Patent Application
20190292229
The invention pertains to the domain of virology of Paramyxoviruses. The invention 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 invention also pertains to the nucleocapsid-like particles obtainable by the method of the invention and their use for biotechnological and pharmaceutical applications.
Paramyxoviridae comprise dangerous human pathogens, for example Measles, an important human pathogen that is responsible for 150000 deaths per year, but also Nipah and Hendra viruses, whose infection of humans has been exacerbated by urbanization of the natural hosts (fruitbats) due to the destruction of their natural habitats (deforestation). Paramyxoviruses, and the closely related filoviruses (including Ebola) therefore present an increasingly serious risk to human health. Paramyxoviruses are non-segmented negative strand RNA viruses that express their own machinery for transcription and replication. Paramyxoviral genomes are packaged into large helical nucleoprotein assemblies, termed nucleocapsids (NC), comprising a multitude of copies of the nucleoprotein (N) that bind the entire sequence of the viral genome1. The N-RNA complex provides the template for replication and transcription by the viral polymerase complex consisting of the large protein (L) carrying the enzymatic activity and its co-factor phosphoprotein (P). N is a two-domain protein, comprising an RNA binding domain (NCORE), approximately 400 amino acids (aa), and a 125 aa intrinsically disordered C-terminal domain (NTAIL)2.
Paramyxoviral NCs have been studied in great detail using structural biology techniques such as electron microscopy3-7, X-ray crystallography8 and nuclear magnetic resonance spectroscopy9,10 They are now understood quite well in terms of three-dimensional structure. While knowledge of the three dimensional structures can help in rational drug design, successful attempts to crystallize monomeric nucleoproteins of paramyxoviruses have required the cleavage of flexible regions of the protein8, to enhance packing in a crystalline lattice. However, these constructs do not assemble into NCs.
NC-like assemblies have been purified from paramyxoviruses, or expressed in E. coli or insect cells11-13. These expressed particles do not specifically encapsulate a given RNA of interest but they assemble on cellular RNA. It has not yet been possible to reproduce the assembly process in vitro. The mechanism of assembly of NCs from these viral families remains one of the outstanding questions and potentially most exciting avenues for pharmaceutical intervention. The rational development of inhibitors of NC assembly is not possible if one cannot observe the process of assembly.
The inventors have now described an in vitro system wherein the self-assembling of the NC can be observed. They have designed new constructs of the so-called “NºP complex”, a complex comprising elements from N and P, that can be solubilized in a heterodimeric form in vitro (
The present invention thus holds great promise for the development of targeted drugs against paramyxoviral diseases and also provides NC-like particles that could be useful in the development of vaccines.
The present invention relates to the process of assembling NCs of Paramyxoviruses in vitro.
The inventors have successfully produced the NºP complex, a molecular association of N with a peptide derived from the P protein, in vitro. They have demonstrated that, provided that monomeric N is stabilized in an appropriate soluble form, NC-like particles can be assembled from recombinantly-expressed protein and RNA alone, in the absence of other viral or cellular partners.
Although NC-like particles from paramyxoviruses can be purified by over-expressing N in recombinant systems,4 for example in E. coli, where N assembles on cellular RNA, the rational development of inhibitors of replication or transcription has been severely hampered by the inability to solubilize NºP and subsequently assemble supramolecular NC-like particles in vitro as multiple copies of N bind to the RNA.
The present invention relies on the demonstration that the 300 aa disordered N-terminal domain of P binds NCORE, to chaperone monomeric RNA-free N prior to NC assembly on the RNA genome in the so-called NºP complex (
A first object of the present invention consists of a method for producing self-assembling paramyxoviral NC-like particles.
The method for producing self-assembling paramyxoviral NC-like particles of the invention comprises the steps of:
(a) co-expressing recombinant N and P peptides in order to allow the formation of NºP complexes where the N peptide comprises at least the NCORE domain including the CTD arm and NTD arm, and the P peptide comprises at least the N-binding domain;
(b) adding RNA molecules to the NºP complex, wherein said RNA molecules comprise at least 6 nucleotides; and
(c) recovering the resulting NC-like particles.
As demonstrated by the inventors, CTD arm and the NTD arm are required to interact with the neighboring N molecules within the NC and thus, the presence of the two arms of the NCORE protein is crucial for assembly of the N proteins into NC-like particles.
In an embodiment, the invention relates to the method for producing self-assembling paramyxoviral
NC-like particles as described above, wherein the recombinant N peptide comprises the full-length nucleoprotein or at least the amino acids 1-405 corresponding to the NCORE domain of the nucleoprotein.
In particular, the recombinant N peptide is from 405 to 532 amino acids in length, preferably from 405 to 525 amino acids in length. For example, the recombinant N peptide comprises at least the NCORE domain of the nucleoprotein and has a length of 405, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 525, 530 or 532 amino acids.
In an embodiment, the invention relates to the method for producing self-assembling paramyxoviral NC-like particles as described above, wherein the recombinant P peptide comprises the full-length phosphoprotein or at least the amino 1-50 of the phosphoprotein.
In particular, the recombinant P peptide is from 50 to 709 amino acids, preferably from 50 to 507 amino acids in length, more preferably from 50 to 300 amino acids in length. For example, the recombinant P peptide comprises at least the amino 1-50 of the phosphoprotein and has a length of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 307, 350, 375, 400, 450, 500, 507, 550, 600, 650, 700 or 709 amino acids.
In the preferred embodiment, the NºP complexes are purified before adding RNA. In this case, the NC-like particles contain a chosen RNA. In this embodiment, the addition of RNA to the NºP complex is not achieved in cells since RNA is added to a solution of the purified NºP complex. This embodiment is appropriate for any application wherein the nature of RNA has to be controlled, including any method requiring NC-like particles with known RNA-content. Thus, the method of the invention will comprise a step of purification of NºP complexes for applications using analytical methods or where clearly defined NC-like particles are desired, as for instance for biotechnological applications.
Thus, in a preferred embodiment, the method for producing self-assembling paramyxoviral NC-like particles as described above is defined as a method wherein step (a) further comprises the purification of NºP complexes.
As used herein, the term “coexpression of N and P proteins” refers to the simultaneous expression of the two proteins in order to allow the formation of the NºP complex and consequently to avoid the formation of aggregates of insoluble N proteins. In the context of the present invention, the coexpression of N and P also encompasses situations where N is expressed in a medium where P is already present, provided that N will not form aggregates but NºP complexes with the available P proteins.
Concerning the length of the RNA molecule, it can vary, as the inventors have shown that both a short version of RNA with 6 nucleotides or a long molecule with 60 nucleotides allow the formation of the NC-like particles. Thus, the RNA length is not a determinant parameter as long as 6 nucleotides are present. A preferred RNA molecule corresponds to the 6 first nucleotides of the 5′ end of the viral RNA. In a particular embodiment, the RNA molecule is not the native RNA of the virus. Further, in another preferred embodiment, the RNA terminates by a protecting group both on the 3′ and 5′ ends. In a more preferred embodiment, the RNA terminates by OH groups both on the 3′ and 5′ ends. Alternatively, the RNA molecule can for example be tagged at one extremity and bear an OH group at the other extremity. Appropriate tags are known by the skilled person in the art, as for example fluorescein.
In an embodiment, the invention relates to the method for producing self-assembling paramyxoviral NC-like particles as described above, wherein the RNA molecules comprises at least 6 nucleotides, preferably from 6 to 100 nucleotides, more preferably from 6 to 60 nucleotides.
For example, the RNA molecules has a length of 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides.
In an embodiment, the method applies to the production of NC-like particles derived from measles virus. In this particular embodiment, N comprises at least the NCORE domain including the CTD arm and NTD arm, which corresponds to the folded domain of N and is represented by SEQ ID NO: 5. In other words, N comprises at least the amino acids 1-405 of SEQ ID NO: 4. Alternatively, N comprises the full-length N protein including the folded domain and the disordered C-terminal domain (NTAIL) and is represented by SEQ ID NO: 4. Further, P comprises at least the minimal N-binding sequence, which is represented by SEQ ID NO: 2. In a particular embodiment, P consists of SEQ ID NO:2. In other words, P comprises at least the amino acids 1-50 of SEQ ID NO: 1.
Still regarding measles virus, in a particular embodiment, the RNA molecule comprises a sequence selected from the group comprising:
In a further embodiment, the RNA molecule consists of a sequence selected from the group comprising: SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 19, ACCAGA, AAAAAA, ACCUGA, UCCAGA, ACUAGA, and ACCAGA.
In an embodiment, the invention relates to the method for producing self-assembling paramyxoviral NC-like particles as described above, wherein the RNA molecules are not poly-U homopolymers, in particular not UUUUUU.
The production of NC-like particles from measles virus is illustrated in the experimental part. However, the method is directly applicable to the production of other Paramyxoviruses since viruses of this family are strongly related in terms of nucleoprotein/nucleocapsid sequence and architecture. However, it will be understood that other viruses being highly infectious pathogens, such as Nipah and Hendra viruses, the demonstration of the mechanism is safer with the measles virus.
Examples of other Paramyxoviruses are Nipah, Hendra, Mumps and human Parainfluenza viruses. Table 1 lists the paramyxoviruses for which the claimed method is directly applicable.
For Nipah, the claimed method can be carried out using the following sequences: For N peptide, the full-length protein corresponds to SEQ ID NO: 15. One can use either the amino acids 1-532 of the full-length protein or at least the amino acids 1-400 corresponding to the folded domain. For P peptide, one can use a peptide including at least 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.
Another object of the invention relates to a NºP complex comprising recombinant N and P peptides, wherein the N peptide comprises or consists of at least the NCORE domain including the CTD arm and NTD arm, and the P peptide comprises or consists of at least the N-binding domain.
In an embodiment, the invention relates to the NºP complex as described above, wherein the recombinant N peptide comprises the full-length nucleoprotein or at least the amino acids 1-400 corresponding to the NCORE domain of the nucleoprotein.
In particular, the recombinant N peptide is from 405 to 532 amino acids in length, preferably from 405 to 525 amino acids in length. For example, the recombinant N peptide has a length of 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 525, 530 or 532 amino acids.
In an embodiment, the invention relates to the NºP complex as described above, wherein the recombinant P peptide comprises the full-length phosphoprotein or at least the amino 1-50 of the phosphoprotein.
In particular, the recombinant P peptide is from 50 to 709 amino acids in length, preferably from 50 to 507 amino acids in length. For example, the recombinant P peptide has a length of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 507, 550, 600, 650, 700 or 709 amino acids.
In an embodiment, said NºP complex does not contain RNA.
In an embodiment, said NºP complex is purified.
Another object of the invention consists of a NC-like viral particle derived from a Paramyxovirus which comprises an RNA of interest, wherein the nature of RNA is controlled.
As used herein, the “control of the RNA” refers to the situation wherein the nature of the RNA that is embedded into the particles is known or predictable. It is indeed a specific characteristic of the invention to be able to control the nature of RNA that is embedded into NC particles. It is of great interest to be able to determine the conditions of entry of the RNA and thus the nature of the RNA. In particular, when the nature of the RNA is controlled, the sequence of the RNA embedded into the particles is known. The sequence of the RNA is chosen depending on the application of the NC-like particles. In order to obtain a homogenous population of NC-like particles with one type of RNA, the NºP complex can be purified to eliminate any contamination with cellular RNA, before adding the RNA of interest to a solution of NºP complex.
In an embodiment, the NC-like particles contain a synthetic RNA molecule.
In an embodiment, the NC-like particles contain only one given RNA molecule, in particular which is not viral RNA.
In an embodiment, the NC-like particles contain only one given RNA molecule, in particular which is not cellular RNA.
In an embodiment, the RNA is not a poly-U homopolymer, in particular is not UUUUUU.
In a preferred embodiment, the NC-like particles are obtainable by the method of production described above using recombinant N and P peptides and an RNA molecule of interest. Required for this assembly: (i) the N peptide comprising at least the NCORE domain including the CTD arm and NTD arm, (ii) a P peptide comprising at least the N-binding domain and (iii) an RNA molecule comprising at least 6 nucleotides.
The P peptide dissociates as the NC-like particle assembles, the latter comprises N peptide and RNA.
Aquaparamyxovirus
Avulavirus
Avian
paramyxovirus
2
Avian paramyxovirus
3
Avian paramyxovirus
4
Avian paramyxovirus 5
Avian paramyxovirus
6
Avian paramyxovirus
7
Avian
paramyxovirus
8
Avian
paramyxovirus
9
Avian
paramyxovirus
10
Avian
paramyxovirus
11
Avian
paramyxovirus
12
Newcastle disease virus
Ferlavirus
Fer-de-Lance paramyxovirus
Henipavirus
Cedar henipavirus
Ghanaian bat henipavirus
Hendra virus
Mojiang henipavirus
Nipah virus
Morbillivirus
Canine distemper virus
Cetacean morbillivirus
Feline morbillivirus
Measles virus*
Peste-des-petits-ruminants
virus
Phocine distemper virus
Rinderpest virus
Respirovirus
Bovine parainfluenza virus 3
Human parainfluenza virus 1
Human parainfluenza virus 3
Porcine parainfluenza virus 1
Sendai virus*
Rubulavirus
Human parainfluenza virus 2
Human parainfluenza virus 4
Mapuera virus
Mumps virus
Parainfluenza virus 5
Porcine rubulavirus
Simian virus 41
Regarding the nature of the RNA molecule, it is contemplated to use any RNA of interest.
As used herein, the term “RNA of interest” refers to any RNA molecule of known sequence that allows the formation of the NC-like particles. Such RNA of interest can be chosen depending on the desired application of the NC-like particles. It can thus correspond to a viral RNA, from native or foreign virus, or to a non-viral RNA. The RNA of interest can be one type of molecule or be constituted by a mix of different types of RNA.
In an embodiment, only one specific RNA of interest is used.
In a particular embodiment, the invention consists of NC-like viral particles as previously defined wherein the N protein comprises a disordered N-terminal domain (NTAIL) which is functionalized.
The functionalized group can be any biological molecule, any chemical compound or any group that provides a particular function to the particle. It can be one or more diversity elements to promote interactions with different protein targets in cells either for inducing a reaction or for tagging purposes. Examples of functionalized groups include ligands 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, a protein chimerically attached to NTAIL . . . . The skilled person in the art is familiar with collection of functionalized groups that can be added to a particular molecule or particles depending on the desired effect.
A functionalized particle is useful for numerous applications, in particular to target the particle to a desired organ or cellular type, for example to cancer cells, or to include domains of proteins attached to the flexible domain for the raising of antibodies for the development of vaccines.
It is indeed one of the aspects of the invention to be able to produce NC-like particles containing the same RNA molecule. This result could not have been achieved previously since NC particles obtained in cells, for example in E. coli or insects cells, have integrated the cellular RNA leading to a heterogeneous population of particles bearing each a particular RNA molecule. Providing NC-like particles with a defined RNA permits to better understand the replication cycle of Paramyxoviruses. It is indeed necessary to have a homogenous sample of particles to study the interaction between the RNA and the nucleocapsid for example by solid state NMR or high resolution cryo-electron microscopy. The present invention thus opens the way for new discovery regarding the mechanism of replication and transcription of these viruses.
Another object relates to a composition comprising NC-like viral particles as previously defined.
In a particular embodiment, said composition comprises a homogeneous population of particles bearing the same RNA.
Another object of the invention consists in the use of a NC-like viral particle as previously defined, as a medicament in the form of a drug or a vaccine.
Moreover, the ability to isolate integral NºP in a stable heterodimeric state that can be triggered to initiate NC-like assembly in the presence of RNA provides a powerful physico-chemical tool, not only to the study of the molecular basis of assembly, including the dynamics of NC-like assembly and possible dependence on RNA sequence and length, but more importantly to establish a tool for the development and testing of inhibitors of viral replication and transcription.
Another object of the invention consists of 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 NC-like particles.
Such screening method comprises the steps of:
In a particular embodiment, the screening method consists in the method as defined above wherein step (a) further comprises the purification of NºP complexes. It can, however, also be performed on cell lysates containing NºP.
The quantification of assembled NC-like particles can be observed by any appropriate method including NMR or electron microscopy. Fluorescence based assays are appropriate for the development of rapid and efficient high-throughput assays.
For example, in a particular embodiment, dyes can be placed at the 3′ terminus of an RNA molecule in a way that does not perturb assembly. This could be a 10-base RNA conjugated with a fluorescein derivative (
In these kind of assays, the absence of NC-like particles or the decrease in quantity of NC-like particles produced directly results from the presence of a compound able to inhibit the assembly of the NC-like particles of Paramyxoviruses. As used herein, the “inhibition of the assembly of NC-like particles” may correspond to a total or partial inhibition.
The invention is further illustrated by the following examples that should not be considered as limiting the scope of the invention.
Materials and Methods
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 P1-50 corresponds to SEQ ID NO 2.
P1-100: 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.
Escherichia coli Rosetta™ (λDE3)/pRARE strain (Novagen) were used for the production of P1-100. The protein was purified by nickel resin chromatography, followed by size exclusion chromatography.
Results
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.8 This region was targeted as the putative N-binding site. The inventors therefore co-expressed MeV P1-100 with N, resulting in a soluble and stable complex NCOREºP1-100. The binding site was then delineated more precisely by titrating 15N/13C labeled P1-150 into NCOREºP1-00, showing localized exchange in the region comprising residues 1-50.
This entire procedure resulted in identification of the minimal P peptide (P1-50 of SEQ ID NO: 2) required to form the complex.
Materials and Methods
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.
Constructs
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:
The resulting PCR products were purified on an agarose gel.
The combined product was then purified (PCR purification kit—Qiagen™) and PCR amplified with the external primers used previously:
Forward: 5′-(SEQ ID NO: 9) and Reverse: 5′-(SEQ ID NO: 12) to yield N1-5250P1-50. For N1-4050P1-50, SEQ ID NO: 12 was replaced by SEQ ID NO: 13 in the final amplification step.
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.
Expression and Purification of NºP
Escherichia coil Rosetta™ (λDE3)/pRARE strain (Novagen) were also used for the production of N1-5250P1-50 and N1-4050P1-50.
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.
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).
Cleavage with TEV gave the following proteins in complex NºP for N1-5250P1-50:
P1-50 with a TEV peptide amino acid sequence is represented by SEQ ID NO: 3.
N1-525 with an Histidine tag is represented by SEQ ID NO: 6.
And for N1-4050P1-50, cleavage with TEV gave the following proteins:
P1-50 with a TEV peptide amino acid sequence is represented by SEQ ID NO: 3.
N1-405 with an Histidine tag is represented by SEQ ID NO: 7.
Results
Coexpression of constructs containing the P1-50 peptide and either full length N (N1-525) or the folded domain of N (N1-405), 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 N1-405ºP1-50 or N1-525ºP1-50 demonstrate that both constructs contain a heterodimer of monomers of P1-50 and N1-525 or N1-405 (
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.
Materials and Methods
RNA molecules added to the NºP complex
RNA was added to the NºP complexes by titrating a solution of the two following particular RNA sequence:
NC-like particles can then be further purified using the methods described in reference 4.
NMR Spectroscopy
The assembly of NC-like particles was followed by real-time NMR using a series of SOFAST 1H-15N HMQC experiments using a sample of 200 μM NºP complex (N1-405ºP1-50 or N1-525ºP1-50) The formation of NC-like particles was initiated by addition of RNA6 or RNA60 (SEQ ID NO: 8) reaching a total concentration of 20 μM (RNA60) or 150 μM (RNA6). 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 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 (
Electron Microscopy
The formation of NC-like particles was followed by negative staining electron microscopy. Initially, a control measurement was performed of the N1-405ºP1-50 complex in the absence of RNA6 showing no formation of NC-like particles. The N1-405ºP1-50 was then mixed with RNA6 and the sample was visualized by electron microscopy. The sample contained 20 μM of N1-405ºP1-50 and 50 μM of RNA6 in the same buffer as used for the NMR experiments (see above).
Fluorescence Spectroscopy
N1-405ºP1-50 or N1-525ºP1-50 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. RNA10-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
with the G-factor correcting for detection differences between parallel (I1) and perpendicular (I⊥) 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.
Protocol for Application to Other Paramyxoviruses
For Nipah, the claimed method can be carried out using the following sequences:
For N peptide, the full-length protein (1-532) corresponds to SEQ ID NO: 15.
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.
Results
Analysis by NMR Spectroscopy
Using NMR spectroscopy, the inventors were able to follow the evolution in real time of the NMR spectra as the P1-50 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 P1-50 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
Analysis by Electron Microscopy
Electron microscopy was carried out on samples immobilized at increasing times after addition of small (6 or 60 nucleotides in length) RNA molecules.
The N1-405ºP1-50 mixed with RNA6 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 (
Analysis by Fluorescence Spectroscopy
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.
Different RNA sequences were tested for their ability to facilitate NC-like particle assembly in vitro from P1-50N1-405. Homopolymers comprising purines or pyrimidines alone, UUUUUU (polyU-RNA6), AAAAAA (polyA-RNA6) were compared, as well as genomic 5′-RNA6. Remarkably, efficiency of assembly is sequence-dependent (
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16305658.3 | Jun 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/063735 | 6/6/2017 | WO | 00 |
