Alphavirus NSP3 helical tubular scaffolds

Abstract

Alphaviruses, such as chikungunya virus (CHIKV), are a group of emerging / re-emerging mosquito-borne viruses that represent an ongoing challenge to medicine and public health. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling molecule whose structure and function remain elusive. NsP3 is a component of the plasma membrane-bound viral RNA replication complexes (vRC) and is also found in large cytoplasmic aggregates of unknown functions. Here, the inventors report the cryo-electron microscopy structure of the CHIKV nsP3 at 2.6 Å resolution. NsP3 forms highly organized tubular structures made by a helical arrangement of its alphavirus unique domain (AUD) which acts as an oligomerization module. NsP3 helices organize into a cytoplasmic ring above the nsP1 capping pores at the vRCneck. These helices assemble into cytoplasmic tubular structures organized in a honeycomb network that contains viral genomic RNA and capsid as well as host factors required for productive infection. CHIKV harboring mutations in the AUD disturbing nsP3 helices have a strong defect in viral replication, assembly and release. Altogether, the results reveal a unique nsP3-dependent molecular organization required for alphavirus infection and suggest that blocking nsP3 function may represent a potent antiviral strategy.

Description

[0001]ALPHAVIRUS NSP3 HELICAL TUBULAR SCAFFOLDS FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular virology and nanotechnology. BACKGROUND OF THE INVENTION: Alphaviruses possess a single-stranded 5′-capped and 3′-polyadenylated RNA genome organized in two open reading frames (ORFs) 4,5. The 5’ ORF encodes the P1234 polyprotein that is proteolytically processed to generate four non-structural proteins (nsP1- to 4) involved in viral replication and immune evasion 4,5. The 3’ ORF is transcribed into a sub-genomic positive-stranded RNA and encodes the structural proteins (C-E3-E2-6K-E1) important for virus assembly and budding 4,5. Alphavirus genome replication and transcription is mediated by a viral replication complex (vRC) formed by co-assembly of the four nsPs and host factors within specialized virus-induced membrane organelles called spherules 4,6,7. These compartments not only create an optimal micro-environment for viral replication by concentrating viral nsPs and the genomic RNA, but also shield the double stranded RNA (dsRNA) replication intermediates from innate immune sensors 4,5. Beside its contribution to vRC assembly, alphavirus nsPs play poorly characterized yet essential functions. NsP1 associates as a ring-shaped dodecamer to form the neck of the replication spherule, mediates capping of the viral RNA and counteracts the IFN-I response by interacting with the cyclic GMP-AMP synthase8,9. NsP2 possesses both helicase and protease activities important for the maturation of the RC and translocates into the nucleus of infected cells to shut down antiviral gene transcription 10,11. Acting as the viral RNA-dependent RNA polymerase, nsP4 localizes within the spherule where the viral RNA synthesis occurs 6. The nsP3 molecule is probably the most enigmatic alphavirus-encoded protein. NsP3 is composed by three domains, a macrodomain (MD) which recognizes and hydrolyses ADP-ribosylated proteins, a conserved Alphavirus Unique Domain (AUD) required for RNA synthesis 12, and a hypervariable domain (HVD) mediating extensive host interactions important for viral replication and adaptation to the host 2,4. During the early phase of the alphavirus life cycle, nsP3 localizes to the viral spherules where it contributes to RNA replication 5. NsP3 also accumulates in large cytoplasmic granules (referred here as “alpha-granules”) distinct from the vRC which contain the host factors G3BPs 13. The nsp3-G3BPs interaction is thought to inhibit stress granule assembly promoting efficient infection 3,14,15 . However, the function of the cytoplasmic nsP3 alpha- granules remain poorly understood. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, to alphavirus nsP3 helical tubular scaffolds. DETAILED DESCRIPTION OF THE INVENTION: Alphaviruses, such as chikungunya virus (CHIKV), are a group of emerging / re-emerging mosquito-borne viruses that represent an ongoing challenge to medicine and public health 1. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling molecule whose structure and function remain elusive. NsP3 is a component of the plasma membrane-bound viral RNA replication complexes (vRC) and is also found in large cytoplasmic aggregates of unknown functions 2,3. Here, the inventors report the cryo-electron microscopy structure of the CHIKV nsP3 at 2.6 Å resolution. NsP3 forms highly organized tubular structures made by a helical arrangement of its alphavirus unique domain (AUD) which acts as an oligomerization module. NsP3 helices organize into a cytoplasmic ring above the nsP1 capping pores at the vRCneck. These helices assemble into cytoplasmic tubular structures organized in a honeycomb network that contains viral genomic RNA and capsid as well as host factors required for productive infection. CHIKV harboring mutations in the AUD disturbing nsP3 helices have a strong defect in viral replication, assembly and release. Altogether, the results reveal a unique nsP3-dependent molecular organization required for alphavirus infection and suggest that blocking nsP3 function may represent a potent antiviral strategy. Main definitions: As used herein, the term “alphavirus” has its general meaning in the art and refers to a family of viruses characterized by one or more of the following: (i) a positive sense, single-stranded RNA genome; (ii) RNA that is 5’ capped and 3’-polyadenylated; (iii) viral particles that are enveloped, have a 70 nm diameter, and have a 40 nm isometric nucleocapsid; (iv) viral replication in the cytoplasm of host cells; (v) lack a DNA phase during the viral replication cycle; and (vi) maintain virions that mature by budding through the plasma membrane. A non- limiting list of exemplary alphaviruses includes Aura virus (AURA), Babanki (BAB), Barmah Forest virus (BF), Bebaru virus (BEB), Buggy Creek, Cabassou virus (CAB), Chikungunya virus (CHIK), Eastern equine encephalitis virus (EEE), Everglades virus (EVE), Fort Morgan virus (FM), Getah virus (GET), Highlands J virus (HJ), Kyzylagach virus (KYZ), Mayaro virus (MAY), Middelburg virus (MID), Mosso das Pedras virus (78V3531), Mucambo virus (MUC), Ndumu virus (NDU), O'nyong-nyong virus (ONN), Pixuna virus (PIX), Rio Negro virus (AG80), Ross River virus (RR), Sagiyama virus (SAG), Salmon pancreas disease virus (SPDV), Semliki Forest virus (SF), Sindbis virus (SIN), Southern elephant seal virus, Tonate virus, Trocara virus, Una virus (UNA), Venezuelan equine encephalitis virus (VEE), Western equine encephalitis virus (WEE), and Whataroa virus (WHA). Sindbis, Semliki Forest, O'nyong'nyong, Chikungunya, Mayaro, Ross River, Barmah Forest, Eastern Equine Encephalitis, Western Equine Encephalitis, and Venezuelan Equine Encephalitis viruses are particularly relevant for medical intervention in humans. Alphaviruses are evolutionarily differentiated based on nucleotide sequence of the nonstructural proteins, of which there are four (nsP1, nsP2, nsP3 and nsP4). As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. Within the specification, the mutation are references according to the standard mutation nomenclature. In particular the term “mutation” encompasses “naturally- occurring mutations” and “non-naturally occurring mutations”. As used herein, the term “nsP3” refers to nonstructural protein 3, which is found in alphaviruses. Nsp3 is a multifunctional protein that plays a crucial role in the replication and assembly of alphaviruses. It is a component of the viral replication complex and is involved in various steps of the viral life cycle. Nsp3 is typically produced as part of a polyprotein precursor that is processed into individual nonstructural proteins, including nsp3, by viral proteases. Nsp3 consists of multiple domains with distinct functions. Exemplary amino acid sequences for Nsp3 are represented by SEQ ID NO:2-11. SEQ ID NO:2> Chikungunya virus (strain S27-African prototype) (CHIKV) APSYRVKRMDIAKNDEECVVNAANPRGLPGDGVCKAVYKKWPESFKNSATPVGTAKTVMCGTYPVIHAV GPNFSNYSESEGDRELAAAYREVAKEVTRLGVNSVAIPLLSTGVYSGGKDRLTQSLNHLFTAMDSTDAD VVIYCRDKEWEKKISEAIQMRTQVELLDEHISIDCDVVRVHPDSSLAGRKGYSTTEGALYSYLEGTRFH QTAVDMAEIYTMWPKQTEANEQVCLYALGESIESIRQKCPVDDADASSPPKTVPCLCRYAMTPERVTRL RMNHVTSIIVCSSFPLPKYKIEGVQKVKCSKVMLFDHNVPSRVSPREYRPSQESVQEASTTTSLTHSQF DLSVDGKILPVPSDLDADAPALEPALDDGAIHTLPSATGNLAAVSDWVMSTVPVAPPRRRRGRNLTVTC DEREGNITPMASVRFFRAELCPVVQETAETRDTAMSLQAPPSTATELSHPPISFGAPSETFPITFGDFN EGEIESLSSELLTFGDFLPGEVDDLTDSDWSTCSDTDDELRLDRAGG SEQ ID NO:3> O'nyong-nyong virus (strain Gulu) (ONNV) APSYRVKRMDIAKNTEECVVNAANPRGVPGDGVCKAVYRKWPESFRNSATPVGTAKTIMCGQYPVIHAV GPNFSNYSEAEGDRELASVYREVAKEVSRLGVSSVAIPLLSTGVYSGGKDRLLQSLNHLFAAMDSTDAD VVIYCRDKEWEKKITEAISLRSQVELLDDHISVDCDIVRVHPDSSLAGRKGYSTVEGALYSYLEGTRFH QTAVDMAEIYTMWPKQTEANEQVCLYALGESIESVRQKCPVDDADASFPPKTVPCLCRYAMTPERVARL RMNHTTSIIVCSSFPLPKYKIEGVQKVKCSKALLFDHNVPSRVSPRTYRPADEIIQTPQTPTEACQDAQ LVQSINDEAVPVPSDLEACDATMDWPSIGTVSTRQRHDSSDSEYSGSRSNIQLVTADVHAPMYAHSLAS SGGSMLSLSSEPAQNGTMILLDSEDTDSISRVSTPIAPPRRRLGRTINVTCDEREGKILPMASDRFFTA KPYTVALSVSTADMTVYPIQAPLGLIPPPTLEPITFGDFAEGEIDNLLTGALTFGDFEPGEVEELTDSE WSTCSDTDEELRLDRAGG SEQ ID NO:4> Ross river virus (strain NB5092) (RRV) APSYRVRRTDISGHAEEAVVNAANAKGTVGVGVCRAVARKWPDSFKGAATPVGTAKLVQANGMNVIHAV GPNFSTVTEAEGDRELAAAYRAVAGIINASNIKSVAIPLLSTGVFSGGKDRVMQSLNHLFTAMDTTDAD VVIYCRDKAWEKKIQEAIDRRTAVELVSEDISLESDLIRVHPDSCLVGRKGYSITDGKLHSYLEGTRFH QTAVDMAEISTLWPKLQDANEQICLYALGESMDSIRTKCPVEDADSSTPPKTVPCLCRYAMTAERVARL RMNNTKAIIVCSSFPLPKYRIEGVQKVKCDRVLIFDQTVPSLVSPRKYIPAAASMHADTVSLDSTVSTG SAWSFPSEATYETMEVVAEVHHSEPPVPPPRRRRAQVTMHHQELLEVSDMHTPIAARVEIPVYDTAVVA ERVAIPCTSEYATPIPTPRAVRVVPVPAPRIQRASTYRVSPTPTPRVLRASVCSVTTSAGVEFPWAPED LEVLTEPVHCEMREPVELPWEPEDVDIQFGDFETPDKIQFGDIDFDQF SEQ ID NO:5> Mayaro virus (strain Brazil) (MAYV) APAYAVKRADIATAIEDAVVNAANHRGQVGDGVCRAVARKWPQAFRNAATPVGTAKTVKCDETYIIHAV GPNFNNTSEAEGDRDLAAAYRAVAAEINRLSISSVAIPLLSTGIFSAGKDRVHQSLSHLLAAMDTTEAR VTIYCRDKTWEQKIKTVLQNRSATELVSDELQFEVNLTRVHPDSSLVGRPGYSTTDGTLYSYMEGTKFH QAALDMAEITTLWPRVQDANEHICLYALGETMDNIRARCPVEDSDSSTPPKTVPCLCRYAMTPERVTRL RMHHTKDFVVCSSFQLPKYRIPGVQRVKCEKVMLFDAAPPASVSPVQYLTNQSETTISLSSFSITSDSS SLSTFPDLESAEELDHDSQSVRPALNEPDDHQPTPTAELATHPVPPPRPNRARRLAAARVQVQVEVHQP PSNQPTKPIPAPRTSLRPVPAPRRYVPRPVVELPWPLETIDVEFGAPTEEESDITFGDFSASEWETISN SS SEQ ID NO:6> Eastern equine encephalitis virus (strain Florida 91-469) (EEEV) (Eastern equine encephalomyelitis virus) APAYRVVRGDITKSNDEVIVNAANNKGQPGSGVCGALYRKWPGAFDKQPVATGKAHLVKHSPNVIHAVG PNFSRLSENEGDQKLSEVYMDIARIINNERFTKVSIPLLSTGIYAGGKDRVMQSLNHLFTAMDTTDADI TIYCLDKQWESRIKEAITRKESVEELTEDDRPVDIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFH QTAKDIAEIYAMWPNKQEANEQICLYVLGESMNSIRSKCPVEESEASSPPHTIPCLCNYAMTAERVYRL RMAKNEQFAVCSSFQLPKYRITGVQKIQCSKPVIFSGTVPPAIHPRKFASVTVEDTPVVQPERLVPRRP APPVPVPARIPSPPCTSTNGSTTSIQSLGEDQSASASSGAEISVDQVSLWSIPSATGFDVRTSSSLSLE QPTFPTMVVEAEIHASQGSLWSIPSITGSETRAPSPPSQDSRPSTPSASGSHTSVDLITFDSVAEILED FSRSPFQFLSEIKPIPAPRTRVNNMSRSADTIKPIPKPRKCQVKYTQPPGVARVISAAEFDEFVRRHSN SEQ ID NO:7> Venezuelan equine encephalitis virus (strain 3880) (VEEV) APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAV GPNFNKVSEIEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDAD VAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLE GTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPE RVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPTVEENQSTEGT PEQPTLITVGETRTRTPEPIIIEEEEDSISLLSDGPTHQVLQVEADIHGPPSASSSSWSIPHASDFDVD SLSILDTLEGASVTSEEASVETNSHFARSMEFLARPVPAPRTVFRNPPQPAPRTRTPSLAPSRASSRIS LVSNPPGVNRVITREELEALTPSRAPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQ SEQ ID NO:8> Barmah forest virus (BFV) APAYRVKRGDISNAPEDAVVNAANQQGVKGAGVCGAIYRKWPDAFGDVATPTGTAVSKSVQDKLVIHAV GPNFSKCSEEEGDRDLASAYRAAAEIVMDKKITTVAVPLLSTGIYAGGKNRVEQSLNHLFTAFDNTDAD VTIYCMDKTWEKKIKEAIDHRTSVEMVQDDVQLEEELVRVHPLSSLAGRKGYSTDSGRVFSYLEGTKFH QTAVDIAEMQVLWPALKESNEQIVAYTLGESMDQIRGKCPTEDTDASTPPRTVPCLCRYAMTPERVYRL KCTNTTQFTVCSSFELPKYHIQGVQRVKCERIIILDPTVPPTYKRPCIRRYPSTISCNSSEDSRSLSTF SVSSDSSIGSLPVGDTRPIPAPRTIFRPVPAPRAPVLRTTPPPKPPRTFTVRAEVHQAPPTPVPPPRPK RAAKLAREMHPGFTFGDFGEHEVEELTASPLTFGDFAEGEIQGMGVEFE SEQ ID NO:9> Sindbis virus (SINV) APSYRTKRENIADCQEEAVVNAANPLGRPGEGVCRAIYKRWPTSFTDSATETGTARMTVCLGKKVIHAV GPDFRKHPEAEALKLLQNAYHAVADLVNEHNIKSVAIPLLSTGIYAAGKDRLEVSLNCLTTALDRTDAD VTIYCLDKKWKERIDAALQLKESVTELKDEDMEIDDELVWIHPDSCLKGRKGFSTTKGKLYSYFEGTKF HQAAKDMAEIKVLFPNDQESNEQLCAYILGETMEAIREKCPVDHNPSSSPPKTLPCLCMYAMTPERVHR LRSNNVKEVTVCSSTPLPKHKIKNVQKVQCTKVVLFNPHTPAFVPARKYIEVPEQPTAPPAQAEEAPEV VATPSPSTADNTSLDVTDISLDMDDSSEGSLFSSFSGSDNSITSMDSWSSGPSSLEIVDRRQVVVADVH AVQEPAPIPPPRLKKMARLAAARKEPTPPASNSSESLHLSFGGVSMSLGSIFDGETARQAAVQPLATGP TDVPMSFGSFSDGEIDELSRRVTESEPVLFGSFEPGEVNSIISSRSAVSFPLRKQRRRRRSRRTEY SEQ ID NO:10> Aura virus (AURAV) APSYRVKRMNIADCTEEAVVNAANARGKPGDGVCRAIFKKWPKSFENATTEVETAVMKPCHNKVVIHAV GPDFRKYTLEEATKLLQNAYHDVAKIVNEKGISSVAIPLLSTGIYAAGADRLDLSLRCLFTALDRTDAD VTIYCLDKKWEQRIADAIRMREQVTELKDPDIEIDEGLTRVHPDSCLKDHIGYSTQYGKLYSYFEGTKF HQTAKDIAEIRALFPDVQAANEQICLYTLGEPMESIREKCPVEDSPASAPPKTIPCLCMYAMTAERICR VRSNSVTNITVCSSFPLPKYRIKNVQKIQCTKVVLFNPDVPPYIPARVYINKDEPPVTPHTDSPPDTCS SRLSLTPTLSNAESDIVSLTFSEIDSELSSLNEPARHVMISSFKLRYTAIQALPQKLSWMREDRTPRQP PPVPPPRPKRAAKLSRLANQLNELRRHATISSVQAEVHYNSGFTPEAELNERGSILRKPPPVPPLRPKQ TTNLSRLANQLSMPITFGDFAEGELDRLLTPSPTPTFGDFSQEEMDRFFGNRQY SEQ ID NO:11> Salmon pancreas disease virus (SPDV) APGYRVLNRNIITAEEEVLVNAANSNGRPGDGVCGALYGAFGDAFPNGAIGAGNAVLVRGLEATIIHAA GADFREVDEETGARQLRAAYRAAATLVTANGITSAAIPLLSTHIFSNGRNRLEQSFSALVEAFDTTECD VTIYCLANNMAARIQQLIDAHAREEFDEEVVVEEEEEHEADAMSDTETLSSFGDETVWVPKHSTLAGRP GYSAYYGDRRSLFVGTKFHRAAVAMSSIEAAWPKTKEANAKLIEYIRGQHLVDVLKSCPVDDIPVGRPP SSLPCGCIYAMTPERVTVLKQRPQEGFVVCSAFKLPLTNIQDVTKVECTVRAPAEEPRPVRHLQERRPA QAAVRQLRPAAVAASVAASHTASRTSTASSRRTPAPGSVQVRLLPPRDGTESRSSRMGSQSSVTSSAGS VPPAPRRAPAVSAASLASSAHSRSVRSAPAMRAASAGARSVRSAQSGSTGHRAGAFSVAGSVRQPSGPP SSVSTPAAIRGLTRDQFDAVRVRARRNLELEGSEHGSQSSFHSGSLAVGSSASSYSQRSDDQDTGTEPS SRGAAVRTRRRGQRDGLGG As used herein, the term “alpha unique domain” or “AUD” has its general meaning in the art and refers to the central domain of Nsp3. The domain is termed as “alphavirus unique domain” because it is unique to the genus of alphaviruses and not present in other related viruses. Exemplary amino acid sequences for Nsp3 are represented by SEQ ID NO:11-20. SEQ ID NO:12> Chikungunya virus (strain S27-African prototype) (CHIKV) DVVRVHPDSSLAGRKGYSTTEGALYSYLEGTRFHQTAVDMAEIYTMWPKQTEANEQVCLYALGESIESI RQKCPVDDADASSPPKTVPCLCRYAMTPERVTRLRMNHVTSIIVCSSFPLPKYKIEGVQKVKCSKVM SEQ ID NO:13> O'nyong-nyong virus (strain Gulu) (ONNV) DIVRVHPDSSLAGRKGYSTVEGALYSYLEGTRFHQTAVDMAEIYTMWPKQTEANEQVCLYALGESIESV RQKCPVDDADASFPPKTVPCLCRYAMTPERVARLRMNHTTSIIVCSSFPLPKYKIEGVQKVKCSKAL SEQ ID NO:14> Ross river virus (strain NB5092) (RRV) DLIRVHPDSCLVGRKGYSITDGKLHSYLEGTRFHQTAVDMAEISTLWPKLQDANEQICLYALGESMDSI RTKCPVEDADSSTPPKTVPCLCRYAMTAERVARLRMNNTKAIIVCSSFPLPKYRIEGVQKVKCDRVL SEQ ID NO:15> Mayaro virus (strain Brazil) (MAYV) NLTRVHPDSSLVGRPGYSTTDGTLYSYMEGTKFHQAALDMAEITTLWPRVQDANEHICLYALGETMDNI RARCPVEDSDSSTPPKTVPCLCRYAMTPERVTRLRMHHTKDFVVCSSFQLPKYRIPGVQRVKCEKVM SEQ ID NO:16> Eastern equine encephalitis virus (strain Florida 91-469) (EEEV) (Eastern equine encephalomyelitis virus) ELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFHQTAKDIAEIYAMWPNKQEANEQICLYVLGESMNSI RSKCPVEESEASSPPHTIPCLCNYAMTAERVYRLRMAKNEQFAVCSSFQLPKYRITGVQKIQCSKPV SEQ ID NO:17> Venezuelan equine encephalitis virus (strain 3880) (VEEV) ELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSI RSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPI SEQ ID NO:18> Barmah forest virus (BFV) ELVRVHPLSSLAGRKGYSTDSGRVFSYLEGTKFHQTAVDIAEMQVLWPALKESNEQIVAYTLGESMDQI RGKCPTEDTDASTPPRTVPCLCRYAMTPERVYRLKCTNTTQFTVCSSFELPKYHIQGVQRVKCERII SEQ ID NO:19> Sindbis virus (SINV) ELVWIHPDSCLKGRKGFSTTKGKLYSYFEGTKFHQAAKDMAEIKVLFPNDQESNEQLCAYILGETMEAI REKCPVDHNPSSSPPKTLPCLCMYAMTPERVHRLRSNNVKEVTVCSSTPLPKHKIKNVQKVQCTKVV SEQ ID NO:20> Aura virus (AURAV) GLTRVHPDSCLKDHIGYSTQYGKLYSYFEGTKFHQTAKDIAEIRALFPDVQAANEQICLYTLGEPMESI REKCPVEDSPASAPPKTIPCLCMYAMTAERICRVRSNSVTNITVCSSFPLPKYRIKNVQKIQCTKVV SEQ ID NO:21> Salmon pancreas disease virus (SPDV) ETVWVPKHSTLAGRPGYSAYYGDRRSLFVGTKFHRAAVAMSSIEAAWPKTKEANAKLIEYIRGQHLVDV LKSCPVDDIPVGRPPSSLPCGCIYAMTPERVTVLKQRPQEGFVVCSAFKLPLTNIQDVTKVECTVRA As used herein, the term “scaffold” is used to denote a specific, conformationally and thermodynamically stable proteinaceous molecule with a specific, fixed three-dimensional structure consisting by a plurality of the polypeptides of the present invention. The scaffold of the present invention can be permissive to a variety of single and multiple amino acid substitutions at a variety of amino acid residue positions. As used herein, the term “helical tubular scaffold” denotes that the scaffold of the present invention adopts the structure of a helical and tubular conformation as described in the EXAMPLE. In particular, the helical tubular scaffold of the present invention comprises a well-defined internal cavity delimited by a weakly structured matrix. According to the present invention, the helical tubular scaffold of the present invention displays a helical arrangement of the nsP3 AUD with an axial rise of 2.782 Å and a helical twist of -164.175 ° as described in the EXAMLE. The assembly generates interstitial positively charged pores connecting the inner cavity with the exterior, which could accommodate small negatively charged molecules like RNA. More particularly, the helical tubular scaffold of the present invention consists of four alpha helixes (a1-a4) with extensions at the N and C terminus acting as oligomerization modules (C and N O-modules) which are responsible for lateral and axial contacts with the AUD of neighboring protomers, respectively. As used herein, the term “self-assembly” refers to the ability of the polypeptide of the present invention to interact to each other in a predicted manner and without external control so as to form the helical tubular scaffold of the present invention. As used herein, the term “nanocarrier” refers to a carrier with a nanosized structure in which at least one of its phases has one or more dimensions (length, width or thickness) in the nanometer size range. As used herein, the term “payload” refers to any compound of interest that can be incorporated into the tubular helical scaffold of the present invention. Scaffolds: The first object of the present invention relates to a helical tubular scaffold formed by self- assembly of a plurality of polypeptides having the consensus amino acid sequence as set forth in SEQ ID NO:1: SEQ ID NO:1> consensus AUD sequence EXVRX5HPXSXLXGRXGX17STXXGXXYSYXE GTXFHQXAXDMAEIXXLWPXXXEANEQICXY XLGX64XMEXXRXKCPVX76DXXXSXPPXTXPC LCXYAMTPERXXRLXXXXXXXXXVCSSFXLP KYXIXXVQKX130XCXXXX wherein: - X represents any amino acid residue - X5 represents V or I - X17represents Y or F - X64represents E or Q - X76 represents D or E - X130represents V or I. According to the present invention, the consensus amino acid sequence can be permissive to a variety of single and multiple amino acid mutation (especially substitutions) at a variety of amino acid residue positions excepting for the amino acid residue at position 27 (Y) and optionally for the amino acid residues at position 74 (P) and 75 (V). In particular, surface residues can be varied by making use of standard protein engineering methods. Making use of the skilled person's knowledge, appropriate selection methods can be applied for the purpose of identifying variants (scaffold derivatives, specific molecular compounds) with highly desired properties (e.g., binding properties, release properties). However, some amino acid residues are critical. In particular, the amino acid residues at position 27 (Y) and optionally the amino acid residues at position 74 (P) and 75 (V) must not be mutated. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:12. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:13. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:14. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:15. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:16. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:17. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:18. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:19. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:20. In some embodiments, the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:21. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:2. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:3. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:4. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:5. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:6. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:7. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:8. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:9. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:10. In some embodiments, the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:11. In some embodiments, the polypeptides are labelled with a detectable molecule. According to the invention, said detectable molecule may consist of any substance or substance that is detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful detectable molecules include radioactive substance (including those comprising 32P, 25S, 3H, or 125I), fluorescent dyes (including 5-bromodesosyrudin, fluorescein, acetylaminofluorene or digoxigenin), fluorescent proteins (including GFPs and YFPs), or detectable proteins or peptides (including biotin, polyhistidine tails or other antigen tags like the HA antigen, the FLAG antigen, the c-myc antigen and the DNP antigen). According to the invention, the detectable molecule is located at, or bound to, an amino acid residue located outside the binding sites of the polypeptides, in order to minimise or prevent any artefact for the binding between said polypeptides or between the test substance and or any of said polypeptides. The helical tubular scaffold of the present invention can be prepared as described in the EXAMPLE. Briefly, the polypeptide of the present invention is expressed in eukaryotic cells. Cells are resuspended in buffer A (20mM Tris pH 8, 500mM NaCl, 2mM TCEP, 5% glycerol, 20mM imidazole) supplemented with 2mM PMSF, 10 ^ ^g / ml DNAse and 5 mM MgSO4, and lysed by sonication. Lysates are centrifuged at 18,000g for 1h to remove unbroken cells and debris. The clarified lysate is incubated with Ni-NTA resin (GE Healthcare) in batch at 4oC for 30 minutes. Resin is washed with 10 column volumes of buffer A and the protein eluted over 1.5 column volumes with buffer A containing 500mM imidazole. Elution fractions containing the protein are pooled and concentrated (Amicon 30k MW C / O) for size exclusion chromatography. Samples are applied to a Supedex 200 10 / 300 increase column (GE Healthcare) pre-equilibrated with the size exclusion buffer: 20 mM HEPES pH = 8.0 ; 500 mM NaCl; 5 % glycerol and 2 mM TCEP. The monomeric polyeptide can then be used for the in vitro assembly of the scaffolds by lowering the salt concentration by dilution or dialysis. The final protein and salt concentration will determine the number and the length of the assemblies yielded. In particular, the concentration of the polypeptides ranges from 0.05 to 1 mg / ml. The concentration of NaCl is inferior to 200mM. More particularly, the concentration of NaCl ranges from 0.05 to 1 M NaCl. The temperature ranges from 4 to 30°C. Screening methods: A further object of the present invention relates to a method for identifying a substance useful for inhibiting the replication capacity of an alpha virus comprising the steps of (a) determining whether a test substance is capable of perturbate the assembly of the tubular helical scaffold of the present invention and (b) selecting the substance that perturbates the assembly of the helical tubular scaffold. In some embodiments, the screening method comprises the steps that consist in i) contacting the test substance with a plurality of the polypeptides of the present invention in conditions that allow the assembly of the tubular helical scaffold of the present invention and ii) determining whether the test substance is capable of inhibiting the assembly of the tubular helical scaffolds and iii) selecting the test substances that is capable of inhibiting the assembly of the tubular helical scaffolds. In some embodiments, the screening method comprises the steps that consist in i) contacting the test substance with one or more tubular helical scaffold(s) of the present invention in conditions that allow the maintenance of the assembly of the tubular helical scaffold and ii) determining whether the test substance is capable of disassembling the tubular helical scaffolds and iii) selecting the test substances that is capable of disassembling the tubular helical scaffolds. Different assays that are routinely used in the art can be used for detecting the assembly of the helical tubular scaffolds. For instance, the methods described in the EXAMPLE can be implemented for determining the assembly of the helical tubular scaffolds. In particular, the assembly can be detected by optical microscopy by immunofluorescence or intrinsic fluorescence when a fluorescent tag is inserted in the polypepitde, negative stained electron microscopy, Dynamic Light Scattering (DLS) and by sedimentation over ultracentrifugation. Typically, the screening method of the present invention may be performed in vitro (outside a cellule) or in cellulo in cells that are infected by an alphavirus or that is genetically engineering to express a polypeptide of the presentation, i.e. a polypeptide having the consensus amino acid sequence as set forth in SEQ ID NO:1. The test substance of the invention may be selected from a library of substances previously synthesised, or a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesised de novo. The test substance may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical substances. In some embodiments, the method of the present invention further comprises the step (d) consisting in determining whether the substance selected at step (c) inhibits the replication of an alphavirus in a host cell and a step (e) that consists in positively selecting the test substance capable of inhibiting the replication of said alphavirus in said host cell. In some embodiments, the method comprises the steps consisting of i) infecting said host cell with said alphavirus and ii) culturing said infected cell in presence of the test substance, iii) comparing the replicating capacity of the virus with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the virus. The term "inhibiting the replication capacity," as used herein with reference to a viral phenotype, means that the virus grows to a lower titer in the presence of a substance as above described relative to the virus grown in the absence of said substance. In some embodiments, the presence of said substance which will inhibit the ability of an alphavirus to replicate in a host cell by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by at least about 100%, or by at least about 200%, or by at least about 300%, or by at least about 400%, or by at least about 500% when compared to said alphavirus grown in the absence of said substance. Said replication capacity may be typically determined by any routine technique well known in the art. According to the present invention, any alphavirus strain can be used. Preferably, said alphavirus strain corresponds to one clinical isolate of at least one circulating strain of alphavirus. According to the invention, any eukaryotic cell may be used in the screening method of the invention. In some embodiments the cell is a human cell. In some embodiments, the cell is a cell line. Non-limiting examples of cell lines that can be suitable for the invention include but are not limited to BS-C-1, CV-1, Vero, Vero 76, Vero C1008, Vero 76, Cos-1, Cos-7, Huh7, FR11K-4, LLC-MK2 original, LLC-MK2 derivative, MDCK, RD, A549, MRC-5, KB, PER.C6, HEK-293 and CaCo-2 cells. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and C02 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Suitable serum free media are described, for example, in U.S. Provisional Application No. 60 / 638,166, filed Dec.23, 2004, and in U.S. Provisional Application No.60 / 641,139, filed Jan.5, 2005, each of which is hereby incorporated by reference in its entirety. Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and / or additional nutrients, such as L-glutamine, sodium pyruvate, nonessential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, (3-mercaptoethanol, and the like. In some embodiments, the infection of the cells with alphavirus is carried out at an m.o.i. (multiplicity of infection) of about 0.0001 to 10, preferably of 0.002 to 0.5. Typically, the MOI is 0.1, 0.01 or 0.001 for Vero cells or 0.1, 0.05, 0.01 or 0.001 for Huh7 cells while preferably an MOI is used of 0.001 for Vero cells or 0.05 for Huh7 cells. Typically, the cells can be grown in culture under conditions permissive for replication and assembly of viruses. In some embodiments, cells can be cultured at a temperature below about 37° C, preferably at a temperature equal to, or less than, about 35° C. Typically, the cells are cultured at a temperature between about 32° C. and about 35° C. In some embodiments, the cells are cultured at a temperature between about 32° C. and 34° C, e.g., at about 33° C. As described above, the methods of the present invention are particularly useful for screening a plurality of substances that may be used for the treatment or prevention of alphavirus infections as described infra. In some embodiments, the substances selected by the above mentioned screening method may be used in the treatment of alphavirus infection. For example, therapeutic treatments includes the reduction or amelioration of the progression, severity and / or duration of alphavirus infections, or the amelioration of one or more symptoms (specifically, one or more discernible symptoms) of alphavirus infections, resulting from the administration of at least one substance selected by the above mentioned screening method. In some embodiments, the therapeutic treatment includes the amelioration of at least one measurable physical parameter of a alphavirus infection. In some embodiments, the therapeutic treatment includes the inhibition of the progression of an alphavirus infection, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In some embodiments, the therapeutic treatment includes the reduction or stabilization of alphavirus infections. In some embodiments, the substances selected by the above mentioned screening method may be used in a prophylactic treatment. The terms "prophylaxis" or "prophylactic use" and "prophylactic treatment" as used herein, refer to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a subject with the disease. As used herein, prophylactic use includes the use in situations in which an outbreak has been detected, to prevent contagion or spread of the infection in places where a lot of people that are at high risk of serious complications live in close contact with each other (e.g. in a hospital ward, daycare center, prison, nursing home, etc). Prophylactic use may also include treating a person who is not ill with the alphavirus or not considered at high risk for complications, in order to reduce the chances of getting infected with the alphavirus and passing it on to a high-risk person in close contact with him (for instance, healthcare workers, nursing home workers, etc). Typically, the substances selected by the above mentioned screening method are administered to the subject in an effective amount. As used herein, an "effective amount" refers to an amount sufficient to elicit the desired biological response. In the present invention the desired biological response is to inhibit the replication of alphavirus, to reduce the amount of alphavirus or to reduce or ameliorate the severity, duration, progression, or onset of a alphavirus infection, prevent the advancement of an alphavirus infection, prevent the recurrence, development, onset or progression of a symptom associated with an alphavirus infection, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against alphavirus infections. The precise amount of compound administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When co- administered with other anti-viral agents, e.g., when co-administered with an anti-alphavirus medication, an "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed. For example, compounds described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment. In some embodiments the substances selected by the above mentioned screening method are used in combination with an additional suitable therapeutic agent, for example, an antiviral agent or a vaccine. When "combination therapy" is employed, an effective amount can be achieved using a first amount of a substance selected by the above mentioned screening method and a second amount of an additional suitable therapeutic agent (e.g. an antiviral agent). As used herein, the terms "in combination" or "co-administration" can be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of the terms does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject. Specific examples that can be co- administered with a substance selected by the above mentioned screening method include non- steroidal anti-inflammatory drugs (NSAIDS). Examples of Aspirin, Naproxen, Sulindac, Ibuprofen, Indomethacin, Valproic acid, Fenamic acid, Flurbiprofen, Diclofenac, Diflunisal, Salsalate, Choline Magnesium Trisalicylate, Dexibuprofen, Fenoprofen, Detoprofen, Dexketoprofen, Oxaprozin, Loxoprofen, Tolmetin, Etodolac, Ketorolac, Aceclofenac, Nabumetone, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, Selective COX-2 inhibitors, and Licofelone. The substances selected by the above mentioned screening method can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present invention relates to a pharmaceutical composition comprising a substance selected by the above mentioned screening method described above, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. Nanocarriers: A further object of the present invention relates to the use of the tubular helical scaffold of the present invention as a nanocarrier. The tubular helical scaffold of the present invention can be indeed used as a nanocarrier, similarly to many other documented nanocarriers (reviewed in Wang, J., Li, Y. & Nie, G. Multifunctional biomolecule nanostructures for cancer therapy. Nat Rev Mater 6, 766–783 (2021); Bhaskar, S., Lim, S. Engineering protein nanocages as carriers for biomedical applications. NPG Asia Mater 9, e371 (2017); and Sun X, Hong Y, Gong Y, Zheng S, Xie D. Bioengineered Ferritin Nanocarriers for Cancer Therapy. Int J Mol Sci.2021 Jun 29;22(13):7023). The tubular helical scaffold of the present invention can present numerous advantages, for example, their relatively small size, high structural stability and easy assembly and disassembly. In particular, these features considerably facilitate their synthesis, purification and storage. The scaffolds of the present invention embody all of aforementioned features, thereby rendering them particularly well-suited for scaffold-based applications, in particular for incorporate and release some payload of interest. Thus a further object of the present invention relates to a nanocarrier comprising a tubular helical scaffold of the present invention and a payload. Typically, the payload of the present invention is a negatively charged molecule. As used herein, the term “negatively charged molecule” includes molecules such as naturally occurring and chemically modified nucleic acid molecules (e.g., RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like), peptides (e.g., polyaminoacids, polypeptides, proteins and the like), nucleotides, pharmaceutical and biological compositions, that have negatively charged groups that can ion-pair with the positively charged pore of the tubular helical scaffold of the present invention. In some embodiments, the payload consists in a small molecule. In some embodiments, the payload consists of a polynucleotide. As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single- stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA. In some embodiments, the polynucleotide of the present invention is a messenger RNA (mRNA). In some embodiments, the polynucleotide is an aptamer. As used herein, the term “aptamer” refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. In some embodiments, the polynucleotide is an antisense nucleic acid. As used herein, the term “antisense nucleic acid” refers to a non-enzymatic nucleic acid molecule that binds to target nucleic acid by means of RNA-RNA or RNA-DNA or RNA-PNA interactions and alters the activity of the target nucleic acid . Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and / or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. In addition, antisense DNA can be used to target nucleic acid by means of DNA-RNA interactions, thereby activating RNase H, which digests the target nucleic acid in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target nucleic acid. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. Typically, the nanocarrier of the present invention allows the release of the payload when the tubular helical scaffold disassembles. In particular, the release occurs when the tubular helical scaffold of the present invention is placed in saline conditions as described in the EXAMPE. As used herein, the term “release” means that all or substantially all the payload associated with the nanocarrier becomes dissociated from the nanocarriers and its constituent polymers, frequently, but not always, accompanying the disassembly / degradation of the nanocarrier. As used herein in this instance, the “substantial release” may refer to the release of approximately 50% or more; the release of approximately 60% or more; the release of approximately 70% or more; the release of approximately 80% or more; the release of approximately 90% or more; the release of approximately 95% or more; or the release of approximately 99% or more of the payload associated with the nanocarrier. The nanocarriers of the present invention can be used to administer the payload (e.g. negatively charged molecule) which acts as a pharmaceutical agent. Thus the nanocarriers of the present invention may be suitable as therapeutic compounds. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient. The present invention thus also includes pharmaceutically acceptable formulations of the nanocarriers described above, preferably in combination with the payload to be delivered. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the payload is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. In some embodiments, the nanocarriers of the present invention can be suitable for vaccination. As used herein, the term “vaccination” or “vaccinating” means, but is not limited to, a process to elicit an immune response in a subject against a particular antigen. Thus, a further object of the present invention relates to a vaccine composition that comprises a therapeutically effective amount of a nanocarrier of the present invention. As used herein, the term "vaccine composition" is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular APCs, T lymphocytes and B lymphocytes. In some embodiments, the nanocarrier of the present invention incorporated a RNA polynucleotide that encodes for an antigen. Some embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines that include at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). The vaccines of the present disclosure may be used to induce an immune response against an alphavirus. The vaccines of the present disclosure may be used to induce an immune response against Malaria (e.g., P. falciparum, P. vivax, P. Malariae and / or P. ovale), Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Chikungunya Virus (CHIKV), Dengue Virus (DENV), Zika Virus (ZIKV) and / or Yellow Fever Virus (YFV). The vaccines of the present invention may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As used herein, the terms “live vaccine composition”, “live vaccine”, “live bacterial vaccine”, and similar terms refer to a composition comprising a strain of live CHIKV that provides at least partial protective immunity against a disease, condition, or disorder. As used herein, the term “adjuvant” refers to a compound that can induce and / or enhance the immune response against an antigen when administered to a patient or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both the innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dendritic cells (DCs). Attenuated viruses of the present invention: The inventors show that the mutant Y200A abrogated the overall synthesis of RNA. Y200A is a mutation capable of both interfere the scaffold formation and the interaction with the protease in the uncleaved polyprotein. In consequence, the mutation could be affecting the function of the early uncleaved replication complex. The Mutants P247A / V248A and K302A / V303A partially disrupting nsP3 assemblies and prevent the crowns to be fully functional, resulting in the reduction of sgRNA synthesis. A further object of the present invention relates to an attenuated alphaviruses (e.g. an attenuated chikungunya virus (CHIKV)) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that harbors one of the following mutation Y200A, K302A / V303A or P247A / V248A. In some embodiments, the attenuated alphaviruses (e.g. an attenuated CHIKV) of the present invention is engineered to express a nsP3 mutein wherein the nsP3 proteins harbors the Y200A mutation. In some embodiments, the attenuated alphaviruses (e.g. an attenuated CHIKV) of the present invention is engineered to express a nsP3 mutein wherein the nsP3 proteins harbors the K302A / V303A mutation. In some embodiments, the attenuated alphaviruses (e.g. an attenuated CHIKV) of the present invention is engineered to express a nsP3 mutein wherein the nsP3 proteins harbors the P247A / V248A mutation. The attenuated alphaviruses (e.g. an attenuated CHIKV) of the present invention can be derived from any genotype of chikungunya virus, such as the African or Asian genotype, that has been genetically modified to introduce one or more mutations in the nsP3 protein. In some embodiments, the attenuated CHIV of the present derives from a strain isolated in the Ile de la Réunion (France). The mutation(s) can be introduced by any suitable method, such as site-directed mutagenesis, or reverse genetics. Methods for introducing deletion in the viral genome are well known in the art. For example, homologous recombination may be used, in which a transfer vector is created in which the relevant gene(s) are missing and used to transfect virus-infected cells. Recombinant viruses expressing the new portion of sequence may then be selected. Thus, certain aspects of the present invention are drawn to a method of producing a vaccine comprising the steps of introducing the engineered virus into host cells and allowing the virus to replicate in the host cells to produce a viral vaccine. The resulting attenuated alphaviruses (e.g. an attenuated CHIKV) can be propagated in cell culture and purified by standard techniques. The attenuated viruses of the present invention can be propagated in various cell lines that are permissive for chikungunya virus infection. Examples of such cell lines include, but are not limited to, Vero cells, C6 / 36 cells, BHK-21 cells, HeLa cells, 293T cells, CHO cells, COS cells, and SF9 cells. The choice of the cell line may depend on the efficiency of virus replication, the yield of virus production, and the safety of the cell culture. The cell line may also be modified to express certain viral proteins or factors that can enhance the virus propagation. For the cell line may also be genetically engineered to have reduced expression or activity of host factors that can restrict the virus infection, such as interferons, PKR, or OAS. The cell line may also be treated with chemicals or drugs that can modulate the cellular environment or metabolism to favor the virus propagation. For example, the cell line may be treated with cycloheximide, puromycin, or actinomycin D, which can inhibit the host protein synthesis and reduce the antiviral response. Alternatively, the cell line may be treated with sodium butyrate, trichostatin A, or valproic acid, which can induce histone acetylation and increase the viral gene expression. The attenuation of the alphavirus (e.g. CHIV) can be confirmed by measuring its reduced replication and virulence in vitro and in vivo compared to the wild-type virus. The attenuated alphaviruses (e.g. an attenuated CHIKV) can also be tested for its immunogenicity and protective efficacy against chikungunya infection in animal models. A further object of the present invention provides a method of vaccinating a patient in need of such treatment, comprising the steps of administering the viral vaccine of the present invention to the patient and allowing the vaccine to produce viral proteins for immune surveillance and / or to stimulate the immune system for antibody production in the patient. In particular, the present invention relates to a method of vaccinating a patient in need of such treatment, comprising the steps of administering an attenuated alphaviruses (e.g. an attenuated chikungunya virus (CHIKV)) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that harbors one of the following mutation Y200A, K302A / V303A or P247A / V248A. In particular, the present invention relates to a method of vaccinating a patient in need of such treatment, comprising the steps of administering an attenuated alphaviruses (e.g. an attenuated In particular, the chikungunya virus (CHIKV)) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) wherein the nsP3 proteins harbors the Y200A mutation. present invention relates to a method of vaccinating a patient in need of such treatment, comprising the steps of administering an attenuated alphaviruses (e.g. an attenuated chikungunya virus (CHIKV)) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) wherein the nsP3 proteins harbors the K302A / V303A mutation. present invention relates to a method of vaccinating a patient in need of such treatment, comprising the steps of administering an attenuated alphaviruses (e.g. an attenuated chikungunya virus (CHIKV)) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) wherein the nsP3 proteins harbors the P247A / V248A mutation. It is contemplated that the vaccine composition of the invention may be combined with one or more additional components to form a more effective vaccine. Non-limiting examples of additional components include, for example, one or more additional antigens, immunomodulators or adjuvants to stimulate an immune response to the vaccine composition of the present invention and / or the additional component(s). For example, it is contemplated that immunomodulators can be included in the vaccine to augment a cell or a patient's (e.g., an animal's) response. Immunomodulators can be included as purified proteins, nucleic acids encoding immunomodulators, and / or cells that express immunomodulators in the vaccine composition. Adjuvants that are known to those skilled in the art can be used in the administration of the viruses of the invention. Adjuvants that can be used to enhance the immunogenicity of the viruses include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Although these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live vaccines. In the case of a virus delivered via a mucosal route (for example, orally) mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants. The vaccine composition of the present invention may also be mixed with one or more additional components (e.g., excipients, salts, etc.) that are pharmaceutically acceptable and compatible with at least one active ingredient (e.g., antigen). Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations thereof. The vaccine composition of the present invention may be formulated into a neutral or salt form. A pharmaceutically acceptable salt, includes the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl group also may be derived from an inorganic base such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2 ethylamino ethanol, histidine, procaine, and combinations thereof. In addition, if desired, the vaccine composition may comprise minor amounts of one or more auxiliary substances such as for example wetting or emulsifying agents, pH buffering agents, etc. that enhance the effectiveness of the vaccine composition or vaccine. The attenuated alphaviruses (e.g. an attenuated CHIKV) of the present invention can be administered as primary prophylactic agents in patients at risk of infection, or can be used as secondary agents for treating infected patients. Examples of patients who can be treated using the CHIKV-related vaccines and methods of the invention include (i) patients in areas in which CHIKV is endemic, such as Asia, and Africa, (ii) foreign travelers, or (iii) military personnel,. Moreover, inhabitants of regions where the disease has been observed to be expanding (e.g., Southern Europe), or regions where it may be observed to expand in the future (e.g., regions infested with Aedes aegypti or Aedes albopictus) can be treated according to the invention. The vaccine compositions of the present invention can be administered using methods that are well known in the art, and appropriate amounts of the vaccines administered can readily be determined by those of skill in the art. For example, the viruses of the invention can be formulated as sterile aqueous solutions containing between 102 and 107 infectious units (e.g., plaque-forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be administered by, for example, intramuscular, subcutaneous, or intradermal routes. Further, the vaccine compositions of the present invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art. The manner of administration of an vaccine compositions of the present invention may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. For example, a vaccine may be conventionally administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, or topically. The vaccination schedule and dosages may be varied on a patient-by-patient basis, taking into account, for example, factors such as the weight and age of the patient, the type of disease being treated, the severity of the disease condition, previous or concurrent therapeutic interventions, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art. For instance a suitable dosage range may be, for example, of the order of several hundred micrograms active ingredient per vaccination. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per vaccination, and any range derivable therein. In non- limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. A suitable regime for initial administration and booster administrations (e.g., inoculations) are also variable, but are typified by an initial administration followed by subsequent inoculation(s) or other administration(s). The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos.3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed—and assays of protection from challenge with the CHIKV can be performed following immunization. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1. Sequence alignment of the AUD from different alphaviruses. ESPript3 sequence alignment representation of CHIKV (UniProt: Q8JUX6), O'nyong-nyong virus (ONNV) (UniProt: P13886), Ross river virus (RRV) (UniProt: P13887), Mayaro virus (MAYV) (UniProt: Q8QZ73), Eastern equine encephalitis virus (EEEV) (UniProt: Q4QXJ8), Venezuelan equine encephalitis virus (VEEV) (UniProt: P36327), Barmah forest virus (BFV) (UniProt: P87515), Sindbis virus (SINDO) (UniProt: P03317), Aura virus (AURAV) (UniProt: Q86924) and Sleeping disease virus (SDV) (UniProt: Q8JJX1). The secondary structures of the AUD are indicated in the top and residue numbering corresponds to CHIKV. At the bottom the consensus sequence is displayed, the residues maintaining hydrogen bonds in the lateral and axial interfaces are indicated by yellow and red triangles, respectively, and cysteine residues coordinated with the Zn atom are labelled by stars. Those residues maintaining contacts in the lateral and axial interfaces are indicated by yellow and red bars, respectively. The contacts were calculated by the PISA server and alignment done with TCoffee (EBI). The mutated residues in this study with drastic effects on replication and alpha-granule formation are indicated by black dots. The regions forming the O-modules and O-loop are indicated by thick bars at the bottom of the alignment. Figure 2. nsP3 helical scaffolds are required for CHIKV infection. a-c, Characterization of nsP3 AUD mutants. U2OS cells were transfected with plasmids encoding FLAG-tagged wild- type CHIKV nsP3 or mutants harboring substitutions of amino acids involved in AUD-AUD contacts (Y200A, P247A / V248A, and K302A / V303A). a, At 48 h after transfection, cells were fixed and nsP3 sub-cellular localization was assessed by immunofluorescence using an anti- FLAG mAb. Images were acquired by confocal microscopy and are representative of 2 independent experiments. White arrows indicate the presence of nsP3 aggregates. Scale bars, 20 μm. b. The number of nsP3 aggregates per cell was assessed using ImageJ. Each symbol represents one cell. (n= 20 cells for each condition). One-way ANOVA with Dunnett’s multiple comparison test (****P<0.0001). c, Negative staining micrographs of nsP3 in vitro assembly experiments after incubation at 0.1M NaCl for 1h showing the assembly of HSs WT, absence of HSs formation for Y200A mutant, and in comparison with the wt, reduced formation of HSs for the P247A / V248A mutant and formation of long collapsed HSs for the K302A / V303A mutant. White scale bars are 100 nm long. d-e, HEK293T (d) or U2OS (e) cells were transfected with the indicated in vitro-transcribed and capped CHIKV RNA. Viral infectious particles in the supernatents of infected cells was quantified by plaque assays 48h after RNA transfection. f, HEK293T were transfected with the indicated in vitro-transcribed and capped SINV RNA. The amount of viral infectious particles was quantified by plaque assays 48h after RNA transfection in the supernatents of infected cells. Data in d-f are mean ± s.e.m. n = 3 independent experiments performed in duplicate. One-way ANOVA with a Dunnett’s multiple comparison test (***P=0.0003) g, 293T were transfected with CHIKV-D-Luc-SGR wild typoe (WT) and the indicated mutant RNA and harversted for both RLuc and Fluc assays at the indicated times. GAA : inactive mutant of nsP4 polymerase) Data are mean ± s.e.m. n = 3 independent experiments performed in quadruplicate. Two-way ANOVA with a Dunnett’s multiple comparison test (****P<0.0001). EXAMPLE: Methods: Recombinant expression and purification of CHIKV nsP3 protein. Nsp3 was expressed in Hi5TM cells (Thermofisher) using BactoBac system (Thermofisher). The coding sequence of nsp3 (corresponding to residues 1334-1863 of the non-structural polyprotein sequence from the S27 African prototype (Uniprot ID Q8JUX6) was synthesized by Gen9 as a codon optimized gene and subcloned into a pFastBac vector (Thermofisher) with a C-terminal hexahistidine tag. Site directed mutagenesis was performed on the pFB-nsP3 construct by Q5 Site-Directed Mutagenesis Kit Quick Protocol (NEB) and the whole nsP3 sequence was verified by sanger sequencing. Recombinant bacmids and viruses were produced in YFP-DH10 Bac cells and Sf21 cells, respectively according to the manufacturer’s protocols (Thermofisher). For protein expression, Hi5 cells were grown in suspension at a density of 0.5- 1.0x 106 ml-1 and then infected with baculoviruses at 2% volume of the culture. Cells were harvested at day 2-3 post cell arrest. Cells were resuspended in buffer A (20mM Tris pH 8, 500mM NaCl, 2mM TCEP, 5% glycerol, 20mM imidazole) supplemented with 2mM PMSF, 10 ^ ^g / ml DNAse and 5 mM MgSO4, and lysed by sonication. Lysates were centrifuged at 18,000g for 1h to remove unbroken cells and debris. The clarified lysate was incubated with Ni-NTA resin (GE Healthcare) in batch at 4oC for 30 minutes. Resin was washed with 10 column volumes of lysis buffer and the protein eluted over 1.5 column volumes with buffer A containing 500mM imidazole. Elution fractions containing the protein were pooled and concentrated (Amicon 30k MW C / O) for size exclusion chromatography. Samples were applied to a Supedex 200 10 / 300 increase column (GE Healthcare) pre-equilibrated with the size exclusion buffer: 20 mM HEPES pH = 8.0 ; 500 mM NaCl; 5 % glycerol and 2 mM TCEP. Grid preparation for cryo-EM imaging. Quantifoil grids R2 / 1 were treated with plasma glow discharge (Quorum) two times for 25 seconds at 15mA with a 2 min pause between the pulses. 3.5µl of Purified nsP3 protein at 1mg / ml in freezing buffer (100mM NaCl, 20mM HEPES pH7.5 and 2mM TCEP) were applied on the prepared grids and left to incubate for 1 min for binding at RT. The grid was transferred in Leica GP plunge freezer and then blotted for 10s from the back side. Grids were plunged into liquid ethane and then transferred and stored in liquid nitrogen until the imaging session. For data collection on nsP3 grids we used the Titan Krios microscope in ESRF CM01 beamline 26 at Grenoble, France. Vitrified nsP3 grids were loaded into the microscope equipped with a Gatan K3 direct electron detector and a Gatan Bioquantum LS / 967 energy filter. Cryo-EM data were acquired with EPU software (FEI) at a nominal magnification of ×81,000, with a pixel size of 1.06 Å and a total dose of 42 e / Å2 (see Table S1).13,046 image frames were acquired in super-resolution mode with a flux of 18.1 electrons per square-pixel, fractionated into 40 frames with a defocus range from −0.6 μm to −2.6 μm. Using the CM01 real-time processing setup, within the SCIPION wrapper 27 , the imported movies were drift-corrected using MotionCor2 and CTF parameters were estimated using Gctf for real-time evaluation. Further data processing was conducted using the cryoSPARC suite 28 and Relion4. Cryo-EM Image processing: 13,046 motion-corrected and dose-weighted micrographs were imported into cryoSPARC for further processing and CTF estimated. After exposure curation 12809 micrographs remained. Combining manual picking plus two rounds of 2D classification template picking we obtained an initial set of templates for template-based filament tracing. Filament tracing was performed with a segment diameter of 130 Å and a distance of 32.5 Å based on measurements within 2D classes and resulted in 6,283,414 particle images. Subsequent picking inspection (NCC score > 0.180, local power 83.0-108.0, curvature, 0.0012 (1 / Å) and sinuosity <1.18) reduced the selection to 831,539 particles (5,451,875 particles excluded).807,973 particles were extracted with an extraction crop size of 408 pixels and 2D- classified into 200 classes. Helical refinement without prior estimation of helical twist and helical rise was not successful. Accordingly, helical twist and rise estimates were found by power spectrum analysis in helixplorer software (https: / / rico.ibs.fr / helixplorer / helixplorer / see supplementary information). Helical parameters were refined during 3D reconstruction and converged to a twist of -164.175 degrees and a helical rise of 2.782 Å. The map refined to a nominal 2.52 Å resolution (according to 0.143 threshold) and a B-factor of 106.0 (EMD: 17729). Symmetry expansion (Point group symmetry: C1, helical twist (degrees): -164.175, helical rise (Å): 2.782, helical symmetry order: 12 of all final particles in this map was performed. Symmetry expanded particles (9,695,676 particles) were used in local refinement with a local mask covering an 3x3 array of 9 helical domains and a protruding central domain to cover the potential macrodomain resulted in a map of a nominal 2.36 Å resolution and slightly improved side chains (EMD: 17678). 807,973 non-subtracted particles and reference maps from cryoSPARC were imported from cryosparc into Relion. A first round of unmasked refinement was performed followed by masked refinement giving rise to better defined protruding densities of the potential macrodomain (EMD: 17730). For that a cylindrical mask covering the 30% central section of the helix and the protruding densities was designed from the helix. Different sets of masked 3D classification variants were tested (varying class size, with and without alignment, with and without particle subtraction). Here a smaller mask covering only a threefold array of two neighboring AUD and the protruding density was used. The best class was derived from non-particle subtracted classification into 3 classes (supplementary map in EMD: 17730). Model building, refinement and analysis. The first model of the whole nsP3 was obtained using ColabFold 29 (see supplementary information). The predicted model AUD domain fitted perfectly in the maps with the exception of the O loop which was traced by hand using Coot. A model with five AUD including one protomer and four neighbor contacting protomers was done using Coot and real-space refined using Phenix on the 2,35 Å resolution map (PDB: 8PHZ and EMD: 17678 respectively). The HVD and MD conformations predicted by Alphafold2 where incompatible with the helix formation, in consequence for the interpretation of the MD densities at low resolution the MD was placed manually and fitted into density using ChimeraX. The biological assembly with 120 protomers forming the helix was calculated extending the helical symmetry with ChimeraX setting an axial rise of 2.782 Å and a helical twist of -164.175 ° on the helical reconstruction map at 2.53 Å resolution (PDB: 8PK7, EMD: 17729). For the analysis of interprotomer contacts we used the PISA server (https: / / www.ebi.ac.uk / pdbe / pisa / ). We applied a cutoff of 3.5 Å for assigning H bonds. Cell culture. HEK293T (ATCC, CRL-3216), Vero E6 (gift of Marc Lecuit, Pasteur Institute Paris, France), BHK-21 (ATCC, CCL-10) and U2OS cells (gift of Andres Merits, University of Tartu, Estonia) were cultured in DMEM supplemented with 10% FBS, 1% penicillin–streptomycin, and 25 mM HEPES. Fibroblast BJ-5ta cells (ATCC, CRL-4001) were cultured in DMEM supplemented with 20% Gibco Media 199, 10% FBS, 1% penicillin–streptomycin, 25 mM HEPES and 10 µg / mL Hygromycin D. All cell lines were cultured at 37 °C in 5% CO2. All cell lines were routinely tested to be free of mycoplasma contamination. Virus strains and production. CHIKV21 (strain 06-21 from Reunion Island) was previously described 30 and obtained from Philippe Despres (Institut Pasteur, Paris, France). CHIKV West Africa (strain 37997), CHIKV St Martin (H202352013 Asian), MAYV (strain TC 625) and ONNV (strain Dakar 234) were obtained from the European Virus Archive (EVA) collection. Viruses were propagated with limited passages on Vero E6 cells. CHIKV 181 / 25 molecular clone (gift from Dr Gofinnet Christine, Institute of Virology Charité, Berlin, Germany) was derived from the parental Asian strain AF155561 and attenuated by two amino acid substitutions in the E2 glycoprotein 31. Recombinant CHIKV nsP3-mCherry and CHIKV-Rluc molecular clones were derived from CHIKV isolate obtained during the Mauritius outbreak in 2006 (GenBank accession no. FJ959103) 32. To generate infectious viruses from CHIKV molecular clones, capped viral RNAs were generated from the NotI-linearized CHIKV plasmids using a mMESSAGE mMACHINE SP6 or T7 Transcription Kit (Thermo Fischer Scientific) according to manufacturer’s instructions. Resulting RNAs were purified by phenol:chloroform extraction and isopropanol precipitation, resuspended in water, aliquoted and stored at -80°C until used. Thirty ^g of purified RNAs were transfected in BHK21 with lipofectamine 3000 reagent and supernatants harvested 72 hours later were used for viral propagation on Vero E6 cells. All virus stocks were purified through a 20% sucrose cushion by ultracentrifugation at 80,000g for 2 hours at 4°C. Pellets were resuspended in HNE1X pH7.4 (Hepes 5 mM, NaCl 150 mM, EDTA 0.1 mM), aliquoted and stored at -80°C. Viral titers were determined on Vero E6 cell by plaque assay and are expressed as PFU per mL and also by flow cytometry as previously described 23. Briefly, Vero E6 cells were incubated for 1 h with 100 ^L of 10-fold serial dilutions of viral stocks. The inoculum was then replaced with 500 ^L of culture medium and the percent of E2 expressing cells was quantified by flow cytometry at 8 hpi. Virus titers were calculated using the following formula and expressed as FACS Infectious Units (FIU) per mL. [Titer (FIU / mL) = (average % of infection) x (number of cells in well) x (dilution factor) / (mL of inoculum added to cells)]. Plasmid constructions. All N-terminal FLAG-tagged full-length nsP3 constructs derived from CHIKV Sri Lanka / 2006 / SL15649 strain (Accession number: MK028838.1), the nsP3 macrodomain (MD; aa A1-T160), the alphavirus unique domain (AUD; aa Q161-Y324) and the hypervariable domain (HVD; aa R325-L523) were synthesized and cloned into pcDNA 3.1 (-) mammalian expression vector by GenScript. The WT FLAG-tagged nsP3 and FLAG-tagged AUD constructs were subcloned into a pLVX-EF1a-IRES-puromycin (Takara) using NotI and BamHI restriction enzymes. pMC-GTU CHIKV P1234 WT and mutant replicase plasmids were described elsewhere21 and kindly provided by Pr. Andres Merits ( University of tartu, Estonia). FLAG-tag ONNV-nsP3 (isolate UVRI0804, accession number: ON595759.1), FLAG-tag MAYV-nsP3 (isolate Haiti-1 / 2014, accession number: KY985361.1) and FLAG-tag EEEV-nsP3 (isolate USA / R131399b / 2019, accession number: MT782295.1) were synthesized and cloned into pcDNA 3.1 (-) mammalian expression vector by GenScript. To establish pCHIKV with an Rluc gene insertion in the nsP3 gene (pCHIKV-Rluc), two PCR fragments were amplified from pCHIKV-M 32. Part of the CHIKV nsp3 sequence was amplified using primers 5009F and Bo426, and a CHIKV sequence encoding parts of nsP3 and nsP4 was amplified using Bo427 and Bo409. Finally, the Rluc gene was amplified from phRL-CMV (Promega) using primers Bo425 and Bo428. The three fragments were fused by PCR amplification using the outer primer pairs 5009F and Bo409. The resulting fragment was cut with NheI and AgeI and fused together with an NgoMIV-NheI fragment derived from pCHIKV-M into pCHIKV-M digested with NgoMIV-AgeI. For pCHIKV-Rluc mutants Y200A, two PCR fragments encompassing the desired change were amplified from pCHIKV- Rluc using primers Bo408 and Bo1646 or Bo1645 and Bo409 (see above). The resulting fragments were fused together using the outer primers Bo408 and Bo409. For P247A / V248A and K302A / V303A mutants, the primers encompassing the mutations (Bo1646 and Bo1645) were replaced by Bo1651 and Bo1652, or Bo1654 and Bo1653 respectively. The PCR fragments containing the mutations were cut with SacII and AgeI and fused together with a BamHI - SacII derived fragment of pCHIKV-RLuc into pCHIKV-Rluc cut with BamHI and AgeI. Lentiviral production. Lentiviral plasmids containing FLAG-tagged full-length nsP3 or AUD were packaged as previously described 23. Supernatants were collected 48 h and 72 h after transfection, cleared by centrifugation (750g for 10 min), filtered using a 0.45-μm filter and purified through a 20% sucrose cushion by ultracentrifugation (80,000g for 2 h at 4 °C). Pellets were resuspended in HNE 1X pH 7.4, aliquoted and stored at −80 °C. Fibroblasts were transduced by spinoculation (750g for 2 h at 33 °C) using these lentiviruses and selected with puromycin (2 µg / mL). Antibodies. The following primary antibodies were used: anti-Flag M2 mouse monoclonal antibody (F1804, Sigma), anti-dsRNA J2 mouse monoclonal antibody (Scicons), anti-nsP1, anti-nsP2, anti-nsP3 and anti-Capsid rabbit polyclonal antibodies (in-house from Andres Merits’s laboratory), anti- CHIKV E2 mouse monoclonal antibody (3E4), anti-GAPDH mouse monoclonal antibody (SC- 47724, Santa Cruz Biotechnology), anti-FHL1 mouse monoclonal antibody (MAB5938, R&D Systems) and anti-G3BP1 mouse monoclonal antibody (H-10 sc-365338, Santa Cruz Biotechnology). The following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (A11001, Invitrogen), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (A31573, Invitrogen), Alexa Fluor 647-conjugated donkey anti-mouse IgG (A31571, Invitrogen), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (21207, Invitrogen), peroxydase-conjugated donkey anti-rabbit IgG (711-035-152, Jackson ImmunoResearch), and anti-mouse / HRP (P0260, Dako Cytomotion). Immunofluorescence. For nsP3 aggregates characterization in infectious context, human fibroblasts (1.1 x 104 cells per well) were grown on Lab-Tek CC2 chamber slides (Nunc) for 24 hours. Cells were inoculated with CHIKV21 or CHIKV nsP3-mCherry at the indicated MOIs. For immunofluorescence in the context of transient transfection, U2OS (3 x 105 cells) were grown on Lab-Tek CC2 chamber slides for 24 hours. Cells were transfected with 250 ng of DNA expression plasmid using jetOPTIMUS® DNA Transfection Reagent (PolyPlus) according to the manufacturer’s instructions. At 7 and 24 hpi, or after 48 h of transfection, cells were washed with PBS and fixed with 2% (v / v) of PFA diluted in PBS for 15 min at room temperature. Fixed cells were washed with PBS for 5 min, then incubated with 0.1 mM of glycine in PBS for 30 min at room temperature. Cells were permeabilized and blocked with 0.1% Triton X-100 and 3% (w / v) BSA in PBS for 45 min. Cells were incubated for 1 h at room temperature with primary antibodies, and washed with PBS. Cells were labelled with corresponding Alexa Fluor- conjugated secondary antibodies (1 ^g.ml-1) for 30 min at room temperature. All antibodies were diluted in PBS supplemented with 3% (w / v) BSA. Slides were mounted with ProLong Gold antifade reagent containing DAPI for nuclear staining (Thermo Fisher Scientific). Fluorescence microscopy images were acquired using a LSM 800 confocal microscope with the following objectives: Plan-Apochromat 63x / 1.40 Oil DIC M27 and Plan-Apochromat 40x / 1.3 Oil DIC (UV) VIS-IR M27 (Zeiss). RNA-Fluorescent In Situ Hybridization. A pool of 48 Custom StellarisTM RNA FISH Probes was designed against the 5’ open reading frame of CHIKV (strain LR2006-OPY1, GenBabk: DQ443544.2) by utilizing the Stellaris RNA FISH Probe Designer (LGC, Biosearch Technologies, Petaluma, CA) available online at www.biosearchtech.com / stellarisdesigner. Each probe was labelled with QuasarTM 570 dye (LGC, Biosearch Technologies, Petaluma, CA) and hybridized to CHIK21-infected human fibroblasts, according to the manufacturer’s instructions available online at www.biosearchtech.com / stellarisprotocols. Briefly, following viral protein immunostaining (see section ‘Immunofluorescence’), cells were washed three times with PBS and fixed with 2% (v / v) of PFA diluted in PBS for 10 min at room temperature. Cells were washed twice in PBS and incubated with Stellaris Wash Buffer A at room temperature for 10 min. Cells were incubated with RNA probes diluted at 0.125 nM final concentration in hybridization buffer supplemented with 0.5 mM vanidyl ribonucleoside complex, for 16 h in humidified chamber at 37°C. Cells were first washed in Stellaris Wash buffer A for 30 min at 37°C, and then with Stellaris Wash buffer B for 10 min. Slides were mounted with ProLong Gold antifade reagent containing DAPI for nuclear staining (Thermo Fisher Scientific). Of note, all buffers and reagents used were RNase-free. Fluorescence microscopy images were acquired using a LSM 800 confocal microscope (Zeiss). Genomic viral RNA transfection and viral infection analysis. To assess wildtype and mutant CHIKV RNA replication, we transfected HEK293T cells with capped genomic viral RNA generated from pCHIKV-Rluc molecular clones (see ‘Virus strains and production). Cells were plated on 48 well plates (5 x 104 cells) and transfected with 50 ng of purified RNA using the Lipofectamine MessengerMax reagent according to the manufacturer’s instructions (Thermo Fisher Science). At 3 h after transfection, medium was removed and fresh medium was added. At specific time intervals, cell supernatant was collected and frozen at -80°C for plaque assay on VeroE6 cells, whilst cells were washed once with PBS and lysed with Passive lysis buffer. Lysates were kept at −20 °C until all samples were collected. Luciferase activity was measured using the Renilla Luciferase Reporter Assay system (Promega) on a TriStar2 LB 942 with 20 ^L of cell lysate, 20 ^L of substrate and 2 s integration time. Each experiment was performed in quadruplicate. Immunoblot. U2OS (3 x 106 cells) were grown on 6-well plates for 24 hours. Cells were transfected with 2.5 ^g of DNA expression plasmid using jetOPTIMUS® DNA Transfection Reagent (PolyPlus) according to the manufacturer’s instructions. At 48 h after transfection, cells were washed with PBS, collected with cell scrapper and pelleted by centrifugation (400g for 5 min). Cell pellets were lysed in Pierce™ IP Lysis Buffer (Thermo Fisher Scientific) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fischer Scientific) for 30 min at 4°C. Equal amount of protein, determined by DC™Protein Assay (BioRad), were prepared in LDS Sample Buffer 4X (Pierce™) containing 25 mM dithiothreitol (DTT) and heated at 95°C for 5 min. Samples were separated on Bolt™ 4-12% Bis-Tris gels in Bolt® MOPS SDS Running Buffer (Thermo Scientific), and proteins were transferred onto a PVDF membrane (BioRad) using the Power Blotter system (Thermo Fischer Scientific). Membranes were blocked with PBS containing 0.1% Tween-20 and 5% non-fat dry milk and incubated overnight at 4°C with primary antibody. Staining was revealed with corresponding horseradish peroxidase (HRP)- coupled secondary antibodies and developed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) following manufacturer’s instructions. The signals were acquired through Fusion Fx camera (VILBERT Lourmat). Transmission electron microscopy. Cells were fixed for 24 h in 1% glutaraldehyde, 4% paraformaldehyde, (Sigma, St-Louis, MO) in 0.1 M phosphate buffer (pH 7.2). Samples were then washed in phosphate-buffered saline (PBS) and post-fixed for 1 h by incubation with 2% osmium tetroxide (Agar Scientific, Stansted, UK). Cells were then fully dehydrated in a graded series of ethanol solutions and propylene oxide. Impregnation step was performed with a mixture of (1∶1) propylene oxide / Epon resin (Sigma) and then left overnight in pure resin. Samples were then embedded in Epon resin (Sigma), which was allowed to polymerize for 48 hours at 60°C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Sections were stained with 2% uranyl acetate (Agar Scientific), 5% lead citrate (Sigma) and observations were made with a transmission electron microscope (JEOL 1011, Tokyo, Japan). Immunogold labelling of cryosections. Cells were fixed for 2 hours with 4% paraformaldehyde in phosphate buffer (pH 7.6), washed with PBS (pH 7.6) for 2 x 5 min and centrifuged at 300g for 10 min. After removing the supernatant, cell pellets were included in gelatin 12% and infused with sucrose 2.3 M overnight at 4°C. 80 nm ultra-thin cryosections were made a −120°C on a LEICA FC7 cryo- ultramicrotome. Ultra-thin sections were retrieved with Methylcellulose 2% / Sucrose 2.3 M mixture (1:1) and collected onto formvar / carbon coated nickel grids. After removal of gelatine at 37°C, sections were incubated on drops of either 1:100 anti-nsP3 rabbit serum, anti-Capsid rabbit serum, anti-FHL1 monoclonal or anti-G3BP1 monoclonal antibody diluted in PBS. After six washes of five minutes each, grids were incubated on drops of PBS containing 1:30 gold- conjugated goat anti-rabbit or anti-mouse Ab (6 nm) (Aurion, Wageningen, Netherlands). Grids were finally washed with six drops of PBS (five minutes each), post-fixed in 1% glutaraldehyde and rinsed with three drops of distilled water. Contrasting step was performed by incubating grids on drops of uranyl acetate 2% / methylcellulose 2% mixture (1:10). The sections were imaged in a transmission electron microscope at 100 kV (JEOL 1011). Image processing and analysis. All confocal microscopy images were processed and analyzed with ImageJ (version: 2.9.0 / 1.53t). For aggregate quantification, cell boundaries were traced to delimitate each signal- positive cell. Aggregates’ channel was extracted and automatic signal thresholding was applied to locate objects of interest. Images were binarized and Image J “Analyze particles” macro was used to quantify aggregates based on size (0.05-5 µm) and circularity (0.01-1) discrimination. For colocalization analysis, images were processed and Pearson’s coefficient was calculated using BIOP JACoP plugin as described in https: / / imagej.net / plugins / jacop). Statistical analysis. Graphical representation and statistical analyses were performed using Prism 9 software (GraphPad software). Unless otherwise stated, results are shown as mean ± s.e.m. from at least two independent experiments in duplicates. Differences were tested for statistical significance one-way or two-way ANOVA with multiple comparison post hoc test. Results: Atomic structure of alphavirus nsP3 helical scaffolds (HSs). We expressed the CHIKV nsP3 in Hi-5 insect cells (see methods). Purification of nsP3 by consecutive affinity and size exclusion chromatography at 0.5 M NaCl yielded both monomeric and high molecular-weight nsP3 species. Analysis by negative staining electron microscopy revealed that the latter correspond to tubular macro-complexes harboring a well-defined internal cavity delimited by a weakly structured matrix and surrounded by a jelly-like shield (data not shown). The nsP3 monomers can re-assemble in vitro by lowering low salt concentration (data not shown). We also determined the structure of the nsP3 tubes by cryo-electron microscopy (cryo-EM) at 2.52 Å resolution (data not shown). Local refinement allowed to reach 2.36 Å on which we refined the AUD structure from an Alphafold2 prediction (data not shown) and reconstitute the helical assembly (PDB: 8PK7). We found that the tubes are made by a helical arrangement of the nsP3 AUD with an axial rise of 2.782 Å and a helical twist of -164.175 ° (data not shown). Lateral views clearly reveal the existence of two-start parallel helixes (hA and hB) with a right-handed arrangement of the AUD (data not shown). Each helix has 11.37 protomers per turn (data not shown). The assembly generates interstitial positively charged pores connecting the inner cavity with the exterior, which could accommodate small negatively charged molecules like RNA (data not shown). Although barely visible in the structure due to their high flexibility, the N-terminal MD and the C-terminal HVD, are clearly projected to the outside of the tubular structure (data not shown). Refinement using a larger mask yielded a lower resolution map of the helical particles (2.98 Å resolution, EMD: 17730) with discernable globular appendices that can be assigned to the MD by main-chain connectivity (data not shown). The MD of each nsP3 protomer is lying on the next AUD (A n+1) of the same helix. A 3D focused classification provided one 3D class clearly showing the beginning of the HVD interacting with the MD of the n-1 protomer (data not shown). Because of the low resolution of the map (6.84 Å) we could not deduce the exact position of contacts between the MD and HVDs, which are connected to the AUD through long flexible linkers. Beside these limitations, the structure clearly shows that MD-MD and HVD-MD interactions build a flexible and structured shield on the exterior of the tube which contributes to the interprotomer interactions. The nsP3 AUD is an oligomerization module activated by the nsP2-3 polyprotein cleavage. The AUD core is a hallmark of alphavirus nsP3 proteins and on the light of the helical structure its folding acquires a new sense (Figure 1). It consists of four alpha helixes ( ^1- ^4) with extensions at the N and C terminus acting as oligomerization modules (C and N O-modules) which are responsible for lateral and axial contacts with the AUD of neighboring protomers, respectively. The lateral and axial interfaces bury 320 Å2 and 530 Å2 of surface area, respectively (data not shown). The O-modules are rich in positively charged residues and hold the core of neighboring complexes both contacting with two regions including residues Y200 to K222 and R243 to P257 (data not shown). The R243 to P257 region contains a loop flanked by C246 and P257 residues (O-loop) rich in acidic residues which adopts a slightly different conformation as compared to the predicted Alphafold216 structure, consistent with previous structures reported on Sindbis virus (SINV)14 (data not shown). The O-loop mediates extensive interactions with both N- and C O-modules from hA n-1 and hB n-protomers, respectively. Even if no stable salt bridges are observed in the structure, the charge distribution is suggestive of an interaction of electrostatic nature (data not shown) which is in line with findings that high salt concentrations promote filament disassembly in vitro (data not shown). In addition, a network of hydrogen bonds woven by residues A251, S254, V211, Y200 with residues H207, Y266, K304, Q301 and K302 of hB n-protomer seals the axial contacts. Likewise, residues M219, K222, D249 and C246 coordinate h-bonds with R187, A185, G203 and R205, stabilizing the lateral interface (data not shown). The cleavage of the P1234 precursor at the boundary between nsP2 and nsP3 is important for viral infection and pathogenesis 5,14. Interestingly, the superposition of AUD domains of HSs and uncleaved nsP2- nsP3 protein reveals that in the uncleaved polyprotein the O-loop is completely buried by the nsP2 protease domain preventing HSs formation (data not shown). Therefore, nsP2 protease- mediated cleavage of the nsP2-3 junction might release the O-loop and trigger the nsP3 AUD scaffold formation (data not shown). NsP3 helical scaffolds are part of the viral replication complex. NsP3 is a component of the vRC 5. Tomographic reconstruction of CHIKV spherule necks revealed the vRC architecture with a molecular pore delineated by a nsP1 dodecamer ring connected to a larger tubular structure (crown) in its cytoplasmic side 6,7. Superposition of nsP3 HSs into the crowns shows similar dimensions and shape 6,7 (data not shown). The tomographic reconstruction of the vRC reported by Laurent et al.7 shows perimetral volumes which correlate well in the superposition with the nsP3 MDs (data not shown). This is lost in higher resolution volumes reported by Tan et al 6 (data not shown). For both tomographic reconstructions a 12-fold symmetry (C12) was imposed to the calculated volumes which is consistent with nsP1 capping pores. However, this conflicts with the helical symmetry that we find now for nsP3 scaffolds (with 11.37 nsP3 protomers per turn). Interestingly, mismatched symmetries of ring-like complexes as the observed in icosahedral viruses vortex and bacterial flagellar rotors are associated to dynamic processes essential for virus assembly and DNA packing or bacterial motion, respectively17,18. One can speculate that symmetry mismatches between the nsP1 capping pores and nsP3 scaffolds might underlie motions occurring during transcription and replication. Helical arrangements of nsP3 AUD form tubular structures that define the architecture of alpha-granules. In infected cells, CHIKV nsP3 accumulates in large cytoplasmic alpha- granules that do not co-localize with the dsRNA and are distinct from vRCs 2,3,19,20(data not shown). During the early phases of infection, the alpha-granules appear globular and spherical (data not shown) while assembling into irregular internal structures up to 0.8 µm long at later stages. (data not shown). Alpha-granule formation is a hallmark of alphavirus biology (data not shown). Of note, alpha-granules length and morphology varies depending on the CHIKV strain or lineage (data not shown). An average of 40 alpha-granules are formed per cell after 24 h post-infection. Of note, the expression of nsP3 alone, independently of the presence of other viral components, is sufficient to induce alpha-granule formation. (data not shown). Alpha-granule formation depends on nsP2-nsP3 processing. Indeed, expression of the P1234 precursor harboring mutations affecting the nsP2-nsP3 cleavage21,22 prevents assembly of alpha-granules (data not shown). Transmission electron microscopy studies performed on either CHIKV-infected primary fibroblasts or cells stably expressing nsP3 revealed that the alpha-granules are made by a honeycomb arrangement of tubular structures consistent in size and shape with nsP3 HSs (data not shown). Immunogold labeling of nsP3 confirmed the identity of these scaffolds (data not shown). NsP3 tubular structures are found in cells either infected by several alphaviruses such as Mayaro virus (MAYV) or O’Nyong Nyong virus (ONNV) or in cells transfected with plasmids encoding Eastern Equine Encephalitis virus (EEEV), MAYV or ONNV nsP3 (data not shown). Confocal and electron microscopy studies show that the nsP3 tubes contain G3BPs 3 and FHL123 , two host factors that bind the nsP3 HVD (data not shown). Considering the density of nsP3 scaffolds in the granules (400 helices / µm2) and the number of nsP3 protomers per µm in the HSs (3870 nsP3 / µm), we estimate that 1.55 million nsP3 protomers would be present in 1 µm3 of alpha-granules. This indicates that nsP3 tubular structures are formidable molecular traps sequestering host factors important for infection. Importantly, in infected cells, most of the nsP3 alpha-granules colocalize with both viral capsid (data not shown) and the viral genomic RNA (data not shown) but contain neither E2 nor nsP1 proteins (data not shown), suggesting that they are the site of preassembly of the viral nucleocapsids. This idea is in line with a study describing the imaging of alphavirus capsid protein during virus replication in living cells 24. Since the tubular structures of in vitro-purified or in vitro-assembled nsP3 (data not shown) do not show any honeycomb arrangement, one can speculate that host or viral factors besides nsP3 could mediate this organization through interactions with the exposed MD or HVD. This unique nsP3 mediated internal architecture, combining solid ordered scaffolds with intrinsically disordered regions, challenges our current understanding of the architecture of biomolecular condensates25. NsP3 helical structures are important for both CHIKV RNA replication and infectious virion production. The nsP3 AUD has an intrinsic oligomerization capacity and forms filaments when expressed in human cells (data not shown). Consistently, nsP3 molecules lacking the AUD domain do not form alpha-granules and are diffusively distributed in the cell cytoplasm when compared to WT or nsP3 deleted from the MD or HVD. We generated a panel of structure-based mutations aiming to disturb nsP3 oligomerization in the context of the AUD alone (data not shown) or the full nsP3 (data not shown). We expressed the nsP3 variants by transfection in U2OS cells and investigated their ability to form filaments and alpha- granules (data not shown). All the nsP3 mutants were similarly expressed but presented different phenotypes (data not shown). Mutants R187A / R205A, A252P, P255A, R187A, S254A / S255A, H207A, Q301A, A250W, D249G / A250G / D251G showed AUD filaments and nsP3 alpha- granule formation like their WT counterpart (data not shown). Interestingly, the AUD or nsP3 proteins harboring K302A / V303A or P247A / V248A mutations are homogeneously distributed through the cytoplasmic with a strong reduction of alpha-granule formation (Figure 2a-b). Finally, the Y200A mutant failed to form alpha-granules and presented a homogeneous distribution in the cell cytoplasm (data not shown). We next purified the recombinant K302A / V303A, P247A / V248A and Y200A nsP3 mutants as monomers and tested their in vitro oligomerization capacity by negative staining electron microscopy in comparison with WT nsP3. At the same protein concentration (0.2 mg / ml), K302A / V303A and P247A / V248A nsP3 molecules showed altered oligomerization properties with the formation of long aggregates and a weak formation of scaffolds, respectively. In agreement with the observations in cells, the nsP3 Y200A mutant was unable to form tubes (Figure 2c). To investigate the role of nsP3 HSs during viral infection, we introduced the most disruptive AUD mutations (K302A / V303A, P247A / V248A and Y200A) in a full length CHIKV Renilla Luc (Rluc) molecular clone 23. We transfected in vitro transcribed viral RNA into 293T or U2OS cells (Figure 2d) and monitored viral replication by measuring the Rluc accumulation in cell lysates as a function of time. The Y200A mutation, which prevents the formation of nsP3 helices, abolished viral replication in both 293T and U2OS cells, recapitulating the phenotype of the replication-deficient CHIKV containing a GDD to GAA mutation in the viral RNA-dependent-RNA polymerase nsP4 (Figure2d). Viruses expressing the K302A / V303A or P247A / V248A mutations showed a slight but significant reduction in viral replication in 293T but not in U2OS (Figure 2d). Importantly, both mutant viruses also formed smaller plaques than their WT counterparts, accompanied by a drastic reduction in viral titer in plaque assays, indicating a strong impairment of infectious virus particle release (Figure 2e,f). Finally, we compared the subcellular localization of nsP3 and capsid in U2OS transfected with WT, K302A / V303A or P247A / V248A genomic RNA. Interestingly, both K302A / V303A and P247A / V248A mutated viruses displayed both an altered intracellular distribution of the structural capsid protein and a strong reduction in nsP3 / capsid foci formation when compared to the WT virus (Figure 2g). Overall, these data show that partial perturbations of the nsP3 scaffold assembly affects alpha- granule formation, with drastic consequences for viral particle production. Inhibiting scaffold assembly (Y200A mutation) causes a total lack of viral replication. Together, these data suggest that nsP3 tubular structures appear to link viral RNA synthesis and viral particle production. Conclusion: In this study, we show that CHIKV nsP3 auto-assembles into HSs important for viral infection. To our knowledge, nsP3 tubular structures represent a new form of viral biomolecular condensates that contribute to the spatiotemporal organization of alphavirus replication cycle. We propose that, during the early steps of infection, nsP3 may induce membrane invaginations during the spherule formation by recruiting host factors towards the vRC. As infection progresses, nsP3 accumulates into alpha-granules organized by a highly-ordered networks of helical tubular scaffolds that contain host factors, genomic RNA and viral capsid proteins (data not shown). It is tempting to speculate that the nsP3 tubular structures may act as a platform that guide the assembly of RNA with capsids from vRCs to alpha-granules, thus connecting viral replication to virion assembly (data not shown). Their structural feature suggests that nsP3 tubes may sort the naked viral RNA through the interstitial positively charged pores. Further studies such as cryo-electron tomography are required to determine the three- dimensional architecture of nsP3 tubes in infected cells and decipher how these structures orchestrate viral particle production. The inner architecture of alpha-granules also challenges our current understanding of the structural organization of non-membranous organelles by combining both intrinsically disordered regions and highly stable scaffolds. Further in situ structural studies of other biomolecular condensates will reveal if this is a general trend or a specific feature of alpha-granules. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Zaid, A. et al. Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses. Lancet Infect Dis 21, e123–e133 (2021). 2. Götte, B., Liu, L. & McInerney, G. M. The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last. Viruses 10, 1–26 (2018). 3. Fros, J. J. et al. Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci. Journal of Virology 86, 10873–10879 (2012). 4. Kril, V., Aïqui-Reboul-Paviet, O., Briant, L. & Amara, A. New Insights into Chikungunya Virus Infection and Pathogenesis. Annu Rev Virol 8, 327–347 (2021). 5. Ahola, T., McInerney, G. & Merits, A. Alphavirus RNA replication in vertebrate cells. Adv Virus Res 111, 111–156 (2021). 6. Tan, Y. B. et al. Molecular architecture of the Chikungunya virus replication complex. Sci Adv 8, eadd2536 (2022). 7. Laurent, T. et al. Architecture of the chikungunya virus replication organelle. Elife 11, e83042 (2022). 8. Jones, R., Bragagnolo, G., Arranz, R. & Reguera, J. Capping pores of alphavirus nsP1 gate membranous viral replication factories. Nature 589, 615–619 (2021). 9. Webb, L. G. et al. Chikungunya virus antagonizes cGAS-STING mediated type-I interferon responses by degrading cGAS. PLoS Pathog 16, e1008999 (2020). 10. Jose, J., Snyder, J. E. & Kuhn, R. J. A structural and functional perspective of alphavirus replication and assembly. Future Microbiol 4, 837–856 (2009). 11. Akhrymuk, I., Kulemzin, S. V. & Frolova, E. I. Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II. J Virol 86, 7180–7191 (2012). 12. Gao, Y., Goonawardane, N., Ward, J., Tuplin, A. & Harris, M. Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription. PLoS Pathog 15, e1007239 (2019). 13. Meshram, C. D. et al. Multiple Host Factors Interact with Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode. J. Virol. (2018) doi:10.1128 / JVI.00838-18. 14. Shin, G. et al. Structural and functional insights into alphavirus polyprotein processing and pathogenesis. Proc Natl Acad Sci U S A 109, 16534–16539 (2012). 15. 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Claims

CLAIMS:

1. A helical tubular scaffold formed by self-assembly of a plurality of polypeptides having the consensus amino acid sequence: EXVRX5HPXSXLXGRXGX17STXXGXXYSYXEGTXFHQXAXDMAEIXXLWPXXXEANE QICXYXLGX64XMEXXRXKCPVX76DXXXSXPPXTXPCLCXYAMTPERXXRLXXXXXXX XXVCSSFXLPKYXIXXVQKX130XCXXXX (SEQ ID NO:1) wherein: - X represents any amino acid residue - X5represents V or I - X17 represents Y or F - X64represents E or Q - X76represents D or E - X130 represents V or I.

2. The helical tubular scaffold of claim 1 wherein: - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:12, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:13, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:14, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:15, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:16, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:17, or- the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:18, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:19, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:20, or - the polypeptides comprise an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:

21.

3. The helical tubular scaffold of claim 1 or 2 wherein - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:2, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:3, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:4, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:5, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:6, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:7, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:8, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:9, or- the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:10, or - the polypeptides consist of an amino acid sequence having at least 90% of identity with the amino as set forth in SEQ ID NO:

11.

4. The helical tubular scaffold according to any one of claims 1 to 3 wherein the polypeptides are labelled with a detectable molecule.

5. A method for identifying a substance useful for inhibiting the replication capacity of an alpha virus comprising the steps of (a) determining whether a test substance is capable of perturbate the assembly of the tubular helical scaffold according to any one of claims 1 to 4 and (b) selecting the substance that perturbates the assembly of the helical tubular scaffold.

6. The screening method of claim 5 that comprises the steps that consist in i) contacting the test substance with a plurality of the polypeptides of the present invention in conditions that allow the assembly of the tubular helical scaffold and ii) determining whether the test substance is capable of inhibiting the assembly of the tubular helical scaffolds and iii) selecting the test substances that is capable of inhibiting the assembly of the tubular helical scaffolds.

7. The screening method of claim 5 that comprises the steps that consist in i) contacting the test substance with one or more tubular helical scaffold(s) of the present invention in conditions that allow the maintenance of the assembly of the tubular helical scaffold and ii) determining whether the test substance is capable of disassembling the tubular helical scaffolds and iii) selecting the test substances that is capable of disassembling the tubular helical scaffolds.

8. The screening method according to any one of claims 5 to 7 that further comprises the step (d) consisting in determining whether the substance selected at step (c) inhibits the replication of an alphavirus in a host cell and a step (e) that consists in positively selecting the test substance capable of inhibiting the replication of said alphavirus in said host cell.

9. The method of claim 8 that comprises the steps consisting of i) infecting said host cell with said alphavirus and ii) culturing said infected cell in presence of the test substance,iii) comparing the replicating capacity of the virus with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the virus.

10. A nanocarrier comprising a tubular helical scaffold according to any one of claims 1 to 4 and a payload 11. The nanocarrier of claim 10 wherein the payload is a negatively charged molecule 12. The nanocarrier of claim 11 wherein the payload consists of a polynucleotide 13. The nanocarrier of claim 12 wherein the polynucleotide is a messenger RNA (mRNA).

14. A pharmaceutical composition comprising an amount of the nanocarrier according to any one of claims 10 to 13.

15. The use of the nanocarrier according to any one of claims 10 to 13 for vaccinating purposes.

16. An attenuated alphaviruses that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that harbors one of the following mutation Y200A, K302A / V303A or P247A / V248A.

17. The attenuated alphaviruses of claim 16 wherein the alphavirus is chikungunya virus (CHIKV).

18. A method of vaccinating a patient in need of such treatment, comprising the steps of administering an attenuated alphaviruses that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that harbors one of the following mutation Y200A, K302A / V303A or P247A / V248A.

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