Method for producing self-assembling paramyxoviral nucleocapsid-like particles and their uses

Abstract

Embodiments of the disclosure relate to the domain of virology of Paramyxoviruses. The disclosure concerns a method for producing self-assembling paramyxoviral particles and a method for identifying a compound able to inhibit the replication or the transcription of a Paramyxovirus. The disclosure also pertains to the nucleocapsid-like particles obtainable by the method of the invention and their use for biotechnological and pharmaceutical applications.

Claims

1. A method for producing self-assembling paramyxoviral nucleocapsid-like particles comprising the steps of: a. co-expressing recombinant nucleoprotein (N) and phosphoprotein (P) proteins in order to allow the formation of NºP complexes wherein: i. the recombinant N protein comprises an N.sub.CORE domain including a C-terminal domain (CTD) arm and an N-terminal domain (NTD) arm, and ii. the recombinant P protein comprises an N-binding domain; b. purifying NºP complexes, the purified NºP complexes comprising recombinant N and P proteins only and being RNA-free; c. adding RNA molecules of interest to the purified NºP complexes, wherein the RNA molecules of interest comprise at least 6 nucleotides and are not poly-U homopolymers; and d. recovering the resulting nucleocapsid-like particles.

2. The method of claim 1 wherein the recombinant N and P proteins correspond to proteins from measles virus, wherein recombinant N protein corresponds to SEQ ID NO.4 or SEQ ID NO.5 and recombinant P protein comprises SEQ ID NO.2.

3. A paramyxoviral nucleocapsid-like viral particle produced by the method of claim 1, wherein said nucleocapsid-like viral particle comprises only one specific type of RNA molecule of interest, and wherein said RNA molecule of interest is a synthetic RNA molecule.

4. The nucleocapsid-like viral particle of claim 3 wherein the N protein comprises a disordered C-terminal domain which is functionalized with a group selected from a ligand for a receptor, a dye compound, a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins, and a protein chimerically attached to N.sub.TAIL.

5. A method for identifying a compound able to inhibit the replication or transcription of a Paramyxovirus, wherein such compound is identified by its ability to abrogate the assembly of the nucleocapsid-like particles according to claim 1.

6. A method for identifying a compound able to inhibit the replication or transcription of a Paramyxovirus comprising the steps of: a. co-expressing recombinant N and P proteins in order to allow the formation of NºP complex wherein: i. the recombinant N protein comprises an N-core domain including a CTD arm and an NTD arm, and ii. the recombinant P protein comprises an N-binding domain; b. purifying NºP complexes, the purified NºP complex comprising recombinant N and P proteins only and being RNA-free; c. adding a compound to be tested; d. adding a RNA molecule wherein said RNA molecule comprises at least 6 nucleotides and is not a poly-U homopolymer; e. detecting the presence of nucleocapsid-like particles in comparison with a control wherein no compound is present; and f. identifying a compound able to inhibit the replication or transcription of a Paramyxovirus, where the assembly of nucleocapsid-like particles is inhibited in the presence of such compound, compared to control.

7. The method of claim 6 wherein identifying the compound in step (f) is performed using a fluorescent read-out assay, in particular a high-throughput assay.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Characterization of the N.sub.2-405ºP.sub.1-50 complex. (a) SDS-PAGE gel (Coomassie blue staining) showing the purity of N.sub.1-405ºP.sub.1-50 after cleavage by the TEV protease, Ni-affinity and size exclusion chromatography. P.sub.1-50 being very small and weakly stained, only N is visible on the gel. Left lane shows molecular weight markers and numbers indicate the size of the respective bands (kDa). (b) Gel filtration profile of N.sub.1-405°P.sub.1-50 showing the UV absorption profiles at 260 and 280 nm demonstrating that the complex does not contain RNA. (c) Size exclusion chromatography combined with detection by multi-angle laser light scattering (MALLS) and refractometry of N.sub.1-405ºP.sub.1-50 showing a mass of 53.7 kDa (expected mass for a heterodimeric complex: 52.6 kDa).

(2) FIG. 2: Cartoon illustrating NMR signals that appear or disappear upon assembly of NC-like particles. In the Nip complex, both NTD.sub.ARM and CTD.sub.ARM of N are flexible, giving rise to observable backbone resonances in .sup.1H-.sup.15N NMR spectra. P.sub.1-50 is bound to N so that peaks are too weak to be observed. As N assembles, NTD.sub.ARM and CTD.sub.ARM stabilize the NC-like particles so that these peaks disappear, while resonances from P.sub.1-50 grow as more N proteins associate.

(3) FIG. 3: a) Part of the .sup.1H-.sup.15N spectrum of N.sub.1-405ºP.sub.1-50, P.sub.1-50 and N.sub.1-405ºP.sub.1-50 after incubation with 5′-RNA.sub.60 for 24 hours at 25° C. Assignments are shown for the N.sub.1-405ºP.sub.1-50 and P.sub.1-50 resonances. Subscript T refers to residues associated with cleavage or affinity tags. b) Assembly of NC-like particles from N.sub.1-405°P.sub.1-50 with 5′-RNA.sub.6 was initiated from 209 μM N.sub.1-405ºP.sub.1-50, adding 5′-RNA.sub.6 to 471 μM. Circles: intensities measured in SOFAST HMQC NMR spectra and summed over appearing P.sub.1-50 (left) or disappearing N.sub.1-405 (right) resonances (P.sub.1-50: 4, 5, 6, 22, 25, 38, N.sub.1-405: 28, 385, 386, 387, 388, 389, 404, 405), lines: simultaneous fit to a double-exponential with common assembly rates.

(4) FIG. 4: Following NC assembly by electron microscopy. Negative staining electron micrographs of N.sub.1-405ºP.sub.1-50 in the presence of a 6 nucleotide RNA from the 5′ end of the measles virus genome. Images were taken at different time points following mixing of the two components in vitro.

(5) FIG. 5: NC assembly in vitro reproduces NC assembly in vivo. (A) Negative staining electron micrograph of N.sub.1-405ºP.sub.1-50 in the presence of a 6 nucleotide RNA from the 5′ end of the measles virus genome (negative staining). (B) Electron micrographs of trypsin digested nucleocapsids purified from E. coli genetically engineered to overexpress N protein.

(6) FIG. 6: Dependence of NC-like particles assembly on RNA sequence. (a-c) N.sub.1-405ºP.sub.1-50 was incubated with different RNAs for 24 hours at room temperature and visualized by negative stain electron microscopy. Assembly of NC-like particles is observed for a) 5′-RNA.sub.6, b) polyA-RNA.sub.6, while no assembly is observed for c) polyU-RNA.sub.6; (d-f) Fluorescence anisotropy during 2000 seconds after addition of 500 nM for d) 5′-RNA.sub.10-FAM (ACCAGACAAA, SEQ ID NO: 18), e) polyA.sub.10-FAM (AAAAAAAAAA, SEQ ID NO: 19) and f) polyU.sub.10-FAM (UUUUUUUUUU, SEQ ID NO: 20) to 23 μM N.sub.1-405ºP.sub.1-50.

(7) FIG. 7. NC assembly in vitro. N.sub.1-405ºP.sub.1-50 was incubated with different RNAs for >12 hours at room temperature and visualized by electron microscopy. Assembly of NC-like particles is observed for 5′-RNA.sub.6 ACCAGA, 5′-RNA.sub.60 (SEQ ID NO: 8), AAAAAA, ACCUGA, UCCAGA, ACUAGA and AUCAGA while no assembly is observed for UUUUUU.

EXPERIMENTAL PART

Example 1: Constructs for Expression of P Peptide

(8) Materials and Methods

(9) The measles virus (Edmonston strain; UniProt Q83623) phosphoprotein (also called P) used in this work corresponds to SEQ ID NO: 1. The first 50 amino acids of the P.sub.1-50 corresponds to SEQ ID NO 2.

(10) P.sub.1-100: DNA of first 100 amino acids of measles virus P protein were cloned into a pET41c (+) vector between NdeI and XhoI sites resulting in a C-terminal His-tagged construct.

(11) Escherichia coli Rosetta™ (λDE3)/pRARE strain (Novagen) were used for the production of P.sub.1-100. The protein was purified by nickel resin chromatography, followed by size exclusion chromatography.

(12) Results

(13) NMR backbone resonances from the first 300 amino acids of P were assigned using standard three-dimensional heteronuclear NMR. The majority of the chain exhibits conformational propensities that are characteristic of an unfolded chain, with the exception of the first 40 amino acids, that shows a slightly elevated additional α-helical propensity. Although sequence homology is negligible, this N-terminal localization coincides with the position of a P peptide that was recently co-crystallised with an N and C-terminal deleted N protein from Nipah virus by the inventors..sup.8 This region was targeted as the putative N-binding site. The inventors therefore co-expressed MeV P.sub.1-100 with N, resulting in a soluble and stable complex N.sub.COREºP.sub.1-100. The binding site was then delineated more precisely by titrating .sup.15N/.sup.13C labeled P.sub.1-150 into N.sub.COREºP.sub.1-00, showing localized exchange in the region comprising residues 1-50.

(14) This entire procedure resulted in identification of the minimal P peptide (P.sub.1-50 of SEQ ID NO: 2) required to form the complex.

Example 2: In Vitro Co-Expression of N and P Proteins as a Heterodimeric Complex

(15) Materials and Methods

(16) The measles virus nucleoprotein (also called N) (Edmonston strain; UniProt 089933) used in this work comprises 525 amino acids and corresponds to SEQ ID NO: 4.

(17) Constructs

(18) The first 50 amino acids of measles virus phosphoprotein (Edmonston strain; UniProt Q83623) in fusion with the 525 or 405 amino acids of measles virus nucleoprotein (Edmonston strain; UniProt Q89933) with a TEV protease cleavage site inserted between the two (ENLYFQG) were cloned into a pET41c (+) vector following a two step PCR reaction: Step 1: The PCR products for P peptide and N peptide were obtained separately using the following amplification primers.

(19) TABLE-US-00002 For P.sub.1-50: Forward: (SEQ ID NO: 9) 5′-GGAATTCCATATGGCAGAGGAGCAGGCACGCCATGTCA-3′ Reverse: (SEQ ID NO: 10) 5′-CATGCCCTGAAAATACAGGTTTTCGCAGGTGGCTCGCTCC-3′ For N.sub.1-525: Forward: (SEQ ID NO: 11) 5′-GCCACCTGCGAAAACCTGTATTTTCAGGGCATGGCCACACTTTTAA G-3′ Reverse: (SEQ ID NO: 12) 5′-CCGGTCGACGTCTAGAAGATTTCTGTCATTGTACACTATAGGGGT G-3′ For N.sub.1-405: Forward primer identical to SEQ ID NO: 11. Reverse N: (SEQ ID NO: 13) 5′-GCGTCGACCTTGTTCTCAGTAGTATGCATTGCAATCTCTG-3′

(20) The resulting PCR products were purified on an agarose gel. Step 2: The purified PCR products obtained at step 1 were mixed in a stoichiometric ratio (about 10 ng of N with about 50 ng of P), heated to 95° C. during 5 min and cooled down to 20° C. during 30 min. The mix was supplemented with 2× MasterMix (Fermentas™) and submitted to 5 cycles: 95° C. 45 s 72° C. 1.5 min.

(21) The combined product was then purified (PCR purification kit—Qiagen™) and PCR amplified with the external primers used previously:

(22) Forward: 5′-(SEQ ID NO: 9) and Reverse: 5′-(SEQ ID NO: 12) to yield N.sub.1-525.sup.0P.sub.1-50. For N.sub.1-405.sup.0P.sub.1-50, SEQ ID NO: 12 was replaced by SEQ ID NO: 13 in the final amplification step.

(23) The resulting products were purified on an agarose gel, digested with NdeI and SalI enzymes and inserted between the NdeI and XhoI sites of a digested pET41c (+) plasmid.

(24) Expression and Purification of NºP

(25) Escherichia coil Rosetta™ (λDE3)/pRARE strain (Novagen) were also used for the production of N.sub.1-525.sup.0P.sub.1-50 and N.sub.1-405.sup.0P.sub.1-50.

(26) For protein expression, cultures were grown at 37° C. in LB until OD600=0.6 was observed, then the temperature was lowered to 20° C. and expression induced with 1 mM IPTG. Cells were harvested after 12-14 hours. For expression of labelled protein, initial 4 liter cultures were grown in LB medium and the cells transferred into 1 liter M9 medium at an OD600 of 0.6. The cells were then grown for an additional 1 hour at 20° C. before induction.

(27) For protein purification, cells were lysed in 20 mM Tris pH 8, 150 mM NaCl, 1 tablet Roche complete EDTA-free protease inhibitors, 1 spatula tip of lysozyme by sonication. Cell debris was harvested by centrifugation and supernatant loaded on Ni-beads (His-select, Sigma Aldrich™). The flow through was discarded and beads were washed with 20 mM Tris pH 8, 150 mM NaCl, 8 mM imidazole. The protein was eluted from the beads in 20 mM Tris pH 8, 150 mM NaCl, 400 mM imidazol. TEV cleavage was set up overnight and at the same time the protein was dialyzed against 20 mM Tris pH 8, 150 mM NaCl, 5 mM beta-mercaptoethanol (BME). The protein was then purified by gel filtration (Superdex 200 column, GE Healthcare™) equilibrated with either the same buffer or the NMR buffer (50 mM phosphate pH 7, 150 mM NaCl, 5 mM BME).

(28) Cleavage with TEV gave the following proteins in complex NºP for N.sub.1-525.sup.0P.sub.1-50:

(29) P.sub.1-50 with a TEV peptide amino acid sequence is represented by SEQ ID NO: 3.

(30) N.sub.1-525 with an Histidine tag is represented by SEQ ID NO: 6.

(31) And for N.sub.1-405.sup.0P.sub.1-50, cleavage with TEV gave the following proteins:

(32) P.sub.1-50 with a TEV peptide amino acid sequence is represented by SEQ ID NO: 3.

(33) N.sub.1-405 with an Histidine tag is represented by SEQ ID NO: 7.

(34) Results

(35) Coexpression of constructs containing the P.sub.1-50 peptide and either full length N (N.sub.1-525) or the folded domain of N (N.sub.1-405), separated by a TEV cleavage site resulted in high yield, heterodimeric, soluble and stable NºP complex. Small angle X-ray scattering and multi-angle laser light scattering (MALLS) of N.sub.1-405ºP.sub.1-50 or N.sub.1-525ºP.sub.1-50 demonstrate that both constructs contain a heterodimer of monomers of P.sub.1-50 and N.sub.1-525 or N.sub.1-405 (FIG. 1).

(36) This result demonstrates that the inventors have successfully produced a heterodimeric, soluble and stable NºP complex. It constitutes a rational basis for the design of a tool with which to mimic the initial steps of the viral replication cycle, by stabilizing for the first time the chaperoned state of N that precedes NC formation in solution, while respecting the integral native sequence of N. In order to demonstrate the potential of such a tool, the inventors have further investigated the interaction of this complex with RNA as described thereafter.

Example 3: Production of NC-Like Particles

(37) Materials and Methods

(38) RNA molecules added to the NºP complex

(39) RNA was added to the NºP complexes by titrating a solution of the two following particular RNA sequence: 6 nucleotide long RNA: 5′ OH-ACCAGA-OH 3′ (RNA.sub.6) 60 nucleotide long RNA:

(40) TABLE-US-00003 5′ OH-ACCAGACAAAGCUGGGAAUAGAAACUUCGUAUUUUCAAGUUUUC UUUAAUAUAUUGCAA-OH 3′ (naked RNA corresponds to SEQ ID NO: 8) 10 nucleotide long RNA containing a fluorescein attached at its 3′end: 5′ OH-ACCAGACAAA-FAM (naked RNA corresponds to SEQ ID NO: 14).

(41) NC-like particles can then be further purified using the methods described in reference 4.

(42) NMR Spectroscopy

(43) The assembly of NC-like particles was followed by real-time NMR using a series of SOFAST .sup.1H-.sup.15N HMQC experiments using a sample of 200 μM NºP complex (N.sub.1-405ºP.sub.1-50 or N.sub.1-525ºP.sub.1-50) The formation of NC-like particles was initiated by addition of RNA.sub.6 or RNA.sub.60 (SEQ ID NO: 8) reaching a total concentration of 20 μM (RNA.sub.60) or 150 μM (RNA.sub.6). A series of SOFAST HMQC experiments were recorded with 100 complex points in the indirect dimension, a 200 ms recycling delay and 4 transients providing a time resolution of 4 minutes. The spectra were recorded on a Bruker spectrometer operating at a .sup.1H frequency of 950 MHz at 25° C. in a buffer consisting of 50 mM phosphate buffer, 150 mM NaCl, 5 mM BME at pH 7.0. In addition, two additional SOFAST HMQC experiments were recorded in the absence of RNA and at the end of the time course (after 24 h) with 256 complex points in the indirect dimension and 16 transients (FIG. 3A).

(44) Electron Microscopy

(45) The formation of NC-like particles was followed by negative staining electron microscopy. Initially, a control measurement was performed of the N.sub.1-405ºP.sub.1-50 complex in the absence of RNA.sub.6 showing no formation of NC-like particles. The N.sub.1-405ºP.sub.1-50 was then mixed with RNA6 and the sample was visualized by electron microscopy. The sample contained 20 μM of N.sub.1-405ºP.sub.1-50 and 50 μM of RNA.sub.6 in the same buffer as used for the NMR experiments (see above).

(46) Fluorescence Spectroscopy

(47) N.sub.1-405ºP.sub.1-50 or N.sub.1-525ºP.sub.1-50 was diluted to the desired concentration into 50 mM Na-phosphate pH 7, 150 mM NaCl, 5 mM β-mercaptoethanol (β ME) directly into the fluorescence cuvette. RNA.sub.10-FAM (SEQ ID NO: 14) was added to NºP immediately to a final concentration of 500 nM prior to kinetics acquisition, mixed quickly through pipetting and fluorescence kinetics were recorded at an emission wavelength of 520 nm upon excitation with 470 nm light. Parallel and perpendicular polarization directions were recorded alternatingly and used pairwise to calculate the fluorescence anisotropy r

(48) $r=\frac{I_{.Math..Math.}-{\mathrm{GI}}_{\perp }}{I_{.Math..Math.}+2{\mathrm{GI}}_{\perp }}$
with the G-factor correcting for detection differences between parallel (I.sub.1) and perpendicular (I.sub.⊥) polarized fluorescence light. G was determined on a daily basis according to standard protocols, and remained stable between days. Fluorescence spectra were recorded at the end of the assembly kinetics with an excitation wavelength of 460 nm and an emission range of 470-650 nm.

(49) Protocol for Application to Other Paramyxoviruses

(50) For Nipah, the claimed method can be carried out using the following sequences:

(51) For N peptide, the full-length protein (1-532) corresponds to SEQ ID NO: 15.

(52) One could use either the amino acids 1-532 of the full-length protein or the amino acids 1-400 corresponding to the folded domain. For P peptide, one could use a peptide including the minimal N-binding domain which corresponds to residues 1-50 of the total P peptide represented by SEQ ID NO: 16. The peptide consisting of the residues 1-50 of the P peptide is represented by SEQ ID NO: 17.

(53) Results

(54) Analysis by NMR Spectroscopy

(55) Using NMR spectroscopy, the inventors were able to follow the evolution in real time of the NMR spectra as the P.sub.1-50 peptide is released from the NºP complex and the NC-like particles are formed. This result is observed either by following the increasing intensity of peaks corresponding to the P.sub.1-50 peptide, or by observing the reduction in intensity of peaks associated with the N- and C-terminal tails of the N protein that become immobile upon assembly of the NC-like particles, according to the mechanism illustrated in FIG. 2. A time trace of the formation of the NC-like particles was obtained by extracting the NMR signal intensities from each of the SOFAST HMQC spectra and plotted as function of time after addition of the RNA (FIGS. 3B and 3C). After a rapid growth phase (<30 minutes) the assembly process is nearly complete within approximately 400 minutes. This result demonstrates that NMR allows to follow the formation of NC-like particles in vitro using only short RNA molecules and the NºP complex.

(56) Analysis by Electron Microscopy

(57) Electron microscopy was carried out on samples immobilized at increasing times after addition of small (6 or 60 nucleotides in length) RNA molecules.

(58) The N.sub.1-405ºP.sub.1-50 mixed with RNA.sub.6 was visualized by electron microscopy after 25 minutes and 67 minutes as well as after 24 hours showing the presence of NC-like particles of increasing lengths (FIG. 4). These results confirmed the NMR-detected time course. Remarkably, even in the presence of relatively short RNA, NC-like particles were formed whose electron micrographs were indistinguishable from NC-like particles purified from E. coli (FIG. 5). These results demonstrate that EM allows to follow the formation of NC-like particles in vitro using only short RNA molecules and the NT complex.

(59) Analysis by Fluorescence Spectroscopy

(60) Fluorescence anisotropy also reports on assembly, using fluorescein labeled RNA (SEQ ID NO: 14). Fluorescein attached to the RNA alone undergoes fast rotation, and exhibits a small fluorescence anisotropy when the RNA is unbound. Upon binding to N, NC-like particle formation and subsequent elongation, its rotational freedom is significantly hindered, and fluorescence anisotropy increases gradually, giving rise to anisotropy curves that describe the NC-like particle assembly.

(61) Different RNA sequences were tested for their ability to facilitate NC-like particle assembly in vitro from P.sub.1-50N.sub.1-405. Homopolymers comprising purines or pyrimidines alone, UUUUUU (polyU-RNA.sub.6), AAAAAA (polyA-RNA.sub.6) were compared, as well as genomic 5′-RNA.sub.6. Remarkably, efficiency of assembly is sequence-dependent (FIG. 6). No assembly is observed for the polyU-RNA.sub.6 samples (FIG. 6c, f), while regular NC-like particles are obtained for 5′-RNA.sub.6 and polyA-RNA.sub.6 (FIGS. 6a, d and 6b, e respectively) with lengths reaching 1-2 μm. This differential ability to form NC-like particles was verified by NMR (data not shown). With this respect, the inventors have also noted that the 5′ sequences of both genome and anti-genome of paramyxovirinae shows strong conservation of ACCA in the first four nucleotides, followed by positions 5 and 6 occupied mainly by A/G. Indeed, the 5′ sequence of the measles genome leads to formation of NCs with high efficiency.

CONCLUSION

(62) It is therefore possible, using the described method, to both detect and monitor NC assembly in vitro from recombinantly-expressed protein and RNA alone, in the absence of other viral or cellular partners.

REFERENCES

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