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WO2018078369A1 - Signaux d'encapsidation viraux - Google Patents

Signaux d'encapsidation viraux Download PDF

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Publication number
WO2018078369A1
WO2018078369A1 PCT/GB2017/053225 GB2017053225W WO2018078369A1 WO 2018078369 A1 WO2018078369 A1 WO 2018078369A1 GB 2017053225 W GB2017053225 W GB 2017053225W WO 2018078369 A1 WO2018078369 A1 WO 2018078369A1
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WIPO (PCT)
Prior art keywords
nucleotide sequence
seq
composition according
nucleic acid
viral
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PCT/GB2017/053225
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English (en)
Inventor
Reidun TWAROCK
Eric Charles DYKEMAN
Peter Stockley
Simon White
Sarah Butcher
Shabih SHAKEEL
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University of Helsinki
University of Leeds
University of Leeds Innovations Ltd
University of York
Original Assignee
University of Helsinki
University of Leeds
University of Leeds Innovations Ltd
University of York
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the disclosure relates to packaging signals of RNA viruses, in particular viruses of the family Picornaviridae or Retroviridae, pharmaceutical compositions comprising anti-viral agents that mimic viral packaging signals and screening methods enabling the identification of anti-viral agents based on the amino acid co-ordinates of capsid proteins to which said packaging signals interact.
  • RNA viruses have a simple structure comprising RNA enclosed in a protein shell called a capsid or nucleocapsid.
  • a protein container that encapsulates and provides protection for the viral genome is a vital step in most viral life-cycles (M.G. Rossmann and J.E. Johnson, lcosahedral RNA virus structure Annu Rev Biochem. 58, 533-73 (1989)).
  • pro-capsid formation may occur via the self- or assisted assembly of protein subunits and be followed by the introduction of the genomic material via a packaging motor, as seen in many double-stranded DNA viruses (S. Sun, S. Gao, K. Kondabagil, Y. Xiang, M.G. Rossmann, and V.B. Rao. Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci U S A. 109, 817- 22 (2012)).
  • capsid assembly may follow a co-assembly process involving protein subunits and the viral genome encompassing so called packaging signals (PSs).
  • Packaging signals comprise discrete secondary structure elements having affinity for their cognate coat proteins. Multiple PSs dispersed across the genome with a common
  • l recognition motif can act collectively to make viral capsid assembly highly efficient and complete. This is a phenomenon occurring in many single-stranded RNA viruses [5, 6].
  • Picornaviruses are positive-sense single-stranded (ss) RNA viruses and are major pathogens in all kingdoms of life, including human and animal viruses such as polio, human rhinovirus and foot and mouth disease virus. Their virion assembly mechanism(s) has been extensively studied previously. The results of those studies suggest that picornavirus assembly appears to differ from the general capsid co-assembly process as the encapsidation of the RNA is regulated by a viral protein-coat protein interaction that does not involve RNA packaging signals 1"3 . In our co-pending application, US14/916,945 we disclose packaging signals for a variety of RNA viruses and their importance in viral assembly.
  • the present disclosure relates to viral assembly and the interactions of RNA packaging signals with viral capsid/coat proteins in the formation of the virus particle via nucleotide/amino acid interactions at defined sites in the viral capsid.
  • Viral assembly in the family Picornaviridae involves multiple capsid protein- genomic RNA interactions at sequence-degenerate RNA packaging signals.
  • the packaging signals and their capsid protein contact sites are highly conserved throughout the Picornaviridae family. This enables the identification of small organic molecules, for example packaging signal mimetics and small anti-viral compounds that inhibit the interaction of native packaging signals with cognate binding sites in a corresponding capsid binding protein, to inhibit or prevent viral assembly across viral species.
  • nucleic acid loop domain comprising a nucleotide binding motif comprising the nucleotide sequence GXUXXX, wherein X is any nucleotide base; or ii) a nucleic acid loop domain comprising a nucleotide binding motif comprising the nucleotide sequence ACC or CCA ; and
  • nucleic acid stem domain comprising a double stranded region by intramolecular base pairing
  • nucleotide binding motif contacts one or more viral capsid protein[s] in one or more amino acid positions in said viral capsid protein[s] and prevents or inhibits the assembly of the Picornaviridae viral capsid.
  • said nucleotide binding motif comprises or consists of the nucleotide sequence GXUXXX, wherein X is any nucleotide base.
  • the binding motif GXUXXX can also be described as GXU(X)n wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and X is any nucleotide base.
  • said motif GXUXXX is GXC(X)n wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and X is any nucleic acid.
  • said nucleotide binding motif comprises or consists of the nucleotide sequence ACC.
  • said nucleotide binding motif comprises or consists of the nucleotide sequence CCA.
  • said virus of the family Picornaviridae is of the genus Parechovirus (HPeV).
  • said human Parechovirus is selected from the group consisting of: HPeV1 , HPeV2, HPeV3, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeV10, HPeV11 , HPeV12, HPeV13 or HPeV14.
  • said human Parechovirus is selected from the group: HPeV15, HPeV16, HPeV17, HPeV18 or HPeV19.
  • said HPeV is HPeV1 or HPeV3.
  • said Picornaviridae is the genus Parechovirus B (Ljungan virus).
  • said Picornaviridae is of the genus Kobuvirus, for example Aichivirus.
  • said Picornaviridae is of the genus Enterovirus, for example poliovirus.
  • said Picornaviridae is of the genus Aphthovirus, for example foot and mouth disease virus.
  • said Picornaviridae is of the genus Rhinovirus.
  • said Picornaviridae is of the genus Cardio Virus, for example Saffold virus.
  • said stem domain is at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56 or 57 nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 to 92 as set forth in table 6, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 1 to 92 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 93 to 173 as set forth in table 7, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 93 to 173 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 174 to 277 as set forth in table 8, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 174 to 277 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • RNA viruses the sequences of which we specifically disclaim.
  • said stem domain is at least 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54 or 55 nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 278 to 355 as set forth in table 9, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 278 to 355 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said stem domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58 or 59 nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 356 to 498 as set forth in table 10, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 356-498 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said stem domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64 or 65 nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 499 to 582 as set forth in table 1 1 , or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 499 to 582 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said stem domain is at least 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 583 to 703 as set forth in table 12, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 583 to 703 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • composition comprises at least 2, 3, 4 or 5 antiviral agents according to the invention.
  • composition comprises 2, 3, 4 or 5 antiviral agents.
  • said composition comprises a nucleic acid stem loop structure comprising or consisting of a nucleic acid stem loop structure comprising a nucleotide sequence as set forth in SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856 or SEQ ID NO: 857, or a nucleotide sequence that is 95-99% identical over the full length nucleotide sequence as set forth in SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856 or SEQ ID NO: 857.
  • nucleic acid loop domain comprising a nucleotide binding motif comprising the nucleotide sequence GXG, wherein X is any nucleotide base; or ii) a nucleic acid loop domain comprising a nucleotide binding motif comprising the nucleotide sequence RYAR, wherein R is adenine or guanine and Y is cytosine or thymidine/uracil; and/or
  • nucleic acid stem domain comprising a double stranded region by intramolecular base pairing wherein said nucleotide binding motif contacts one or more viral capsid protein[s] in one or more amino acid positions in said viral capsid protein[s] and prevents or inhibits the assembly of the Retroviridae viral capsid.
  • said stem domain is at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86 or 87 nucleotides in length of which all or part of said stem domain is double stranded by complementary base pairing.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 704 to 744 as set forth in table 13, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 704 to 744 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said anti-viral agent comprises a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 745 to 813 as set forth in table 14, or nucleic acid molecules comprising or consisting of a nucleotide sequence that is a polymorphic sequence variant of the nucleotide sequence of SEQ ID NO: 745 to 813 and has a nucleotide sequence identity of 95-99% over the full length nucleotide sequence.
  • said anti-viral agent comprises a nucleic acid molecule that comprises modified nucleotides.
  • modified describes a nucleic acid molecule in which: i) at least two of its nucleotides are covalently linked via a synthetic internucleotide linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide).
  • a synthetic internucleotide linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide.
  • said linkage may be the 5' end of one nucleotide linked to the 5' end of another nucleotide or the 3' end of one nucleotide with the 3' end of another nucleotide; and/or ii) a chemical group, such as cholesterol, not normally associated with nucleic acids has been covalently attached to the single-stranded nucleic acid.
  • Preferred synthetic internucleotide linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
  • modified also encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars, which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified nucleotides may also include 2' substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a- anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
  • 2' substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a- anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses
  • Modified nucleotides include alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8- hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5- fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5 carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; I- methyladenine; 1 -methylpseudouracil; 1 -methylguanine; 2,2-dimethylguanine; 2- methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N
  • Modified double stranded nucleic acids also can include base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996).
  • base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996).
  • the use of modified nucleotides confers, amongst other properties, resistance to nuclease digestion and improved stability.
  • an anti-viral agent according to the invention for use in the treatment of viral infections.
  • a pharmaceutical composition comprising an anti-viral agent and a pharmaceutical excipient.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and supplementary therapeutic agents.
  • compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or trans- epithelial.
  • the compositions of the invention are administered in effective amounts.
  • An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily to enable the host's natural antiviral defences to clear the infection and ideally reversing disease phenotype. This can be monitored by routine methods.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of agent according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the doses of the agent according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • doses of agent of between 1 nM - 1 ⁇ generally will be formulated and administered according to standard procedures. Preferably doses can range from 1 nM- 500nM, 5nM-200nM, and 10nM-100nM. Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing.
  • the administration of compositions to mammals other than humans, is carried out under substantially the same conditions as described above.
  • a subject, as used herein, is a mammal, preferably a human, and including a nonhuman primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • the pharmaceutical preparations of the invention When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents used in the treatment of viral disease.
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration into a human.
  • carrier in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application, (e.g. liposome or immuno-liposome).
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction, which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion or as a gel.
  • Compositions may be administered as aerosols and inhaled.
  • Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of agent, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable dilutent or solvent, for example, as a solution in 1 , 3-butane diol.
  • a non-toxic parenterally-acceptable dilutent or solvent for example, as a solution in 1 , 3-butane diol.
  • acceptable solvents water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectable.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • a combined pharmaceutical composition comprising an agent according to the invention and one or more additional antiviral agents different from said agent according to the invention.
  • the additional anti-viral agent is an anti-retroviral agent.
  • Anti-viral agents are known in the art and include by example Amantadine, deoxythymidine, zidovudine, stavudine, didanosine, zalcitabine, abacavir, lamivudine, emtricitabine, tenofovir, maraviroc, efuvirtide, nevirapine, delavirdine, efavirenz, rilpivirine, Elvitegravir, Lopinavir, Indinavir, Nelfinavir, Amprenavir, Ritonavir, Bevirimat and Vivecon or combinations thereof.
  • Anti-viral agents also include by example: ACH-3102, Arbidol, Boceprevir, Daclatasvir, Faldaprevir, Fluvir, Ledipasvir, Moroxydine, Pleconaril, PSI-6130, Ribavirin, Rimantadine, Setrobuvir, Simeprevir, Sofosbuvir, Taribavirin and Telaprevir.
  • the pharmaceutical composition is adapted to be delivered as an aerosol.
  • an inhaler comprising a pharmaceutical composition according to the invention.
  • a method to screen for antiviral agents that bind to one or more packaging signals and/or one or more viral capsid proteins comprising the steps: i) providing a preparation comprising a combinatorial library of small molecular weight compounds and contacting said library with a preparation comprising: a. a viral capsid protein or part thereof; or
  • a method for the identification and testing of an anti-viral agent comprising the steps: i) providing a virtual viral capsid binding pocket wherein said pocket comprises a protein complex of at least four viral capsid proteins comprising the amino acid sequences set forth in SEQ ID NO: 815 combined with three capsid proteins comprising SEQ ID NO: 814 to form a binding pocket and wherein said complex provides first, second, third and fourth interactive surfaces for a viral packaging signal nucleic acid said interactive surfaces having the following amino acid co-ordinates
  • SEQ ID NO: 815 (Arg 55, Ala 56 and Tyr58);
  • ligands demand various computational analyses, which are necessary to determine whether a molecule is sufficiently similar to the target moiety or structure. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135.
  • the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target.
  • the screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER. Specialized computer programs may also assist in the process of selecting fragments or chemical entities, prior to testing fragments in an infection assay for efficacy.
  • GRID P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28, pp. 849-857 (1985)).
  • GRID is available from Oxford University, Oxford, UK;
  • MCSS A. Miranker et al., "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 1 1 , pp. 29-34 (1991)).
  • MCSS is available from Molecular Simulations, Burlington, Mass; AUTODOCK (D. S. Goodsell et al.
  • CAVEAT P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules". In: "Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)).
  • CAVEAT is available from the University of California, Berkeley, California, 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, California). This is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992); and HOOK (available from Molecular Simulations, Burlington, Mass).
  • target-binding compounds may be designed as a whole or de novo. These methods include: LUDI (H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61 -78 (1992)). LUDI is available from Biosym Technologies, San Diego, California; LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)).
  • LEGEND is available from Molecular Simulations, Burlington, Mass; LeapFrog (available from Tripos Associates, St. Louis, Mo.). Other molecular modelling techniques may also be employed, see, e.g., N. C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990). See also, M. A. Navia et al., “The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992).
  • an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
  • the most efficient ligands should preferably be designed with deformation energy of binding of not greater than about 10 kcal/mol, preferably, not greater than 7 kcal/mol.
  • a ligand designed or selected as binding to a target may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the inhibitor or other ligand and the target, when the inhibitor is bound to the target preferably make a neutral or favourable contribution to the enthalpy of binding.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group.
  • Another approach is the computational screening of small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to a target. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy (E. C. Meng et al. , J. Comp. Chem., 13, pp. 505-524 (1992)).
  • RNA crosslinking and peptide mapping assays RCAP
  • CLIP-SEQ NextGeneration sequencing
  • XFP X-ray synchrotron footprinting of both the genomic RNAs in contact with coat proteins inside infectious virions and the peptides from coat proteins protected from modification by those contacts
  • Figure 1 Ordered RNA segments in the structures of picornaviruses.
  • Four icosahedrally- symmetric structures for parechovirus virions are currently available. These are HPeV1 (Cryo-EM, 8.5 A resolution, EMD-1690) 7 , HPeV1 (X-ray crystallography, 3.1 A resolution, PDB 4Z92) 4 , HPeV3 (Cryo-EM, 4.3 A resolution, EMD-3137) 8 , and Ljungan virus (Cryo-EM, 4.5 A resolution, EMD-6395) 5 .
  • each capsid On the top row, an exterior view of each capsid is shown, viewed perpendicular to an icosahedral 2-fold axis.
  • each virus On the bottom row, each virus is shown in the same orientation, as a 60 A thick central section on the left hand side, and the rear half of the capsid on the right hand side. All panels are coloured with an identical radial colour scheme (red: 98 A, yellow: 111 A, green: 124 A, cyan: 137 A, blue: 151 A).
  • the diameters of all capsids are very similar, with density ascribed to RNA shown in yellow/red in each case.
  • RNA can be seen in the lower resolution HPeV1 EM reconstruction at 8.5 A, than in the X-ray density at 3.1 A resolution, suggesting that only a few nucleotides are identical in all 60 positions, but similar stem-loop occupy all positions;
  • peaks within the Harris strain are termed packaging signals (PSs) and are aligned with equivalent sites in the other strains provided the peak nucleotides lie within 10 nucleotides of the Harris PSs.
  • PSs packaging signals
  • the last row of the table shows the number of such matches from all the strains. Co-localised peaks in more than 50% of the strains are shown in green, the others are in red.
  • the mF 0 -DF c difference map is contoured in green at 2.5 ⁇ .
  • (b) Close-up showing a single copy of the viral RNA and its interactions with the surrounding amino acid residues from the viral coat proteins,
  • (a and b) Protein and RNA are shown in stick representation with the following colours: oxygen, coral; nitrogen, dark blue; phosphorus, orange. Carbon atoms are coloured by subunit as shown in (b) and (c); additionally, carbon atoms in the RNA base G1 are shown in magenta. Putative hydrogen bonds shown as dashed lines,
  • (c) View of the viral capsid from inside the virus showing the arrangement of proteins and viral RNA around the icosahedral 5-fold.
  • VP1 , VP3 and VPO from one asymmetric unit are shown in red, green and yellow respectively.
  • VP3 contains a long N-terminal extension that reaches around the icosahedral 5-fold.
  • the N-terminal residue of each copy of VP3 in the crystal structure (Met15) is marked with a ball on the nitrogen atom and labelled N for VP3 a .
  • the RNA associated with VP1 a and VP3 a is shown in dark blue, with the 5' guanosine in magenta. 5-fold related copies of the RNA are shown in light blue.
  • 5-fold related copies of VP3 a are also coloured in different shades of green and labelled as shown. Note how the VP3 N-terminal regions interdigitate between adjacent RNA copies around the 5-fold, so that each RNA is recognised by VP1 and 3 copies of VP3 (VP3 a , VP3 b and VP3 C for the RNA molecule shown in dark blue);
  • FIG. 4 In vitro pentamer binding to the predicted PSs.
  • MST microscale thermophoresis
  • Panel (a) left shows a view along the five-fold axis into the virion, i.e. the outer surface.
  • the coat protein subunits are coloured, yellow for VPO; red for VP1 and various shades of green for the different VP3 subunits.
  • the right hand panel shows the view along the same axis form the centre of the virus, highlighting the extension of the N-terminal arms of the VP3s around the symmetry axis
  • b) shows a similar view with the ordered RNA segments in dark blue
  • (c) A cartoon of a hypothesized model of capsid assembly based on known aspects of picornavirus morphogenesis. The cartoon is based on Fig. 5 of Jiang et al 2 .
  • Pentamers of the viral CPs are bound by the 2C ATPase , associated with a replication factory at the cell membrane via sequence-specific protein-protein contacts to VP1 or VP3. Here they contact newly replicated genomic RNA.
  • the 2C ATPase is known to play multiple roles in virion replication and morphogenesis. Its RNA helicase activity would allow it to bias RNA folding to short-range contacts favouring the appearance of the PSs in HPeV1. These could then associate with their binding sites on a pentamer. As each protomer binds RNA the genome would form a loop until the next PS sequence appeared. This could attach to a neighboring binding site by chance, or pentamers could rotate relative to 2C to orientate the RNA at unbound sites. Once a pentamer's RNA sites are full, a second pentamer may become associated forming the CP-CP contacts seen in the virion.
  • Such a model accounts both for the data described here and previous models of picornavirus
  • Figure 6 Conservation across the HPeV genus.
  • the selected HPeV1 library was used to derive a Bernoulli plot for a representative of the HPeV3 group (GQ183026), which is most distal from the HPeV1 group from the point of view of VP1-VP4 conservation (Fig. 9).
  • the number of nucleotide matches of the HPeV3 genome to the selected HPeV1 library is remarkable: with up to 15,000 nucleotide matches for higher peaks and smaller peaks between 1000 and 2000, as opposed to a maximum of 8000 for the higher peaks and 1000- 3000 for the lower peaks in HPeV1 , indicating that a similar recognition motif is likely to occur also in HPeV3.
  • Stringency was increased after round 5 by decreasing the number of pentamer-coated Dynabeads by half and increasing the number of washes in rounds 6 to 10 by one wash each round, i.e. by round 10 there were 15 washes.
  • a counter selection was performed against biotinylated whole capsid (blue decagon) whereby the selected RNA library (50 ⁇ ) was mixed with the capsid (50 ⁇ at 0.1 mg/ml) for 5 min at 37°C, and the biotinylated capsid and any associated RNA were then captured on streptavidin Dynabeads. The unbound RNA was then used in a normal round of SELEX.
  • the final round (round 10) was performed as normal but before heat elution of the RNA library from the positive beads, the positive beads were challenged with 100 ⁇ of whole capsid (0.1 mg/mL) for 5 minutes at 37°C. After three washes to remove any remaining capsid/RNA complexes the positive beads were heat eluted as normal.
  • Figure 8 Putative Packaging Signals corresponding to the Bernoulli Peaks in the Harris strain (a) Fragments plus/minus 20 nucleotides around the 21 Bernoulli peaks in Fig. 2a can fold into stem-loops containing the GxU motif in the loop portion, consistent with the HPeV-1 protein:RNA interactions in Fig. 3b. Only the predicted Mfold secondary structures are shown for each PS, with numbers indicating starting and ending nucleotides in the Harris strain.
  • Bernoulli peak positions of PS1 to PS21 are (in nucleotides): 338, 693, 753, 827, 1142, 1344, 1957, 1995, 2322, 2493, 2660, 2873, 2919, 3553, 4037, 5073, 5139, 6194, 6409, 6778, 7264.
  • the two peaks below the cut-off defined by the naive library, marked by asterisks in Fig. 2a can also fold into stem-loops with a GxU motif in the loop portion, suggesting that peaks below the cut-off could also be relevant, even though this cannot be assumed a priori. There are 56 such peaks in total, which is close to the 60 expected from the RNA density distribution in the virion.
  • Figure 9 RNA model in the HPeV1 EM density.
  • the ordered EM density below this fragment would not be seen if the sequences flanking the GxU recognition motif were not capable of forming a base-paired stem.
  • An A-duplex model of such a stem fits well into the EM map (not shown), consistent with the equivalent sites in HPeV3 8 ;
  • Figure 10 Phylogenetic tree based on the structural protein sequences of human parechovirus genomes from groups 1 to 8.
  • a phylogenetic tree based on the VP1 -VP4 coding sequences is shown for all human parechovirus sequences available from GenBank (see Table 2), created using SplitsTree. Even though the structural proteins in the Harris strain in the HPeV1 group (red arrow) are most distal from those in the HPeV3 group (green arrow), a representative viral genome in the HPeV3 group is still showing excellent matches with the selected HPeV1 library and PS recognition motif;
  • Table 2 PS mutants testing in cell culture and in vitro a The nucleotides substituted within each mutant PS are in lower case, the loop region of each PS is italicized and the GxU motifs are underlined.
  • Replication efficiency of each mutant was tested by staining with anti-dsRNA antibody and by quantitative Real time PCR.
  • TCID50 TCID50
  • Seq36 AB433629.1 ; Seq37: DQ315670.1 ; Seq38: AM235750.1
  • Seq41 FJ888592.1 ; Seq42: AB252582.1 ; Seq43: EU077518.1
  • HPeV1 pentamers were prepared by heating biotinylated capsids. Capsids were disrupted by heating to 56°C for 30 minutes and incubating at 4°C for 10 days. Biotinylated pentamers in 10 mM Tris-HCI pH 7.7, 150 mM NaCI and 1 mM MgCI 2 buffer immobilised on Dynabeads (positive beads) were subjected to 10 rounds of in vitro selection with a synthetic combinatorial N40 2 ⁇ RNA library ( ⁇ 10 24 potential sequences, ⁇ 10 15 sequences were used in SELEX) as described previously 35 . The amplified DNA of round 10 was then subjected to NGS on an lllumina platform (see Supplementary Fig. 1 for more details).
  • the plasmids containing the cDNA clones of mutants or wild-type were used as the templates to produce linear cDNA by PCR amplification using the T7 promoter-containing forward primer, 5'- TAATACGACTCACTATAGGGTTTGAAAGGGGTCTCCTAGAGAG-3' and polyT-containing reverse primer 5'-TTTTTTTTTTTGTCATGTCCAATGTTCC-3'.
  • the PCR running conditions were 98°C (5min), [98°C (30 s) + 55°C (30 s) + 72°C (5 min)] x 25, 72°C (10 min), 4°C.
  • the resulting linear cDNA contained the T7 promoter and the appropriate full-length HPeV1 cDNA genome.
  • a plasmid containing the T7 RNA polymerase under the CMV promoter 38 was transfected into GMK cells (kind gifts from Petri Susi) using Lipofectamine 2000 (Life Technologies). Next day, these GMK cells were transfected with the linear cDNA, to generate full-length genomic RNA, which in the control experiments with the wild type cDNA, generates virions. Cell lysate was prepared from the transfected cells on the third day post the second transfection by two cycles of freeze-th awing at -180°C and 37°C.
  • HT29 cells human colorectal adenocarcinoma cell line
  • the viral titres were expressed as 50% tissue culture infectious dose (TCID50) per ml using an end-point titration method described earlier 39 .
  • TCID50 tissue culture infectious dose
  • the HT29 cells are an indicator cell line which shows very clear CPE for HPeV1 compared to GMK. The HT29 and GMK cells were tested for mycoplasma prior to use.
  • the transfected cells were produced as for the above end point assay.
  • cells were infected with HPeV1 virions (MOI 0.1) to serve as a positive control, and the untransfected cells were used as a negative control.
  • HPeV1 virions MOI 0.1
  • an immunofluorescence assay was performed. The cells were fixed at 6 h post second transfection with 4 % paraformaldehyde for 15 min at room temperature (RT).
  • the cells were washed twice with 1 X PBS and permeabilised with 0.1 % Triton X-100 for 20 min at RT, followed by blocking with blocking buffer (3% bovine serum albumin and 0.1 % Tween-20 in 1X PBS) at RT for 1 h.
  • the primary antibody incubation was done at 4°C overnight with mouse J2 mAb against dsRNA (English and Scientific Consulting Kft.; stock: 1 mg/ml) at a 1 : 1500 dilution.
  • the cells were washed 3 times with 1X PBS and incubated with an FITC-labelled anti-mouse secondary antibody (Sigma) at 1 :200 dilution.
  • the cells were washed 3 times with 1X PBS.
  • DAPI (Sigma) at a working concentration of 0.1 ⁇ g/ml was added to the cells.
  • the images were obtained with a Floid Cell Imaging Station (Life Technologies) in the Biocenter Finland Light Microscopy Unit, University of Helsinki.
  • the wild type, mutants, positive virus infection and negative controls from the GMK cells were used for both total RNA extraction with Trizol (Invitrogen) and the production of cell lysates by freeze-thawing.
  • the total RNA was used for cDNA synthesis with random hexamers utilizing a Phusion RT kit (Thermo Scientific).
  • the cDNA was subjected to quantitative real-time PCR (Roche) using primers and a probe designed for a 142bp-long conserved 5' UTR region as described earlier 40 .
  • Plasmids carrying the wild type genome (pHPeVI) or its mutants were transcribed and translated using the TNT Quick Couple Transcription/Translation system (Promega) as per the manufacturer's protocol.
  • a plasmid carrying the luciferase gene was used as the positive control.
  • the proteins were labelled with 35S-methionine. The signal was read out on a phosphoimager after overnight exposure.
  • RNA oligomers based on the genome sequences for the PSs (PS6, PS7, PS9, PS14 and PS21) and their sequence variants and measured their affinities for HPeV1 pentamers, ranging in concentration from ⁇ 0.5 to 900 nM pentamer, using a microscale thermophoresis (MST) Monolith machine (NanoTemper) 41 .
  • MST microscale thermophoresis
  • RNA final concentration 25 nM
  • MST 5 sec LED on (Blue LED), 35 sec laser on, 5 sec recovery.
  • a control was also performed using the MS2 coat protein and TR RNA.
  • the MST signal is a composite of positive and negative thermophoretic effects that are acutely sensitive to the nature and solution environment of the dye-labelled species. If multiple self- association occurs, as was observed here, the signal can reverse preventing accurate Kd determination.
  • the binding data were therefore assessed semi-quantitatively (+/-) noting the amplitude of signal change over the range of pentamer concentrations and where significant binding began to occur. These were compared to a known PS-CP interaction, that of bacteriophage MS2 TR and its CP (Kd ⁇ 1 -4 nM, depending on binding assay used), which was designated as +++++.
  • the highest protein concentration samples from these assays were recovered, negatively-stained and examined by electron microscopy.
  • the coordinates and structure factors for the X-ray crystal structure of human parechovirus were obtained from the PDB (accession code 4Z92) 4 .
  • the un-averaged 2mF 0 -DF c map was of sufficient quality for structural interpretation, without further real-space averaging being required.
  • Close inspection of the deposited model revealed that portions of the RNA molecule and some of the surrounding amino acid side-chains were not optimally modelled into the electron density. These errors were corrected by manual rebuilding in COOT 42 and 10 cycles of refinement in REFMAC 43 with the X-ray weight set to 0.008 and strict 10-fold non-crystallographic symmetry applied.
  • Structures were refined in parallel with either A or G in position 1 of the RNA molecule, to R values of 0.26 and root-mean-square deviations in bond lengths/angles of 0.007A/1.25 0 .
  • the refined structures were validated using the MOLPROBITY server 44 .
  • the Molprobity score for the structures was 1.89 (100% percentile), with 93.8%/5.8%/0.4% of residues lying in favoured/allowed/forbidden regions of the Ramachandran plot.
  • HPeV1 strains used in genome analysis
  • strains 1 -21 referred to in Fig. 2b were retrieved from Genbank with the following Genbank IDs: Harris strain: L02971.1 , strain 2: JX441355.1 , strain 3: JX575746.1 , strain 4: EF051629.2, strain 5: FJ840477.1 , strain 6: GQ183035.1 , strain 7: GQ183025.1 , strain 8: GQ183023.1 , strain 9: GQ183021.1 , strain 10: GQ183019.1 , strain 11 : GQ183034.1 , strain 12: GQ183024.1 , strain 13: GQ183022.1 , strain 14: GQ183020.1 , strain 15: GQ183018.1 , strain 16: HQ696574.1 , strain 17: HQ696572.1 , strain 18: HQ696570.1 , strain 19: HQ696573.1 , strain 20: HQ696571.1 , strain 21 : FM 178558.1.
  • Example 1 Example 1
  • RNA SELEX was then used to isolate preferred binding sequences for these pentamers immobilized on magnetic beads (see Materials & Methods, and Fig. 7). Counter-selection against intact virions was used to remove aptamers with affinity for the exterior surface of the virus, thus biasing the aptamers isolated to those that bind to the interior surface of the capsid where the ordered genome segments are seen.
  • the RNA library used for SELEX encompasses a 40 nucleotide region in which every position is randomized. Fragments of this size exceed the likely RNA content of the ordered EM density (Fig. 1), allowing us to have complete coverage of likely binding motifs.
  • the 10 th round of selected aptamers was converted to cDNA and subjected to Next Generation DNA Sequencing (NGS). This yielded ⁇ 600,000 aptamer sequences, of which ⁇ 400,000 were unique from an initial starting library of ⁇ 10 15 sequences. Individual aptamers in the selected pool occur between 100-10,000 times, compared to at most 1 or 2 occurrences of each sequence in the unselected (naive) library. This outcome, together with the change in nucleotide composition of the starting and final aptamer pools (see inset in Fig. 2a), confirms that efficient selection of pentamer binding aptamers had been achieved. .
  • NGS Next Generation DNA Sequencing
  • RNA SELEX was then used to isolate preferred binding sequences for these pentamers immobilized on magnetic beads (see Materials & Methods, and Fig. 7). Counter-selection against intact virions was used to remove aptamers with affinity for the exterior surface of the virus, thus biasing the aptamers isolated to those that bind to the interior surface of the capsid where the ordered genome segments are seen.
  • RNA library used for SELEX encompasses a 40 nucleotide region in which every position is randomized. Fragments of this size exceed the likely RNA content of the ordered EM density (Fig. 1), allowing us to have complete coverage of likely binding motifs.
  • the 10 th round of selected aptamers was converted to cDNA and subjected to Next Generation DNA Sequencing (NGS). This yielded ⁇ 600,000 aptamer sequences, of which ⁇ 400,000 were unique from an initial starting library of ⁇ 10 15 sequences. Individual aptamers in the selected pool occur between 100-10,000 times, compared to at most 1 or 2 occurrences of each sequence in the unselected (naive) library. This outcome, together with the change in nucleotide composition of the starting and final aptamer pools (see inset in Fig. 2a), confirms that efficient selection of pentamer binding aptamers had been achieved.
  • genomic segments in contact with the CPs in the virions were bound sequence specifically, we would expect these aptamers to share some primary/secondary structure motifs with them. Since the minimal CP recognition motifs within a PS may be discontinuous and/or structurally degenerate, e.g. by differing in the sequences of non-contacted base pairs, the number of identical nucleotide positions might be quite low. Therefore, in order to identify putative genomic sites with affinity for CP, we aligned all the aptamer sequences against every position in the Harris genome using a 1 nt sliding window. We quantified the goodness of any match between an aptamer and the genomic sequence with reference to the probability equivalent of finding a contiguous match.
  • a non-contiguous match across a length of N nucleotides has the same probability as a contiguous match across M ⁇ N nucleotides
  • M known as the Bernoulli score (see Methods)
  • we increased a counter by one at every matching nucleotide position resulting in the histogram plot in Fig. 2a, which corresponds to the summation of the counters in each case for the aptamer pool (green curve) and naive library (red curve).
  • PS1 1 forms an exception in that only 4 nucleotides of the identified GxUxUxxU motif overlap with the loop of the stem-loop.
  • this is due to the repeat occurrence of the GxU motif, which could not be properly positioned based on sequence information alone. Indeed, taking both sequence and structure into account, the second GUU (underlined in Fig. 2c) should be aligned with the GxU consensus motif.
  • the conserved sequence motif is sequence-specifically bound in the virion
  • RNA residues were poorly modelled (Fig. 3a).
  • Fig. 3a We therefore rebuilt and re-refined the structure, adding an atomic model of the PS consensus into the resulting density for RNA yielding a structure with an improved R factor (0.26).
  • the model is an excellent fit (Fig. 3a) and reveals extensive RNA-CP contacts (Table 1 & Table 4), comprising both hydrogen bond as well as van der Waals contacts.
  • the RNA contacts multiple CP subunits, principally in VP3 and VP1 .
  • the contacts include three base-specific hydrogen bonds, two to the initial purine, which is therefore confirmed as the consensus G rather than the previously modelled A, although the latter could also be accommodated in this position.
  • N+3 is modelled as a uridine which stacks on the side chain of Tyr21 , although any base could make this interaction.
  • the neighbouring N+4 position is fixed as U by SELEX and participates in an extended stacking interaction with U4, although why it needs to be uridine is unclear.
  • the remaining fixed nucleotide in the SELEX consensus is outside the X-ray density. The fact that not all PSs contain these latter two fixed bases may indicate that their identities are less critical.
  • RNA buries -1 120 A 2 at the interface of the CPs and each PS i.e. 5600 A 2 /capsomer and 67,200 A 2 /capsid. This is a very large surface area and could easily provide the binding free energy to drive capsid formation and the confinement of the polynucleotide genome.
  • RNA fragments are important for virion assembly.
  • Reverse genetics was used to create mutational variants (PS#-M) of a number of the individual PS sites in the context of an infectious HPeV1 cDNA clone (wild- type, WT) under the control of a T7 promoter.
  • PS#-M mutational variants
  • peak heights in the Bernoulli plot Fig. 2a
  • PSs within both the coding and non- coding regions corresponding to different peak heights namely PS3, PS9, P1 1 and PS21.
  • PS6, PS7, PS14 and PS18 We also altered the highly conserved PSs in the coding region.
  • linear cDNA encoding each mutational change was transfected into a GMK cell line transiently expressing T7 RNA polymerase that then transcribes the viral genome.
  • the viral titres in each case were determined by preparing cell lysates, treating them with RNase and DNase and then assaying their titres on HT29 cells using the 50% tissue culture infectious dose (TCID50) method.
  • TCID50 tissue culture infectious dose
  • HT29 cells were chosen as they give a clear cytopathic effect with TCID50/ml as low as 10 1 being unambiguously detected.
  • TCID50 tissue culture infectious dose
  • PS9-M and PS21 -M each result in 6-log drops in TCID50 with no cytopathic effect (Table 2), whilst PS14-M and PS18-M show less dramatic reductions in viral fitness (2- and 1 -log drops, respectively).
  • the variation in mutational effects is striking, and may be the result of the way the virion is assembled (see below; Fig 5). It is entirely consistent with previous modelling 14,16 with the differing impacts of mutating individual PSs on the overall assembly efficiency being expected in the context of a co-operative process. Given the number of individual putative PS sites that appear to contribute to viral assembly, this is strong evidence in favour of PS-mediated assembly in which multiple PS sites act collectively.
  • capsid protein and PSs mutational results confirm the specificity of these capsid protein- RNA interactions, and further consolidate our hypothesis that specific PS-CP amino acid interactions facilitate HPeV1 assembly.
  • the detailed molecular mechanisms of PS action can vary from virus to virus. In at least one case they overcome repulsive electrostatic contacts between protomers in a capsomer 13 , and in another they act as allosteric effectors to create the quasi-equivalent conformers required to assemble capsids of the correct size and symmetry26,27 26,27 .
  • individual PSs aid the formation of capsomers of the geometry required for capsid formation, albeit via different specific modes of action (e.g. allosteric switching or bridging electrostatic barriers), hinting at the spectrum of effects PSs may have on assembly.
  • stabilization of the HPeV1 virion is very likely to be one such PS function. .
  • MST micro-scale thermophoresis
  • RNA conformations that regulate replication and translational efficiency or assembly. Being able to alter global RNA conformation in response to changes in the viral lifecycle is clearly a powerful way to regulate the timing of assembly.
  • One way to alter folding propensity would be during replication, local secondary structures being more favoured as incomplete transcripts emerge from viral polymerases.
  • nascent genomic transcripts are encapsidated 29 through recognition of their 5 ' IRES structure marking them as mRNAs.
  • the virally encoded 2C protein introduces nascent viral RNAs into the pentameric capsomers 2 .
  • the PS sites identified here are entirely compatible with such systems.
  • FIG. 5 is of an "assemblysome" that combines production of nascent genomes with RNA chaperoning by 2C, resulting in kinetic folding of the PS sites as they emerge from the replicase.
  • the illustration in Fig. 5 depicts the essential molecular challenges the virus must overcome to assemble the virion.
  • Parechoviruses and SELEX thus turn out to be a great combination for identifying PSs.
  • Many other picornaviruses undergo cleavage of VPO that could dislodge the RNA-CP contacts seen in HPeVI .
  • extensive genetic screening has been carried out in other picornavirus systems to look for RNA assembly determinants, the dispersed and sequence- degenerate nature of the PSs identified here, explain some of those negative results 1"3 .
  • the selective advantages of the PS-mediated mechanism are manifold. They include being evolutionarily robust, and ensuring assembly fidelity and efficiency arising out of the collective action of multiple sites of varying affinity, rather than being dependent on a single site.
  • PSs are part of a co-operative assembly process
  • mutation of individual sites can be expected to have different levels of effect from zero to lethality depending on their neighbouring PSs as we saw with our transfection studies.
  • bacteriophage MS2 which was a paradigm of the classical single PS site, shows only a limited decrease on viral titre when that site is mutated 30 , rather than a non-assembling phenotype expected for a unique assembly initiation site.
  • Multiple analyses have now shown that this is because of the presence of dispersed PSs assisting assembly 15,31"33 .

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Abstract

Nous divulguons des signaux d'encapsidation de virus à ARN, en particulier des virus de la famille Picornaviridae ou Retroviridae, des compositions pharmaceutiques comprenant des agents antiviraux et des méthodes de criblage permettant l'identification d'agents antiviraux en fonction des coordonnées de liaison à l'acide aminé, de protéines de capside par rapport auxquelles lesdits signaux d'encapsidation interagissent.
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Citations (2)

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WO2013040577A1 (fr) * 2011-09-16 2013-03-21 The Research Foundation Of State University Of New York Aptamères résistant à la dégradation par la nucléocapside
WO2015033155A1 (fr) * 2013-09-05 2015-03-12 The University Of York Thérapie antivirale

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Publication number Priority date Publication date Assignee Title
WO2013040577A1 (fr) * 2011-09-16 2013-03-21 The Research Foundation Of State University Of New York Aptamères résistant à la dégradation par la nucléocapside
WO2015033155A1 (fr) * 2013-09-05 2015-03-12 The University Of York Thérapie antivirale

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