D. reached 0.6, and protein expression was induced overnight at 20 C. by addition of isopropyl--D-thiogalactoside (IPTG) to a final concentration of 1 mM. For the .sup.13C-.sup.15N labeled P.sub.100, cells were grown in M9 minimal medium supplemented with MEM vitamins (Gibco), with 1.0 g.Math.L.sup.1 of .sup.15NH.sub.4Cl and 4.0 g.Math.L.sup.1 of .sup.13C glucose as previously described.sup.24. Cells were harvested and the pellet was suspended in buffer A supplemented with Complete protease inhibitor cocktail (Roche). Cells were disrupted by sonication, and the crude extract was cleared by centrifugation at 45,000 g at 4 C. for 20 min. The supernatant was loaded onto His Select resin (Sigma) column pre-equilibrated in buffer A. The column was washed with 10 volumes of buffer A containing 500 mM NaCl and 10 mM imidazole (Sigma) and the protein was eluted in buffer A containing 300 mM imidazole. The fractions containing the peptide were pooled and concentrated using Vivaspin concentrators (GE Healthcare) with a 5 kDa cut-off. The solution was loaded onto a S200 Superdex column equilibrated in buffer A at 4 C. Fractions containing the peptide were pooled and concentrated. For NMR experiments, the N.sub.32-402.sup.0-P.sub.100 complex was reconstituted as described above, and buffer A was exchanged with 20 mM Bis-Tris buffer at pH 6.0 containing 150 mM NaCl, 50 mM arginine, 50 mM glutamate and 0.5 mM TCEP.
[0189] To produce a selenomethionine substituted of N.sub.32-383, cells were grown at 37 C. in M9 minimal medium supplemented with MEM vitamins (Gibco), with 1.0 g.Math.L.sup.1 of NH.sub.4Cl and 2.0 g.Math.L.sup.1 of glucose until O.D. reached 0.6. Then, the temperature was lowered to 20 C. and the culture was supplemented with a mix of amino acids containing 100 mg Lys, 100 mg Phe, 100 mg Thr, 50 mg Ile, 50 mg Leu, 50 mg Val and 50 mg SelenoMet per liter of medium and incubated for 45 minutes. Protein expression was induced overnight at 20 C. by addition of isopropyl--D-thiogalactoside (IPTG) to a final concentration of 1 mM. The selenomethionine derivative was purified as described above.
[0190] SEC-MALLS Experiments.
[0191] Size exclusion chromatography (SEC) combined with on-line detection by multi-angle laser light scattering (MALLS) and refractometry (RI) is a method for measuring the absolute molecular mass of a particle in solution that is independent of its dimensions and shape.sup.25. SEC was performed with a S200 Superdex column (GE Healthcare) equilibrated with 20 mM Tris-HCl buffer containing 150 mM NaCl. The column was calibrated with globular standard proteins. Separations were performed at 20 C. with a flow rate of 0.5 mL.Math.min.sup.1. On-line multiangle laser light scattering (MALLS) detection was performed with a DAWN-HELEOS II detector (Wyatt Technology Corp.) using a laser emitting at 690 nm, and protein concentration was measured on-line by the use of differential refractive index measurements using an Optilab T-rEX detector (Wyatt Technology Corp.) and a refractive index increment, do/dc, of 0.185 mL.Math.g.sup.1. Weight-averaged molar masses (Mw) were calculated using the ASTRA software (Wyatt Technology Corp.). For size determination, the column was calibrated with proteins of known Stokes radius (R.sub.S).sup.26.
[0192] Small Angle X-Ray Scattering Experiments.
[0193] Small angle X-ray scattering (SAXS) data were collected at the BioSAXS beamline (BM29) of the ESRF (http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/BM29. The scattering from the buffer alone was measured before and after each sample measurement and was used for background subtraction using the program PRIMUS from the ATSAS package .sup.27. Scattering data were collected at different concentration ranging from 0.3 mg.Math.mL.sup.1 to 0.6 mg.Math.mL.sup.1 for P.sub.100 and from 0.55 mg.Math.mL.sup.1 to 2.4 mg.Math.mL.sup.1 for N.sup.0-P complex. No concentration-dependent inter-particle effect was observed. R.sub.g was estimated at low Q values using the Guinier approximation. Ah initio low-resolution bead models of the N.sup.0-P complex were computed from the distance distribution function P(r) (Dmax=10 nm) using the program DAMMIN.sup.28. 20 low-resolution models, obtained from independent reconstructions, were aligned, averaged and filtered with the program DAMAVER.sup.29. The crystal structure was docked within the envelop using the program SUPCOMB.sup.29.
[0194] NMR Spectroscopy.
[0195] The spectral assignment of P.sub.100 of NiV P protein was obtained at 25 C. in 20 mM Bis-Tris buffer at pH 6.0 containing 150 mM NaCl, 50 mM arginine, 50 mM glutamate and 0.5 mM TCEP using a set of BEST-type triple resonance experiments.sup.30. The NMR experiments were acquired at a .sup.1H frequency of 800 MHz. A total of six experiments were acquired: HNCO, intra-residue HN(CA)CO, HN(CO)CA and intra-residue HNCA, HN(COCA)CB and intra-residue HN(CA)CB. All spectra were processed in NMRPipe.sup.31, analyzed in Sparky.sup.32 and automatic assignment of spin systems was done using MARS.sup.33 followed by manual verification. The .sup.1H-.sup.15N HSQC spectrum of P.sub.100 was compared to the spectrum of purified N.sub.32-383.sup.0P.sub.100 complex. The intensity ratio of the resonances in the two spectra was used for mapping the binding site of N.sup.0 on P.sub.100. Chemical shifts depend on the backbone and dihedral angles, and in disordered systems, they are highly sensitive to the presence of transient secondary structure, commonly expressed in terms of a secondary structure propensity (SSP).sup.34,35. The SSP score for isolated P.sub.100 revealed the presence of several fluctuating -helices.
[0196] Crystallography.
[0197] We used different constructs of N and P to reconstitute N.sup.0-P analogs, but only the N.sub.32-383.sup.0P50 complex crystallized. Initial crystallization conditions for the N.sub.32-383.sup.0P50 complex were identified at the High Throughput Crystallization Laboratory of the EMBL Grenoble Outstation (https://htxlab.embl.fr). Plate clusters obtained in 22% PEG 3350, 0.2M KBr were used to grow crystal of selenomethionine derivative of the N.sub.32-383.sup.0P.sub.50 complex by the microseeding method. A plate cluster of native protein was crushed in 50 l of stabilization solution (20 mM Tris HCl at pH 8 containing 22% PEG 3350, 0.2 M KBr and 0.2 M NaCl,) using the Seed Bead kit (Hampton Research). The seed stock was serially diluted (5, 25, 100, 1000 times), and the drops were set by mixing 0.5 l of the resulting seed stock, 1 l of protein solution and 1 l of precipitant solution. The crystals used for data collection were obtained with protein concentrations of 10 to 20 mg.Math.mL.sup.1 in the presence of 16-18% PEG 3350 and 0.2 M KBr and were frozen with 15% glycerol as cryo-protectant. X-ray diffraction data were collected at the ID29 beamline of the ESRF at a wavelength of 0.9793 and at a temperature of 100 K and were processed with the XDS package.sup.36. Initial phases were obtained using the anomalous scattering from selenium atoms by the SAD method with the program HKL2MAP.sup.37. A model was initially constructed with the Autobuild program.sup.38 from the phenix suite.sup.39 and subsequently refined with the phenix.refine program.sup.40 and Coot.sup.41. The geometry of the final model was checked with MolProbity.sup.42. In the model, 97.0% of residues have backbone dihedral angles in the favored region of the Ramachandran plot, 2.77% fall in the allowed regions and 0.23% are outliers. Part of the .sub.N5-.sub.N6 loop is not visible in the crystal electron density. Figures have been generated with PyMol.sup.43 and Chimera.sup.44. Low frequency normal modes of N.sup.0 were computed with the Elastic Network Model.sup.45. Multiple sequence alignments were performed with MAFFT.sup.46.
[0198] Plasmid Construction.
[0199] Sequences corresponding to residues of NiV P, CDV P or MeV P were cloned in-frame with GFP into the pEGFP-C2 vector (CLONTECH Laboratories) to produce the construct pEGFP-P40 and derivatives (P6-40, P11-40; P1-20; P12-35 and P7-17 of NiV P and P1-22, P9-19 of CDV P and P9-19 of MeV P). NiV P variants P40-G10R and P40-I17R were obtained by site-directed mutagenesis using the QuickChange XL kit (Stratagene). The consensus peptide was produced by PCR cloning into the same pEGFP-C2 vector.
[0200] Intracellular Localization of N and P40.
[0201] HEK 293T cells were obtained from ATCC (HEK 293T/17-ATCC CRL-11268). Cell lines were routinely assayed for mycoplasma contamination. 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, PAA laboratories) supplemented with 10% fetal calf serum (FCS) (PerbioHyclone). For transfection, cells were grown for 24 h to a confluence of 50% and were transfected with 0.5 g of plasmid encoding N, GFP-P.sub.40-wt or both (or empty plasmid as control) using Turbofect transfection reagent (Thermoscientific) at 4:1 ratios of reagent:DNA as recommended. After 48 hours, cells were fixed in 3.7% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 45 min, then treated for 30 min with 50 mmol.Math.L.sup.1 NH.sub.4Cl, and finally, for another 40 min in 0.1% Triton X100-PBS. Immunofluorescence of N was performed with an in-house henipavirus specific rabbit anti-N antibody (at a 1/1000 dilution, antibody specificity was determined by immunofluorescence as shown and by Western blot and Alexa Fluor 555 secondary antibody (Life Technologies) at a 1/1000 dilution. 4,6-diamindino-2-phenylindole (DAPI) diluted in PBS containing 1% bovine serum albumin (BSA) was used for nuclear staining. After several washing steps, pictures were taken using a Zeiss 200M fluorescent microscope. Images were analyzed by Axiovision Software (Zeiss) and ImageJ software.sup.47.
[0202] Inhibition of Viral Replication.
[0203] All experiments with the Nipah virus were performed at INSERM Laboratoire Jean Mrieux (Lyon, France) in a biosafety level 4 (BSL-4) containment laboratory. HEK 293T cells were grown as described above for 24 h to a confluence of 40%. Initially, the cells were transfected with plasmids encoding w.t. P.sub.40 in fusion with GFP, variants of P.sub.40 (P.sub.40-G10R or P.sub.40-I17R in fusion with GFP or pEGFP alone as a control using Turbofect reagent as described above. In each case, the amount of plasmid was varied from 2 g to 0.125 g. 24 h after transfection, cells were infected with NiV (Malaysian isolate UMMC1) at a multiplicity of infection (MOI) of 0.01. 1 h post-infection (p.i.) virus inoculum was removed and replaced with DMEM media containing 3% FCS. Culture supernatants and cell lysates were collected at 48 h p.i. for TCID50 titration and virus growth was assessed visually by inspecting for syncytial formation. Images of GFP fluorescence were taken using a Zeiss 200M fluorescent microscope. Images were analyzed by Axiovision Software (Zeiss). For Krber TCID50 determination, serial ten-fold dilutions of viral culture supernatants were used to infect Vero E6 cells in the same way as described above and read 48 h p.i. Significant differences were calculated using an ANOVA test where applicable, n=6.
Example 2
[0204] Results
[0205] Reconstitution of a Functional NiV N.sup.0-P Core Complex
[0206] The inventors reconstituted several structural variants of NiV N.sup.0-P complex using peptides encompassing the N.sup.0-binding region of P and recombinant N molecules, truncated at the NT.sub.ARM and the CT.sub.ARM and N.sub.TAIL. By size exclusion chromatography (SEC) combined with multi-angle laser light scattering (MALLS) (
[0207] The inventors mapped the region of P that directly interacts with N.sup.0 by nuclear magnetic resonance (NMR) spectroscopy. To this purpose, we expressed and purified a peptide of 100 amino acids corresponding to the N-terminal region of P (P.sub.100) and characterized its structural properties. By SEC-MALLS, we showed that the peptide is monomeric in solution and that both its hydrodynamic radius measured by SEC and its radius of gyration measured by SAXS were larger than expected for a globular protein of this molecular mass. In addition, the poor chemical shift dispersion of amide resonances in the heteronuclear single quantum coherence (HSQC) NMR spectrum was typical of disordered protein, but after assigning the NMR spectrum, the secondary structure propensity (SSP) parameter calculated from C and C secondary chemical shifts, indicated the presence of five fluctuating -helices. We then analyzed the HSQC spectrum of P100 bound to N.sub.32-383. In a complex of this size (50 kDa), NMR signals are strongly broadened in protonated samples, precluding their detection, but in the HSQC spectrum we observed resonances corresponding to residues 50 to 100, indicating that this region remains flexible in the complex and that the N.sup.0-binding region is comprised within the first fifty N-terminal amino acids of P (
[0208] Accordingly, The inventors demonstrated that a peptide corresponding to the first forty residues of P (P.sub.40) is sufficient to maintain N in a soluble form in vitro (
[0209] Crystal Structure of NiV N.sup.0-P Core Complex
[0210] The NiV N.sub.32-383.sup.0-P.sub.50 complex crystallized in space group P2.sub.12.sub.12.sub.1 with three heterodimers in the asymmetric unit. We determined the structure at 2.5 resolution by the SAD method (
[0211] The N-terminal chaperone region of P is stabilized upon binding to its N.sup.0 partner, but only the first 35 residues of P, corresponding to the first fluctuating helix observed in solution (helix .sub.P1), were visible in the crystal structure of the N.sub.32-383.sup.0-P.sub.50 complex. In the complex, this region formed a 2.9 nm-long helix (helix .sub.P1a: aa 1-19) with a 90 kink at residue N20 leading to a short helix (helix .sub.P1b: aa 21-28) (
[0212] NiV N is in an Open Conformation in N.sup.0-P Complex
[0213] By comparing the structure of NiV N.sub.32-383.sup.0-P.sub.50 with that of RSV N in complex with RNA.sup.5, we found that the fold of N is conserved (
[0214] RNA Binding and the Rule of Six
[0215] In RSV NCs, each N interacts with seven nucleotides (nt) and base 1 packs on the flat surface of helix .sub.N9 formed by two glycine residues (G241 and G245) (
[0216] Conservation of the N.sup.0-P Binding Interface
[0217] NNV phosphoproteins vary greatly in length and amino acid sequence.sup.22, with sequence conservation generally becoming undetectable beyond the family level. However, a recent study identified residues in the N-terminal region of P that are conserved among most members of the Paramyxoviridae in spite of an overall distant evolutionary relationship.sup.23. Most of these conserved residues appeared to be key residues for the interaction with N.sup.0 (
[0218] Inhibition of NiV Replication
[0219] The inventors found that expression of GFP-fused P.sub.40 peptide in human cells (HEK293T) prior to infection significantly inhibits viral growth in a dose-dependent manner and abolishes syncytia formation, the latter being a hallmark of NiV infection (
[0220] The Chaperone Functions of P
[0221] To understand the chaperone functions of P.sub.NTR, we used RSV N-RNA complex as a model for NiV N-RNA complex (
DISCUSSION
[0222] The inventors present the structure of the N.sup.0-P core complex of Nipah virus, revealing that unassembled N.sup.0 is maintained in an open conformation and providing experimental evidence that NNV N switches between open and closed conformations during NC assembly. In the N.sup.0-P complex, the N-terminal N.sup.0-binding region of P prevents N polymerization by occupying the binding sites for the exchanged subdomains of adjacent N and prevents RNA encapsidation by bridging N.sub.CTD and hindering closure of the molecule. We propose a possible scenario for the assembly of N.sup.0 molecules along newly synthesized viral RNA by a concerted mechanism of transfer of N.sup.0 from the N.sup.0-P complex to the nascent RNA molecule, which involves the release of P and the closure of the RNA binding groove. In a first step, we assume that encounter complex forms with the RNA molecule loosely inserted in the open cavity. In a second concerted step, P is released and N grasps the RNA molecule. The release of P from the RNA-bound N liberates the binding site for the NT-arm of the next incoming N molecule. Upon formation of the encounter complex with the next N.sup.0-P complex, the NT.sub.ARM of the incoming N can bind to the previously bound N. The CT.sub.ARM of bound N can bind to the incoming N and help in displacing the P peptide. In a second or concomitant process, P is released and N closes on the RNA. The NT.sub.ARM of the first bound N molecule locks the second N in its closed conformation by bridging N.sub.NTD with N.sub.CTD.
[0223] The inventors confirmed that the short N.sup.0-binding region of P is sufficient to chaperone N.sup.0 and to keep it in a soluble form, but P.sub.40 inhibited viral replication, indicating that the N-terminal region of P is not sufficient to enable NC assembly and suggesting the involvement of other regions of P in this process. P is a multifunctional, highly flexible molecule, which also possesses binding sites for L or for NCs, and it is thus plausible that interactions with these other viral proteins are necessary to correctly position the N.sup.0-P complex at the site of viral RNA synthesis. The attachment of N.sup.0-P to the NC, as suggested in
[0224] The successful inhibition of NiV infection by the N.sup.0-binding peptide of P suggests that the P binding cavity in N can be specifically targeted for designing inhibitors of NiV replication. The structure of the N.sup.0-P core complex provides the structural basis for designing small molecules that could prevent the formation of the complex. The strong conservation of the binding interface suggests that NiV N.sub.32-383.sup.0-P.sub.50 structure is a good structural model for the N.sup.0-P complex of other medically-relevant paramyxoviruses and that possibly a broad spectrum drug might be developed against several viruses.
Example 3
[0225] Materials and Methods:
[0226] Plasmid Construction.
[0227] Sequences corresponding to residues of NiV P or CDV P (reference strain, Genbank NC_001921.1) were either cloned in-frame with GFP into the pEGFP-C2 vector (CLONTECH Laboratories) to produce the constructs pEGFP-P40-NIV and pEGFP-P40-CDV, or into the vector pCG (refDifferential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Tanaka M, Herr W. Cell. 1990 February 9; 60(3):375-86. 10.1016/0092-8674(90)90589-7) containing a GFP-IRES-multicloning site. This vector allows the simultaneous expression of both GFP and p40 peptide separately but from the same bi-cistronic RNA transcript after transfection into mammalian cells.
[0228] Inhibition of Viral Replication with NIV and CDV.
[0229] 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, PAA laboratories) supplemented with 10% fetal calf serum (FCS) (PerbioHyclone). All experiments with the Nipah virus were performed at INSERM Laboratoire Jean Mrieux (Lyon, France) in a biosafety level 4 (BSL4) containment laboratory. All experiments with the canine distemper virus were performed at under biosafety level 2 confinement in a cell culture biosafety cabinet reserved for work with infectious material. Cells were grown as described above for 24 h to a confluence of 40%. Initially, the cells were transfected with plasmids encoding w.t. P.sub.40-NIV in fusion with GFP (pEGFP vector) or expressed separately (from the pCG bi-cistronic IRES vector, described above), or w.t. P.sub.40-CDV in fusion with GFP (pEGFP vector) or either pEGFP/pCG alone as control, using Turbofect following manufacturer's recommendations. 24 h after transfection, cells were infected with either NiV (Malaysian isolate UMMC1) or CDV at a multiplicity of infection (MOI) of 0.01. 1 h post-infection (p.i.) virus inoculum was removed and replaced with DMEM media containing 3% FCS. Culture supernatants and cell lysates were collected at 48 h p.i. for TCID50 titration and virus growth was assessed visually by inspecting for syncytial formation. Pictures of GFP fluorescence were taken using a Zeiss 200M fluorescent microscope. Images were analyzed by Axiovision Software (Zeiss). For Krber TCID50 determination, serial ten-fold dilutions of viral culture supernatants were used to infect Vero E6 cells in the same way as described above and read 48-72 h p.i.
[0230] Results:
[0231] Inhibition of NiV or CDV Replication by a N.sup.0-Binding Peptide of P.
[0232] The inventors demonstrated that peptides derived from the N.sup.0-binding region of P (P.sub.40) from NiV inhibit viral growth of both NiV and CDV (
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