CHMP2b mutants for use for treating infection

Abstract

Here, the inventors report the biochemical and functional characterization of a new PTM on a key ESCRT-III member and its impact during viral budding. They discovered that CHMP2B K6 plays a pivotal role in both abscission and HIV viral budding, highlighting the importance of the novel methylation event. Strikingly, expression of unmethylatable CHMP2B mutants (CHMP2B K6A, CHMP2B K6R) perturbed the localization of ESCRT-III at the ICB with delayed recruitment and distribution of CHMP2B to the cleavage site, ultimately leading to impeded abscission. They reported that expression of CHMP2B mutant bearing a specific lysine point mutation at position 6 drastically reduces the number of viral particles released into the supernatant. Additionally, this mutation impairs the infectivity of the produced virions. This is the first description of the antiviral effect of a point mutant of ESCRT-III proteins. Thus, the present invention relates to an isolated polypeptide derived from unmethylatable mutant CHMP2B and its uses for treating infection.

Description

[0001] CHMP2B MUTANTS FOR USE FOR TREATING INFECTION

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular infection.

[0004] BACKGROUND OF THE INVENTION:

[0005] The ESCRT (Endosomal Sorting Complex Required for Transport) machinery is evolutionarily conserved and orchestrates diverse cellular processes that require membrane remodeling and membrane fission1 3. Four distinct ESCRT complexes (ESCRT-0, 1, II, and III) play crucial roles in a range of processes1, such as formation of intraluminal vesicles, reformation of the nuclear envelope, plasma membrane, organelle repair, autophagy, virus budding and release of viral particles. Notably, ESCRT -III proteins are required for the abscission process during cytokinesis in mammals, as well as in Archaea, supporting an ancestral role of the ESCRT pathway in cell division4. Cytokinesis is the final stage of cell division and consists of a rapid step of furrow ingression followed by the abscission process, which lasts for several hours in mammalian cells, until the ESCRT machinery splits the membrane apart5 7. Abscission leads to the physical separation of the two daughter cells through the scission of the intercellular bridge (ICB) which serves as a platform to assemble the abscission machinery8,9. Reconstitution assays in vitro showed that yeast S. cerevisiae Snf7 (CHMP4 ortholog) recruits Vps2-Vps24 (CHMP2-CHMP3 orthologs) followed by Did2 and Istl (CHMP1 and Istl orthologs, respectively)10,11. The AAA+ ATPase Vps4 promotes the turnover of ESCRT -III filaments and subunit exchange to catalyze membrane fission12,13. At the early stage of abscission in human cells, the ESCRT -III members (including CHMP2B) are recruited to form two ring structures surrounding the midbody, at the center of the ICB12,14Subsequently, ESCRT -III proteins polymerize to form hetero-polymer, helical spiral structures with progressively smaller diameters. This leads to membrane constriction at the abscission site, located approximately 1 pm from the midbody, thereby promoting abscission11,15. The precise mechanisms controlling the spatio-temporal dynamics of ESCRT -III components and the timing of abscission are not fully understood. Specifically, the mechanisms regulating the polymerization of ESCRT -III proteins from the initial assembly site at the midbody (rings) to the abscission site remain unclear. Protein interactions and post-translational modifications (PTMs) may facilitate ESCRT -Ill-mediated scission across various spatial and temporal scales. For example, phosphorylation of CHMP4C by Aurora B kinase delays abscission in response to abscission checkpoint activation16,17, and ubiquitination of ESCRT -III is critical to complete abscission in Drosophila^ . The roles of other PTMs, such as methylation, in ESCRT regulation has not been investigated.

[0006] ESCRT -III plays a pivotal role in virus budding, a critical step in the life cycle of many enveloped viruses. During budding, the virus hijacks the host cell's ESCRT machinery to facilitate the release of newly formed viral particles from the cell surface. ESCRT -III components, including CHMP2A / B, CHMP3, CHMP5 and CHMP4 A / B / C, are recruited to the budding site by viral proteins. These ESCRT -III proteins assemble into filamentous structures that drive membrane curvature and neck formation. As the ESCRT -III machinery constricts, it pinches off the budding neck, liberating the newly formed virus particle from the host cell. The proteins of the ESCRT -III complex are cellular factors that promote the replication of many enveloped viruses including retrovirus, filovirus, Flavirirus, Coronavirus, and Togavirus19. CHMP2 and CHMP4 protein families are required and act in concert during the final step of HIV-1 particle budding20. In this case, silencing of CHMP proteins20or overexpression of CHMP truncation mutants21block viral budding at a late stage. As a result, fewer viral particles are released into the extracellular environment, and these particles exhibit a defect in precursor Gag maturation and, consequently, a loss of infectivity.

[0007] The process of virus budding orchestrated by ESCRT-III is essential for viral dissemination and propagation, making it an attractive target for developing antiviral strategies to inhibit viral release and combat infectious diseases.

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention relates to an isolated polypeptide derived from a Charged multivesicular body protein 2b, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 3 to the amino acid residue at position 10 in SEQ ID NO:1 (CHMP2B) and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue. The present invention also relates to a method for treating viral infection in a subject in need thereof comprising administering to the patient a therapeutically effective amount of the polypeptide of the invention.

[0010] In particular, the present invention is defined by the claims.

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Here, the inventors report the biochemical and functional characterization of a new PTM on a key ESCRT-III member and its impact during viral budding. They discovered that CHMP2B K6 plays a pivotal role in both abscission and HIV viral budding, highlighting the importance of the novel methylation event. Strikingly, expression of un-methylatable CHMP2B mutants (CHMP2B K6A, CHMP2B K6R) perturbed the localization of ESCRT-III at the ICB with delayed recruitment and distribution of CHMP2B to the cleavage site, ultimately leading to impeded abscission. They reported that expression of CHMP2B mutant bearing a specific lysine point mutation at position 6 drastically reduces the number of viral particles released into the supernatant (Figure 2A). Additionally, this mutation impairs the infectivity of the produced virions (Fig. 2B). This is the first description of the antiviral effect of a point mutant of ESCRT- III proteins.

[0013] Polypeptide and polynucleotide of the invention

[0014] Accordingly, the first object of the present invention relates to an isolated polypeptide derived from a Charged multivesicular body protein 2b, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 3 to the amino acid residue at position 10 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B mutant”).

[0015] As used herein the term “Charged multivesicular body protein 2b” or “CHMP2B”, also known as “ALS17”, “DMT1”, “FTDALS7”, has its general meaning in the art and refers to the protein encoded by the CHMP2B gene which forms part of the endosomal sorting complexes required for transport (ESCRT-III). ESCRT-III plays a critical role in cytokinesis, facilitating the abscission of the two daughter cells during cell division. Moreover, this protein complex is involved in viral budding, specifically in enveloped virus release from host cells. CHMP2B, as well as other ESCRT-III components such as CHMP2A / B, CHMP3, CHMP5 and CHMP4 A / B / C, are recruited to the budding site by viral proteins. CHMP2B is constituted of 4 Helix alpha and a C-terminal tail. Its Entrez reference is 25978 and Its UniProt reference is Q9UQN3.

[0016] The native variant of CHMP2B is represented by SEQ ID NO: 1.

[0017] SEQ ID NO:1 (UNIPROT ref. Q9UQN3):

[0018] MASLFKKKTVDDVIKEQNRELRGTQRAI IRDRAALEKQEKQLELEIKKMAKI GNKEACKVLAKQLV HLRKQKTRTFAVSSKVTSMSTQTKVMNSQMKMAGAMSTTAKTMQAVNKKMDPQKTLQTMQNFQKENMKMEM TEEMINDTLDDI FDGSDDEEESQDIVNQVLDE IGIE I SGKMAKAPSAARSLPSASTSKAT I SDEEI ERQLK ALGVD As used herein the term “comprise” or a variant thereof (e.g., “comprises”, “comprising”) also encompasses narrower expression “substantially consist of’, further narrower expression “consist of’ and any variants thereof (e.g., “consists of’, “consisting of’), unless otherwise stated.

[0019] In some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 54 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 mutant”)

[0020] In some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 117 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 2 mutant”)

[0021] In some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 136 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 4 mutant”)

[0022] In some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 154 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 4 mutant bis”)

[0023] In some embodiment, the polypeptide of the invention comprises the sequence as set forth as SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B mutant”)

[0024] The inventors discovered that the lysine residue (K) at position 6 in SEQ ID NO: 1 can be methylated which influenced viral budding. The substitution of the lysine residue (K) by a alanine residue (A) or a arginine residue (R) allow to generate methylation and acetylationdeficient mutant. The substitution of the lysine residue (K) by a glutamic acid residue (E) lead to a reversal of the amino acid charge and introduced a shorter side chain. The substitution of the lysine residue (K) by a phenylalanine (F) lead to a suppression of the polarity and charge of the amino acid, and an increase in hydrophobicity and steric hindrance. Thus, in some embodiment, the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of an alanine residue (A), an arginine residue (R), a glutamic acid residue (E) and a phenylalanine (F).

[0025] In particular embodiment, the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an alanine residue (A) (“CHMP2B K6A”)

[0026] Two recent studies described that truncated retrocopies of ESCRT-III member CHMP3 (retroCHMP3), which have independently evolved in New World monkey and mouse species, block ESCRT-dependent virus budding with little effect on critical cellular ESCRT processes in human cells. Remarkably, these truncated retroCHMP3 proteins have acquired mutations to reduce interactions with other ESCRT-III factors and limit effect on other cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. Interestingly, the inventors found that 5 mutations acquired during retroduplication event of retroCHMP3 in titi monkeys (F5L, K59T, K81P, K90R, Q110R), are positioned on residues conserved between CHMP3 and CHMP2B.

[0027] Thus, in some embodiment, the polypeptide of the invention further comprises at 1, 2, 3, 4 or 5 mutations selected from the group consisting of F5L, K59T, K81P, K90R, QI 10R.

[0028] Thus, in some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 6 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of an alanine residue (A), an arginine residue (R), a glutamic acid residue (E) and a phenylalanine (F), and wherein the phenylalanine residue (F) at position 5 in SEQ ID NO: 1 is substituted by leucine residue (L).

[0029] Thus, in some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 54 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of an alanine residue (A), an arginine residue (R), a glutamic acid residue (E) and a phenylalanine (F) and, wherein the phenylalanine residue (F) at position 5 in SEQ ID NO: 1 is substituted by leucine residue (L).

[0030] In some embodiment, the polypeptide of the invention comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 117 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consisting of an alanine residue (A), an arginine residue (R), a glutamic acid residue (E) and a phenylalanine (F) and wherein the polypeptide further comprises 1,2, 3, 4 or 5 mutations selected from the group consisting of : i) the substitution of the phenylalanine residue (F) at position 5 in SEQ ID NO:1 by a leucine residue (L), ii) the substitution of the lysine residue (K) at position 59 in SEQ ID NO:1 by a threonine residue (T), iii) the substitution of the lysine residue (K) at position 81 in SEQ ID NO: 1 by a proline residue (P), iv) the substitution of the lysine residue (K) at position 90 in SEQ ID NO: 1 by an arginine residue (R) and v) the substitution of the glutamine residue (Q) at position 110 in SEQ ID NO: 1 by an arginine residue (R).

[0031] In some embodiments, the polypeptide of the invention is fused to a heterologous moiety. In some embodiments, the heterologous moiety is a cell-penetrating peptide. As used herein, the term "cell-penetrating peptide” refers to a short peptide, for example comprising from 5 to 50 amino acids, which can readily cross biological membranes and is capable of facilitating the cellular uptake of various molecular cargos, in vitro and / or in vivo. In some embodiments, the heterologous polypeptide is an internalization sequence derived from the Drosophila Antennapedia / Penetratin (Antp) protein (amino acids 43-58), the trans-activating transcriptional activator of HIV-1.

[0032] Cell-penetrating peptide to transfer peptide / protein-based and nucleic acids-based therapeutic vaccines against viral infections are well known in the art as disclosed in Khairkhah et al (2023)27or Kardani K, et al. (2021)28

[0033] As used herein, the term “polypeptide” has its general meaning in the art and refers to a polymer of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptides for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. In particular, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. The polypeptides of the invention and fragments thereof according to the invention can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N-linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine / threonine or tyrosine). In some embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0034] A further object of the present invention relates to a nucleic acid that encodes for a polypeptide of the present invention.

[0035] As used herein, the term “polynucleotide” or “nucleic acid” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogues 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 a DNA or RNA. In some embodiments, the nucleic acid comprises a 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 nucleic acid disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the nucleic acid (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.

[0036] As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein” or “encoding nucleic acid”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0037] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0038] In particular embodiment, the nucleic acid is an RNA molecule, and more particularly a mRNA molecule.

[0039] Thus, a further object of the present invention relates to a vector comprising the nucleic acid of the invention.

[0040] As used herein, the term “vector” has its general meaning in the art and refers to the vehicle by which a nucleic acid molecule can be introduced into cells, so as to transform the cell and promote expression (e.g. transcription and / or translation) of the introduced sequence. According to the invention, vectors include viral vectors or non-viral vectors. In some embodiments, the vector according to the invention is a non-viral vector. Typically, the non-viral vector may be a plasmid or an exosome.

[0041] Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non-viral vectors useful in the practice of the present invention has very well known in the art.

[0042] According to the invention, non-viral vectors include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM- 102-based nanoparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al. Biol Pharm Bull (2018). Non viral vectors according to the invention include also the non-viral vectors described in patent WO2017049245, W02018081480 and W02021016430.

[0043] In some embodiments, the method according to the invention, wherein the non-viral vector is cationic a polymers-based nanoparticle, and more particularly is a polyethylenimine(PEI)-based nanoparticle.

[0044] In some embodiments, the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding the polypeptide of the invention.

[0045] In some embodiments, the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding the polypeptide of the invention.

[0046] In some embodiments, the vector according to the invention is a viral vector.

[0047] Examples of viral vector include adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

[0048] Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94 / 19478. In a particular embodiment, the viral vector is an adeno-associated viral (AAV) vector.

[0049] By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV9 etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus. The ITRs need not be the wildtype nucleotide sequences, and may be altered, e. g, by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the CYP46A1 gene) and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5'and Y) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAVITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e. g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. Furthermore, 5'and 3 'ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV- 1, AAV-2, AAV-3, AAV-4, AAV 5, AAV6, etc. Furthermore, 5'and 3'ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

[0050] In another particular embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and all variants of AAV9, including AAV PHP.B (see for example the patent application WO2015038958), AAVPHP.eB, AAV-PHP.N", and "AAV-PHP.B- DGT (see the patent application W02017100671 or Chan Y Ken, Nat Neurosci. 2017 Aug;20(8): 1172-1179.), AAV3B, AAV-2i8, Rh74, AAV capBlO, AAVMacPNSl or AAVMacPNS2 or any other serotypes of AAV that can infect human, monkeys or other species.

[0051] The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally associated with the selected gene.

[0052] Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, neuronal promoters, promoter of Dopamine- 1 receptor and Dopamine-2 receptor, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. , Stratagene (San Diego, CA). Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia andaufin.

[0053] A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid of the invention and / or a vector. As used herein, the term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

[0054] Such nucleic acid or vectors find use when the polypeptide of the invention is intended to be administered to a patient in the frame of a gene therapy. In this case, the nucleic acid is preferably present on an expression vector, on which the sequence coding for the polypeptide is placed under the control of expression signals (e.g. a promoter, a terminator and / or an enhancer) allowing its expression.

[0055] Method for treating pathogen infection of the invention

[0056] In particular, the polypeptide of the invention is able to decreases viral budding involving ESCRT machinery (i.e decrease the viral particle release).

[0057] Accordingly, the present invention relates to a method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of i) the isolated polypeptide of the invention, ii) the nucleic acid encoding for such polypeptide of the invention, or iii) the vector comprising said nucleic acid.

[0058] In other words, the invention refers to i) the isolated polypeptide of the invention or ii) the nucleic acid encoding for such polypeptide of the invention for use for treating an infection in a subject in need thereof.

[0059] In some embodiments, the nucleic acid of the invention (i.e encoding for the polypeptide of the invention) is comprised in a vector.

[0060] In particular embodiments, the vector is a non-viral vector and more particularly a Lipid- based nanoparticles.

[0061] In other words, in some embodiments the nucleic acid of the invention is administered simultaneously, separately or sequentially with a transfection reagent.

[0062] As used herein, the term “transfection reagent” has its general meaning in the art and refers to any agents suitable to introduction of nucleic acid into eukaryotic cells by non-viral agents. According to the invention, transfection reagent includes non-viral vector as described above. It is well known in the art many various non viral methods to introduce nucleic acid as described in Yamano S et al, Mol Biotechno.201022, Kumar et al, Cold Spring HArb Protoc. 201923, Feigner et al, Proc Natl Acad Sci.198724, Rahimi et al, Bratisl Lek Listy. 201825, Pardi et al. J Control Release. 201526. As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. Typically the patient is affected or likely to suffer from a viral infection. In some embodiments, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

[0063] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0064] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. polypeptide and / or nucleic acid and / or vector of the invention) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. In the context of the invention, the subject is administered with the polypeptide of the invention by topical administration. The topical administration is performed by a patch application. In a further embodiment, the subject is administered with the polypeptide of the invention by subcutaneous, nebulization or sublingual administration. In another embodiment, the subject is administered with the polypeptide of the invention by oral administration. The oral administration is performed by tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms.

[0065] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients (i.e the nucleic acid encoding the polypeptide of the invention and transfection reagents) by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients (i.e the nucleic acid encoding the polypeptide of the invention and transfection reagents) at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients (i.e the nucleic acid encoding the polypeptide of the invention and transfection reagents) at different times, the administration route being identical or different.

[0066] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0067] In particular embodiment, the infection is induced by a pathogen involving Endosomal Sorting Complex Required for Transport (ESCRT) machinery to coordinate their vesicle budding.

[0068] Pathogen involving ESCRT machinery has known in the art as described in Rivera- Cuevas et al, 202329

[0069] In particular embodiment, the pathogen is selected from the group consisting of virus, intracellular bacteria, intracellular parasite.

[0070] In particular embodiment, the infection is a viral infection.

[0071] As used herein, the expressions "viral infection" and "infected by a virus" mean that said animal or human has been exposed to a pathogenic RNA or DNA virus and that said virus has attached itself to one or more cells of the host and has then penetrated (or is likely to penetrate) into said cell(s) and has had (or will possibly have) harmful effects for at least one cell of said animal or human. In particular, such a viral infection is capable of evolving into clinical signs of induced pathologies or pathologies accompanying said infection. Accordingly, a "viral infection" within the scope of the present invention includes the earliest phases of viral contamination as well as the latest phases and the intermediate phases of viral contamination. By way of example, in the case of HIV, the infection evolves in several phases which may follow one another over time. Four phases in particular are distinguished: (1) the primary infection corresponds to the phase of seroconversion which follows contamination and is (in 50 to 75% of cases) or is not accompanied by symptoms; it is followed by (2) a latent phase, then (3) a phase with minor symptoms, and finally (4) the phase of profound immunodepression or the AIDS stage, which is generally symptomatic and is generally accompanied by numerous opportunistic infections. The term "viral infection" therefore also includes any clinical sign, symptom or disease that occurs in an animal or human (patient) following contamination of said animal or patient by a virus as described in the present application. Accordingly, the "viral infection" includes both contamination by said virus and the various pathologies which are the consequence of contamination by said virus.

[0072] The viruses that fall within the scope of the present invention include DNA viruses and RNA viruses (riboviruses), in particular viruses responsible for cell deficiencies such as immune deficiencies (such as AIDS), respiratory deficiencies (such as SARS and SARS-CoV- 2), neuronal deficiencies (such as rabies) or epithelial deficiencies (such as haemorrhagic fevers). More specifically, said virus is a virus selected from the following families: the Retroviridae (i.e. Lentiviridae), like HIV (human immunodeficiency virus); the coronaviridae, like endemic human coronaviruses (HCoV-229E, -NL63, -OC43, and -HKU1), Middle-East respiratory syndrome (MERS) coronaviruses and severe acute respiratory syndrome (SARS) such as SARS-CoV-2;

[0073] - the retroviruses, in particular those of the genus lentivirus and those of the genus oncovirus, for example the HTLV-1 virus;

[0074] - the flaviviridae, in particular those of the genus flavivirus, which includes especially the dengue virus, the yellow fever virus and the viruses responsible for viral encephalitis, such as the West Nile virus, the Japanese encephalitis virus and the Saint-Louis encephalitis virus; the Pestiviruses like Bovine viral diarrhea virus (BVDV); or in particular those of the genus hepacivirus, such as Hepatitis C virus;

[0075] - the togavirus (chikungunya virus); the orthomyxoviridae, like influenza virus A, B and C; the paramyxoviridae, in particular those of the genus morbillivirus, especially the measles virus, and the respiratory viruses, in particular those of the genus pneumovirus, for example human respiratory syncytial virus (RSV, type A and type B), metapneumovirus (MPV type A and type B) or parainfluenza virus (e.g., PIV-1, PIV-2, PIV-3, PIV-4);

[0076] - the reoviridae, in particular the virus of the genus rotavirus; - the picornaviridae, in particular the viruses of the genus enterovirus, including the polioviruses and the viruses responsible for viral meningitis, those of the genus aphthovirus, especially the aphthous fever virus, and those of the genus rhinovirus; or in particular the viruses of the genus hepatovirus such as Hepatitis A virus;

[0077] - the filoviridae, in particular the Ebola virus or the Marburg virus; the arenaviridae, in particular the Lassa virus;

[0078] - the rhabdoviridae, in particular those of the genus rhabdovirus, including the rabies virus, and the genus vesiculovirus, which includes the vesicular stomatitis virus; the togaviridae, in particular of the genus Rubivirus, including the rubella virus; the poxviridae, in particular the vaccinia and variola viruses;

[0079] - the herpesviridae, in particular the Herpes type 1 or 2, varicella, cytomegalovirus (CMV) or Human Herpes virus type 6 (HHV-6) and Zoster viruses; and the hepadnaviridae such as the hepatitis B virus; the hepatitis D virus;

[0080] - the plant viruses,

[0081] - Prokaryotic virus such as plant viruses, sulfolobus tuerreted icosahedral virus 1 (STIV), carnation Italian ringspot virus (CIRV), equine infectious anemia virus (EIAV), Rous sarcoma virus (RSV), or the hepeviridae such as the Hepatitis E virus.

[0082] In some embodiments, the patient suffers from an human immunodeficiency virus (HIV) infection. In some embodiments, the patient suffers from Ebola virus infection or from dengue virus infection. In some embodiments, the patient suffers from chikungunya virus infection. In some embodiments, the patient suffers from SARS-Cov 2 infection.

[0083] In some embodiments, the infection is induced by intracellular bacteria.

[0084] The intracellular bacteria involving ESCRT machinery that fall within the scope of the present invention include anaplasma phagocytophilum, brucella abortus, uropathogenic Escherichia coli (UPEC), mycobacterium tuberculosis, salmonella enterica, Coxiella burnetti,

[0085] In some embodiments, the infection is induced by intracellular eukaryote.

[0086] The intracellular eukaryote involving ESCRT machinery that fall within the scope of the present invention include toxoplasma gondii. Pharmaceutical composition of the invention

[0087] In particular embodiment, i) the polypeptide of the invention ii) the nucleic acid of the invention (i.e encoding for the polypeptide), or iii) the vector of the invention (i.e vector comprising the nucleic acid of the invention) are typically combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to be administered in the form of a pharmaceutical composition.

[0088] As used herein, the term "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0089] Thus, another object of the present invention relates to a pharmaceutical composition comprising the polypeptide of the invention as previously defined, or the nucleic acid of the invention.

[0090] In particular embodiment, the present invention relates to a pharmaceutical composition comprising i) the polypeptide of the invention as previously defined, or ii) the nucleic acid of the invention or iii) the vector of the invention for use for treating infection in a subject in need thereof.

[0091] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The GPR50 ligand agonis of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active antibody in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0092] 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.

[0093] FIGURES:

[0094] Figure 1: CHMP2B K6 is involved in the timely progression of CHMP2B to the abscission site. A. Quantification of the abscission time of HeLa cell lines stably expressing CHMP2B GFP or CHMP2B K6A GFP (calculated from furrow ingression formation to the microtubule cut), each dot represents one dividing cells. (N=3, n>50, median (full lines) and quartiles (dotty lines), Mann-Whitney test). B. Live cell imaging of HeLa cell lines stably expressing CHMP2B GFP or CHMP2B K6A GFP with siR-tubulin were observed. Duration of each abscission stage measured from live cell imaging performed in CHMP2B GFP or CHMP2B K6A GFP expressing cells (N=3, n>50, median (full lines) and quartiles (dotty lines), Mann-Whitney test, ns = non-significant (p>0.05)). C. Quantification of the abscission time of HeLa cell lines stably expressing CHMP2B GFP or CHMP2B K6R GFP (calculated from furrow ingression formation to the microtubule cut), (N=3, n>50, median (red lines) and quartiles (dotty lines), Mann-Whitney test). D. Duration of each abscission stage measured from live cell imaging performed in CHMP2B GFP or CHMP2B K6R GFP expressing cells (N=3, n>50, median (red lines) and quartiles (dotty lines), Mann-Whitney test.

[0095] Figure 2: CHMP2B K6 and its methylation contribute to HIV particles release.

[0096] HeLa cells expressing GFP, CHMP2B WT-GFP, CHMP2B K6R-GFP and CHMP2B K6A- GFP were transfected with pNL4-3 HIV-1 pro-viral DNA. Forty-eight hours later, cells were lysed and virus contained in supernatants were pelleted by ultracentrifugation. Viral production was evaluated by western blot and by ELISA quantification of HIV-1 CAp24. A. Western blot analysis of HIV-1 Gag and CAp24 products, GFP and GAPDH in transfected cells and pelleted viruses. B. HIV-1 release index was calculated as the ratio between released CAp24 and cell- associated CAp24. Results were normalized to control cells (set as 100%). C. The virus titer was scored by infection of HeLa P4R6 indicator cells, followed by P-galactosidase activity quantification in the cells. (Statistical analysis using two-tailed unpaired Student’s t test, N=4, mean ± SD. All western blots are representative of at least three independent experiments). D. HeLa cells transfected with indicated siRNA were transfected with pNL4-3 HIV-1 pro-viral DNA. Forty-eight hours later, cells were lysed, and virus contained in supernatants were pelleted by ultracentrifugation. Viral production was evaluated by western blot and by ELISA quantification of HIV-1 CAp24. Western blot analysis of HIV-1 Gag and CAp24 products, SMYD2 and GAPDH in transfected cells and purified viruses. E. HIV-1 release index was calculated as the ratio between released CAp24 and cell-associated CAp24. Results were normalized to control cells (set as 100%). F. The virus titer was scored by infection of HeLa P4R6 indicator cells, followed by P-galactosidase activity (Statistical analysis using two-tailed unpaired Student’s t test, N=4, mean ± SD. All western blots are representative of at least three independent experiments). G. 293 T cells were transfected with either CHMP2B WT-HA, 1- 154 CHMP2B WT-HA and 1-154 CHMP2B K6A-HA in association with pNL4-3 HIV-1 pro- viral DNA. Forty-eight hours later, cells were lysed and virus contained in supernatants were pelleted by ultracentrifugation. Western blot analysis of HIV-1 Gag and CAp24 products, HA and GAPDH in transfected cells and pelleted viruses. H. The virus titer was scored by infection of HeLa P4R6 indicator cells, followed by P-galactosidase activity quantification in the cells. (N=l experiment in duplicate).

[0097] EXAMPLE:

[0098] Material & Methods

[0099] Cell cultures

[0100] HeLa cells CCL-2 (American Type Culture Collection ATCC CCL-2, human cervical carcinoma, female) 293T cells and U2OS cells (ATCC HTB-96, human osteosarcoma, female) were grown in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 5% CO2 at 37 °C. HeLa cell lines stably expressing GFP, CHMP2B GFP, CHMP2B K6A GFP, SMYD2 GFP, SMYD2 Y240A GFP, CHMP2B GFP + Cherry and CHMP2B GFP + Cherry_SMYD2 were generated by lentiviral transduction of HeLa CCL-2 with lentiviruses and selected by FACS sorting to obtain a bulk population of cells expressing the different fusion proteins at comparable level and near to the endogenous protein expression level (lentivectors are reported Table SI). HeLa CHMP2B GFP and CHMP2B K6A GFP were then subject to clonal selection on 3 criteria: CHMP2B GFP expression levels comparable to endogenous expression, similar expression level between CHMP2B GFP and CHMP2B K6A GFP, and CHMP2B localization at the midbody that recapitulates the endogenous one. CHMP2B GFP and CHMP2B K6A GFP are siRNAs resistant cell lines that have been obtained by mutating 6 nucleotides of the siRNAtargeting sequence using Phusion Site-Directed Mutagenesis Kit (Thermoscientific). To generate Lap2_ P_RFP cell lines, GFP or SMYD2 GFP HeLa cells were transfected with pmRFP_ P_IRES_puro2b plasmid58 (plasmid created by Dr Daniel Gerlich lab, IMBA, Vienna), followed by 1 pg / ml puromycin selection and cytometry sorting.

[0101] Transfections and siRNAs

[0102] Plasmids were transfected in HeLa cells for 24h using Lipofectamine 2000 Transfection Reagent (Thermo Scientific) or with Equilibrate X-tremeGENE 9 reagent (ref. 6365787001 Roche). Plasmids were transfected in 293T cells for 24h using PEI (Polyethylenimine, Ref. 23966, Polysciences). For silencing experiments, siRNAs oligonucleotides were complexed with Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM Reduced Serum Medium and HeLa or U2OS cells were plated on the mix in penicillin and streptomycin free DMEM, at a final siRNA concentration of 20nM (Ctrl, CHMP2B, SMYD2 and Rab35 siRNAs) and 10 nM (Nupl53 siRNAs). After 16h00, transfection media was changed and cells were maintained in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin for 48h (for CHMP2B, SMYD2 and Rab35 depletion) or 72h (for Nupl53 depletion). Plasmids and siRNA are reported in Tables S2 and S3.

[0103] Plasmid constructs

[0104] Complementary DNAs (cDNA) coding for the full length human SMYD2 and CHMP2B proteins were obtained from HeLa total cDNA and subcloned into pSUMO (Life Sensor) and pGEX-6P-l (Addgene) vectors respectively in order to produce 6xHis-tagged SMYD2 and GST-tagged CHMP2B proteins. BL21 Hl-Control™ (DE3) E.Coli were then transformed with these plasmids in order to express and purify recombinant proteins. Human SMYD2 and CHMP2B were subcloned into Gateway pDonor207 plasmid and GFP, Cherry or Flag transient expression vectors were generated by LR recombination (Thermofisher) into pDest-eGFP-Cl, pDest-mCherry-Cl or pCiNeo-3xFLAG59 destination vectors. Lentiviral vectors containing GFP, CHMP2B GFP, CHMP2B K6A GFP, SMYD2 GFP, SMYD2 Y240A GFP, CHMP2B GFP, Cherry or Cherry SMYD2 were generated either by classical enzymes digestion followed by ligation, or by using in-fusion HD Cloning Kit (Takara bio). CHMP2B K6A and SMYD2 Y240A point mutations have been generated using Agilent QuikChange XL site-directed mutagenesis kit or Phusion Site-Directed Mutagenesis Kit. Complementary DNAs (cDNA) coding for the full length of human SMYD2, CHMP2B and CHMP3 proteins were obtained from HeLa total cDNA and subcloned into pSUMO (LifeSensor), pGEX-6P-l (Addgene) and pet28 MBP TEV (Addgene) vectors respectively in order to produce 6xHis-tagged SUM0-SMYD2, GST-tagged CHMP2B, and 6xHis-tagged MBP-CHMP3 proteins. A shorter version of CHMP2B (1-154) was also cloned in the pet28 MBP TEV vector in order to produce 6xHis-tagged MBP- CHMP2B 1-154 protein. BL21 HL Control™ (DE3) E.Coli (for pSUMO and pGEX-6P-l plasmids) and BL21 Rosetta 2™ (DE3) E.Coli (for pet28 MBP TEV plasmid) were then transformed with these plasmids in order to express and purify recombinant proteins. Residues 240 of SMYD2 and 6 of CHMP2B were mutated from a tyrosine into alanine and from a lysine into alanine respectively using the Agilent Quickchange XL site-directed mutagenesis kit. Primers used for mutagenesis are reported in Table S4, and mutations was verified through DNA sequence analysis (Eurofins). CHMP2B WT and shorter version of CHMP2B WT and K6A (1-154) were cloned into a pCMV-Tag 4 vector (Clonetech) modified to express the HA Tag at the C-terminus.

[0105] Western blots

[0106] Total proteins were extracted in IX Laemmli lysis buffer (prepared from Laemmli 2X : 4% SDS, 20% glycerol, 120mM Tris HC1 IM pH 6.8, 0,02% bromophenol blue, 10% P mercaptoethanol) and heated 10 min at 95° before loading. Protein extracts were loaded on NuPAGE Bis-Tris 4-12% Gels (Invitrogen) for migration, and transferred to nitrocellulose membranes. Protein transfer was assessed by Ponceau red staining. Membranes were blocked in PBS containing 0.1% Tween-20 and 5% milk for 1 h at room temperature. Incubations with primary antibodies were carried out at 4 °C overnight using the manufacturer recommended dilutions. After 3 washes in PBS-tween 0.1%, membranes were incubated with secondary antibodies coupled with peroxidase (Jackson ImmunoResearch) at room temperature for 1 h. Proteins were detected by chemiluminescence using SuperSignal (Thermo Fisher Scientific) with the LI-COR Odyssey FC Imaging System and Image Studio Lite Software.

[0107] For HIV-1 production assay Cell lysates were lysed in ice-cold DOC buffer (lOmM Tris, pH 8, 150mM NaCl, ImM EDTA, 1% Triton X-100 and 0.1% DOC 10%) containing complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation for 15 min at 13,000 rpm. The protein concentrations were determined using a Bradford protein assay (BioRad), and equal amounts of protein for each sample were used for the following steps.

[0108] Cell lysates were subjected to SDS-PAGE gels. Laemmli 2x concentrate (Sigma) has been used as a sample buffer loading protein sample in SDS-PAGE. Proteins were then transferred onto hydrophobic polyvinylidene difluoride membranes (PVDF, 0.45 pm, Millipore), followed by blocking in milk buffer (Tris-buffered saline [TBS] [0.5 M Tris pH 8.4, 9% {wt / vol} NaCl], 5% [wt / vol] nonfat dry milk, 0.05% [vol / vol] Tween 20) for Ih at room temperature (RT). Membranes were incubated overnight at 4°C with the appropriated primary antibodies in milk buffer (Tris-buffered saline [TBS] [0.5 M Tris pH 8.4, 9% {wt / vol} NaCl], 3% [wt / vol] BSA, 0.05% [vol / vol] Tween 20). Blots were washed with TBS containing 0.05% (vol / vol) Tween 20 and incubated with appropriate HRP-conjugated secondary antibodies in milk buffer for Ih at RT. After washing, protein bands were detected by using Amersham ECL Select Western blotting detection reagent (GE Healthcare).

[0109] Protein-protein interaction studies

[0110] HeLa cells were lysed in lysis buffer (20mM tris HC1, pH8, 150 mM NaCl, 0,4% to 1% NP40-lysis buffer, 2mM EDTA, protease inhibitors) 24 h post transfection, sonicated for 7 minutes and 30 sec (10 secondes on, 50 sec off), and protein lysates were collected after 20 minutes centrifugation at 13 000 rpm. Protein lysates were incubated O / N with GFP antibody Dynabeads magnetic beads (ThermoFisher Scientific). After washing three times, the beads were heated at 95 °C in Lammeli 1 X buffer, and immunoprecipitated products were then analyzed by western blot. Immunofluorescence and image acquisition

[0111] HeLa or U2OS cells were grown on coverslips and then fixed with cold methanol for 5 min on ice. Cells were rinsed twice in PBS and then blocked for 30 min with PBS Tween 0.2%, 1% BSA and 1% SVF to prevent non-specific staining. After three washes in PBS Tween 0.2%, cells were incubated with primary antibodies in PBS Tween 0.2% at room temperature for 40 min -Ih. After three washes in PBS 0.2% Tween, cells were incubated for 30 min with secondary antibodies. After three washes in PBS Tween 0.2% and one last wash in water, coverslips were mounted with VECTASHIELD PLUS Antifade Mounting Medium with DAPI (EuroBio Scientific). Images were acquired with a fluorescent microscope Leica Inverted 6000B using a CCD camera (Photometries) and Metamorph software. Images were analyzed with Imaged software.

[0112] Time-lapse microscopy

[0113] For time-lapse phase-contrast imaging, HeLa cells were plated on p-Dish 35 mm Quad (ibidi), and maintained in a chamber at 37° with 5% CO2 (Life Imaging Service). Time-lapse sequences were recorded every 10 min for 48 h using sCMOS Orca-Flash4 V2+ camera (Hamamatsu) on an inverted Leica DMI8 with HCX Plan APO 40x NA1,3 OIL PH3 CS objective and Metamorph software. For time-lapse fluorescent microscopy, images were acquired on cells treated with SiR-tubulin (spirochrome), using sCMOS Orca-Flash4 V2+ camera (Hamamatsu) on an inverted Leica DMI8 equipped with a CSU-W1 spinning disk confocal scanning unit (Yokogawa - Andor) or using EMCCD Camera (Evolve 512 Delta, Photometries) inverted Eclipse TiE Nikon microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa). Images were acquired every 10 min overnight using HC PL APO 63x NA1.4 OIL CS2 or with ax60 1.4 NA PL-APO VC objective and Metamorph software. Both for phase-contrast and fluorescent imaging, Autofocus hardware (Leica AFC) was used.

[0114] Viability assay

[0115] HeLa cells stably expressing CHMP2B GFP were seeded without treatment, with DMSO (control) or with 0.5, 1, 5, 10 or 20 pM of Bay-598. 48 hours post treatment, cell viability was assessed with the cell counter fluidlab R-300 (anvajo).

[0116] Expression and purification of recombinant proteins

[0117] Expression and purification protocols were the same for wild type or mutant proteins. BL21 bacteria were cultured at 37°C under agitation until reaching an OD of 0.6. Protein expression was then induced by adding 500 pM isopropyl-l-thio-P-D-galactopyranosid (IPTG) and lowering the temperature to 16°C overnight. The bacteria were pelleted by centrifugation (4400 rpm, 20 min), washed with cold PBS, and harvested by centrifugation (4400 rpm, 15 min). Pellets were then stored at 80 °C or directly used.

[0118] In order to purify recombinant protein, bacteria were resuspended in 40 ml of lysis buffer per liter of culture (PBS IX, 300 mM NaCl, pH 8, 1 % Triton X-100, 1 mg / ml lysozyme and protease inhibitor cocktail) and incubated for 30 min at 4°C under agitation. Lysate was then sonicated on ice (10 sec ON, 20 sec OFF, 7 min run and 20% power) and centrifuged (15,000 g, 30 min, 4 °C). The supernatant was incubated 2 h on ice with either 1 ml His-select Nickel resin (Sigma) per liter of culture and 10 mM imidazole for SMYD2 purification or 1 ml of Glutathione- Agarose resin (Sigma) per liter of culture for CHMP2B purification. Beads were then poured into a column and washed successively with washing buffer 1 (PBS IX, 300 mM NaCl, pH 8, 0.1% Triton X-100) and washing buffer 2 (PBS IX, 300 mM NaCl, pH 8). Proteins were eluted in elution buffer (SMYD2: PBS IX, 300 mM NaCl, pH 8, 300 mM imidazole; CHMP2B: 20 mM HEPES, pH 8.3, 20 mM GSH) and SMYD2 was reduced with 10 mM DTT for 20 min on ice. The purified proteins were then exchanged against storing buffer (SMYD2: 25 mM Tris-HCl, 150 mM NaCl, pH 8, 2 mM DTT; CHMP2B: 20 mM HEPES, pH 8.3) using a PD 10 desalting column (GE Healthcare). Protein concentration was measured using the Bradford assay with BSA as standard and protein purity was assessed by SDS-PAGE analysis. For SMYD2 protein, the 6xHis-tag was removed by SENP1 digestion. Briefly, 1 mg of purified protein was mixed with 1 pg of SENP1 SUMO-protease and incubated overnight at 4°C under agitation. The sample was then polished over a Superdex 200 16 / 60 HiLoad (GE Healthcare) column in gel filtration buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8, 2 mM DTT). Protein samples were aliquoted and stored at -80°C.

[0119] In vitro methylation assay

[0120] The lysine methyltransferase reaction was carried out overnight at 30°C in 20 pL methylation buffer (Tris 50 mM pH 8, 50 mM NaCl, 1 mM DTT) containing 2 pg of CHMP2B (WT or K6A mutant), 2 pM of SMYD2 (WT or catalytic dead mutant Y240A) and 1 pCi of 3H-S-Adenosyl methionine (3H-SAM, PerkinElmer). The reaction was stopped by adding 10 pL of Laemmli buffer and heating the samples at 95°C for 5 min. Samples were separated by SDS-PAGE (NuPage 4-12%, Invitrogen) and the gel was blocked for 1 h at room temperature in blocking buffer (50 % ethanol, 5 % acetic acid, 5 % PEG 400). Coomassie staining was used to ensure equal protein loading. The gel was then soaked in Kodak Enlightning Rapid Autoradiography Enhancer solution (PerkinElmer) to optimize the methylation signal for 1 h at room temperature, dried on a Whatman paper and exposed to a ECL Hyperfilm (Cytiva) for different time at -80°C. The film was later developed, and methylated proteins were detected by autoradiography.

[0121] In vitro methylation assay followed by western-blot analysis

[0122] The lysine methyltransferase reaction was carried out overnight at 30°C in 20 pL methylation buffer (Tris 50 mM pH 8, 50 mM NaCl, 1 mM DTT) containing 2 pg of CHMP2B (WT or K6A mutant), 2 pM of SMYD2 (WT or catalytic dead mutant Y240A), 100 pM of S- Adenosyl methionine (SAM, Sigma) and 1 pM of the SMYD2 inhibitors BAY-598 or LLY- 507 (Sigma). The reaction was stopped by adding 10 pL of Laemmli buffer and heating the samples at 95°C for 5 min. Samples were separated by SDS-PAGE (NuPage 4-12%, Invitrogen) and proteins were transferred onto a nitrocellulose membrane (0.2pm, Amersham) at 220 mA for 1 h. Ponceau staining was used to ensure equal protein loading. Membranes were blocked with non-fat milk (5%) in PBS with 1% Tween (PBST) for 1 h at room temperature and incubated with a homemade a CHMP2B-K6mel antibody in 1% non-fat milk PBST over night at 4°C under agitation. After washing 3 times with PBST, the membranes were incubated for 1 h at room temperature with peroxidase-coupled secondary a-rabbit antibody (SantaCruz, sc- 2357). The proteins were then visualized by chemiluminescence detection using ECL reagent on LAS 4000 (Fujifilm) instrument.

[0123] RP-UFLC-based separation and quantification of the fluorescein-labelled peptide

[0124] A 9-amino-acid peptide derived from the sequence of human CHMP2B protein and containing the lysine 6 residue was synthesized and conjugated to fluorescein amide (FAM) on its N-terminus and modified by amidation (NH2) on its C-terminus (Proteogenix). The peptide was as follows: FAM-ASLFKKKTVD (SEQ ID NO:2)-NH2. A monomethylated version of this peptide FAM-ASLFKmeKKTVD (SEQ ID NO:3)-NH2 was also synthesized and used as a standard control. The lysine methyltransferase reaction was carried out overnight at 30°C in 50 pL methylation buffer (Tris 50 mM pH 8, 50 mM NaCl, 1 mM DTT) containing 75 pM of the substrate peptide, 1 pM of SMYD2, 100 pM of SAM and different concentrations of BAY- 598 or LLY-507. The reaction was stopped using 50pL of 15% perchloric acid (HC1O4) (v / v) prior to injection in the instrument. Samples containing CHMP2B-K6 peptide (substrate) and its methylated form (product) were separated by RP-UFLC (Shimadzu) using Kromasil 100-5- C18 column 4.6 x 250 mm, 5 pm particle size at 40 °C. The mobile phase used for the separation consisted of two solvents. Solvent A containing water with 0.1% HC1O4 and solvent B containing acetonitrile with 0.12% trifluoacetic acid (TFA). Separation was performed by an isocratic flow as followed: 79 % A / 21 % B, rate of 1 ml / min, time of run = 30 min. CHMP2B- K6 peptide and its methylated form were monitored by the fluorescence emission ( = 530 nm) after excitation at k = 485 nm and quantified by integration of the peak absorbance area, employing a calibration curve established with various known concentrations of peptides

[0125] 3D modeling of CHMP2B

[0126] Homology model of CHMP2B was generated with Alphafold2 using the full-length sequence of the human CHMP2B protein as template (Q9UQN3). Display and coloring of the structure have been done with Chimera software (version 1.4). Residues have been colored depending on their hydrophobicity according to Eisenberg scale. Red surface indicates the highest Eisenberg hydrophobicity, and blue the lowest.

[0127] Generation of Venn diagram

[0128] The 198 reproducible SMYD2 dependent Kmel sites from Olsen et al. 2016 study were crossed with the flemmingsome (489 proteins found enriched at the midbody) from Addi et al. 2020 study. 16 common proteins were found and 3 of them were discriminated since they presented a SILAC ratio >0.65, considered as non-significant Kmel diminution. Among the remaining 13 proteins, we selected only the 3 with a known cytokinetic function.

[0129] Anti-CHMP2B lysine 6 mono-methylation antibody purification

[0130] Anti-CHMP2B K6mel rabbit polyclonal antibody raised against CHMP2B peptide C+ASLF-K(Me)-KKTVDDV (SEQ ID NO:4) (C for Cysteine added at the N-terminus) was prepared and purified by Eurogentec (Belgium). Antiserum was obtained by immunizing rabbits with keyhole limpet haemocyanin (KLH)-conjugated peptide. The resulting IgG fraction was purified from antiserum by affinity chromatography against the CHMP2B Kmel peptide. This antibody was used for Fig. 2A-E; Fig. 3; Fig. 4; Fig. S2B-C; Fig. S5B,F. Anti-CHMP2B K6mel rabbit polyclonal antibody raised against CHMP2B peptide (ASLF-K(Me)-KKT (SEQ ID NO:5) was prepared and purified by Covalab. Antiserum was obtained by immunizing rabbits with (KLH)-conjugated peptide. The resulting IgG fraction was purified from antiserum by affinity chromatography against the CHMP2B K6mel peptide.

[0131] Samples preparation for MS analysis

[0132] For identification of CHMP2B lysine 6 methylation (K6) on endogenous CHMP2B protein, HeLa cells were lysed in lysis buffer (20mM tris HC1, pH8, 150 mM NaCl, 0,4% to 1% NP40-lysis buffer, 2mM EDTA, protease inhibitors) and CHMP2B was immunoprecipitated (IP) with CHMP2B antibody and Dynabeads magnetic beads (ThermoFisher Scientific). IP product was loaded on SDS PAGE gel and CHMP2B band was excised and in-gel digested by using chymotrypsine (Promega). For label Label Free Quantification (LFQ) analysis of CHMP2B lysine 6 (K6) methylation, HeLa cells were cotransfected with GFP CHMP2B and flag SMYD2 or flag SMYD2 Y240A (dead catalytic mutant). Twenty-four hours post transfection, HeLa cells were lysed in lysis buffer (20mM tris HC1, pH8, 150 mM NaCl, 0,4% to 1% NP40-lysis buffer, 2mM EDTA, protease inhibitors) and GFP CHMP2B was IP with GFP antibody and Dynabeads magnetic beads. The IP product was loaded on SDS PAGE gel and the GFP CHMP2B band was excised and in-gel digested by using chymotrypsine. Peptide extracted from each band are vacuum concentrated to dryness and resuspended in loading buffer (0.3% TFA in miliQ water) before nanoLC-MS / MS analysis.

[0133] LC-MS / MS Analysis

[0134] Peptide extracted from each band were analyzed by coupling a RSLCnano system (Ultimate 3000, Thermo Scientific) to a Q Exactive HF-X (Thermo Scientific) mass spectrometer. Peptides were first trapped onto a Cl 8 column (75 pm inner diameter x 2 cm; nano Viper Acclaim PepMapTM 100, Thermo Scientific) with buffer A (0.1% formic acid) at a flow rate of 2.5 pL / min over 4 min to desalt and concentrate the samples. Separation was performed on a 50 cm nanoviper column (i.d.75 pm, C18, Acclaim PepMapTM RSLC, 2 pm, 100A, Thermo Scientific) regulated to a temperature of 50°C and with a linear gradient from 2% to 30% buffet B (100% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL / min over 91 min. For methylation rate quantification, extracted and synthetic peptides were separated with a linear gradient from 2% to 12% over 45 min. The mass spectrometer was operated in parallel analysis monitoring (PRM) mode and MS2 scan parameters were set to select the m / z ratio of CHMP2B peptides. The MSI scans were acquired in the m / z range 375-1500 with a mass resolution of 120,000, automatic gain control (AGC) target 3 x 106, and maximum ion injection time of 50 ms. The PRM scans were acquired at a resolution of 30,000, AGC target value of 2 x 105, maximum ion injection time of 100 ms or 200 ms, isolation window of 0.7 or 1.6 m / z, and the normalized collisional energy (NCE) at 27 or 30.

[0135] Data processing of MS files

[0136] For identification, the resolved raw files from the Q Exactive HF-X were searched against the GFP-CHMP2B sequence using Mascot. Enzyme specificity was set to chymotrypsin and a maximum of two-missed cleavage sites were allowed. Methionine oxidation, cysteine carbamidomethylation, N-terminal acetylation, methylation, dimethylation and trimethylation of lysine were set as variable modifications. Phosphorylation of serine and threonine were also set as variable modifications for CHMP2B characterization. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS / MS peaks. The resulting files were further processed using myProMS v3.9.3596260. Synthetic peptide was used to validate the presence of methylation For PRM quantification, Skyline was used for processing the data, and extracted fragment ions from each targeted masses and peak areas were integrated. Peak areas of extracted fragment ions from the same peptide ion were then summed and used as proxy for parent ion abundance.

[0137] Site abundance estimation, injection biases and changes in global variance between biological replicates (N=5 for GFP CHMP2B co-expressed with flag_SMYD2, and N=4 for GFP CHMP2B co-expressed with flag_SMYD2 Y240A, (one replicate needed to be removed for SMYD2 Y240A because not enough methylation event was detected)) were corrected by a Median and Scale normalization using a non-modified set of CHMP2B peptides. Label free quantification (LFQ) was performed at site-level following the algorithm as described with the minimum number of peptide ratios set to 1 and the large ratios stabilization feature. A t-test was then used to evaluate the significance of the mean's difference of the log2 transformed abundances between condition SMYD2 and SMYD2 Y240A, for each site.

[0138] To estimate the methylation rate by PRM, Skyline was used for processing the data. The spectral library of the surrogate peptides was built in Skyline based on DDA MS analysis of the synthetic peptide mixture. Peak picking and peak boundaries were carried out by Skyline and manually adjusted based on the overlapping precursor and product peaks. The peak area for each transition was exported to Microsoft Excel. To build the calibration curve, the sum of the peak areas of the fragment ions of each synthetic peptide precursor charge state, 4+ and 3+, at different concentrations, as shown in table S9, were plotted.

[0139] The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository62 with the dataset identifier PXD041760.

[0140] GUV in vitro test

[0141] MBP-CHMP2B 1-154, MBP-CHMP3 and SUM0-SMYD2 were used in this experiment. Methylation of CHMP2B protein was achieved by incubating 20 mg of MBP- CHMP2B with 20 mg of SUMO-SMYD2 in the presence of 1 mM SAM for 6 hours at room temperature. Then, for CHMP2B (WT or methylated) and CHMP3, 6xHis MBP-tag was removed by 6xHis-tagged TEV digestion. Briefly, 20 mg of purified protein was mixed with 1 mg of TEV protease and incubated overnight at 8°C under agitation. For CHMP3, the sample was further incubated with 1 ml of His-select Nickel resin for 2 hours at 4°C under agitation to remove TEV protease, MBP-tag and uncleaved MBP-CHMP proteins. For CHMP2B, cleavage of 6xHis MBP-tag leads to CHMP2B protein aggregation in the form of white precipitates while TEV, cleaved MBP-tag and SUMO-SMYD2 remain in solution. Cleaved CHMP2B was then centrifugated (15,000 g, 30 min, 4 °C) and the protein pellet was washed 3 times with CHMP buffer (20 mM HEPES, pH 8.3) before being resuspended in 20 mM HEPES, pH 8.3, 6 M guanidine. To prevent protein re-aggregation, cleaved CHMP2B was serially diluted by half to reach a guanidine concentration of 375 pM, and the remaining guanidine was removed by buffer-exchange (20 mM HEPES, pH 8.3) using a PD 10 desalting column. Protein concentration was measured using absorbance measurement at 280 nm and protein purity was assessed by SDS-PAGE analysis. Methylation of CHMP2B protein was assessed by two different manners: (i) by western-blot in a similar way as for the in vitro methylation assay, (ii) by mass spectrometry using a standard curve of synthetic CHMP2B K6 peptides displaying different level of methylation. These peptides recapitulate the lysine-6-containing CHMP2B peptide generated when the protein is digested by chymotrypsin. All the protein samples were aliquoted and stored at -80°C.

[0142] GUVs were prepared as previously described using the lipid-covered silica bead method. Briefly, dioleoyil-phosphatidylcholine (DOPC), dioleoyil-phosphatidylserine (DOPS), and dioleoyl-phosphoethanolamine (DOPE), L-a-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and dioleoyl-phosphoethanolamine labeled with Atto 647N (Atto 647N DOPE), all purchased from Avanti Polar Lipids, were dissolved in chloroform and mixed at a molar ratio of 54.9:20:20:5:0.1%, respectively. The mixture was then dried in vacuum for 3 hours in a glass vial. The dried lipid film was then hydrated in buffer solution containing 25 mM HEPES at pH 7.4 to form a suspension of multilamellar vesicles (MLVs) at 0.5 g / L. 20 pL of MLVs were mixed with 2 pL of 40 pm silica beads (Microspheres-Nanospheres, USA), deposited on parafilm and then dried for 2 hours in vacuum. The beads supporting the dried lipid films were hydrated in a 1 M trehalose solution at 60 °C and then transferred to the observation chamber with the working buffer (25 mM HEPES and 150 mM NaCl at pH 7.4). Lastly, the chamber was stirred gently for 20-30 in order to promote detachment of hydrated GUVs from the supporting silica beads. Chemical protein labelling. Purified proteins were chemically labelled with alexa 568 following the instructions provided by the Alexa Fluor 568 NHS Ester Labelling Kit (Thermo Scientific, A20003 catalog number). Free labelling molecules were removed using a PD-10 Desalting Column (GE Healthcare), and the conjugate was dialyzed into the dialysis buffer containing 25 mM HEPES and 150 mM NaCl at pH 7.4. After dialysis, the conjugate was aliquoted with 10% glycerol, frozen in liquid nitrogen and stored at -80 °C. Membrane nanotube pulling from GUVs. Membrane nanotubes were pulled from GUVs by direct contact between the tip of a closed glass micropipette prepared with a P-1000 micropipette puller (Sutter Instruments, USA) and the GUVs. XY micropipette position was controlled using a micro-positioning system (MP -285, Sutter Instrument, Novato, CA, USA). Desired proteins were added in the observation chamber before tube pulling at 1 pM final concentration. Quantification of sorting coefficients. CHMP3 membrane binding was quantified by measuring its integrated fluorescence and normalizing it with the integrated fluorescence of the lipid membrane. Sorting coefficients, defined as the relative change of membrane surface occupied by one protein molecule / complex, were calculated using the ratio between CHMP3 and Atto 647N DOPE integrated fluorescence on the surface of the pulled nanotube and on the GUV, neglecting the polarization factor64, according to the following equation:

[0143] HIV-1 production assay

[0144] For a HIV-1 production assay, HeLa stably expressing GFP, CHMP2B WT-GFP, CHMP2B K6R-GFP or CHMP2B K6A-GFP were transfected with 500ng of HIV- 1 proviral DNA NL4-3. Transfections of HeLa cells with NL4-3 HIV-1 proviral DNA were performed using Lipofectamine LTX with PLUS Reagent (Life technologies), following the manufacturer's instructions.

[0145] 293 T cells were transfected with either 200 ng of CHMP2B WT or shorter version of CHMP2B WT and K6A (1-154) and 600ng of HIV-1 proviral DNA NL4-3 using PEI (Polyethylenimine, Ref. 23966, Polysciences) following the manufacturer's instructions.

[0146] In a single round of infection, HeLa cells were treated with siRNA (25 nM) using Lipofectamine RNAiMAX (Life TechnologiesSpecific). Forty-eight hours after, siRNA-treated Hela cells were transfected with HIV-1 proviral DNA NL4-3 for 4h in OPTIMEM.

[0147] Forty-eight hours after proviral transfection, supernatants were then collected, centrifuged 5 min at 500g, 0.45 pm-filtered and used for HIV-1 CAp24 quantification by ELISA (released CAp24) (Perkin Elmer). Viral particles released into the supernatant were pelleted through a 20% sucrose cushion by ultracentrifugation at 150,000g for 60min and resuspended in laemmli sample buffer. Equal volumes of pelleted viruses were analyzed by western blotting using mouse anti-CAp24. Cell lysates were analyzed by western blotting.

[0148] HIV-1 infectivity assay

[0149] In a single round infectivity assay, the titers of released viruses were determined by infection of the indicator cells HeLa P4R5 in a standardized 96-well titration assay by luminometric analysis of P-galactosidase activity (Kit Galacto-Star™ P-Galactosidase Reporter Gene Assay System, Life Technology) following the manufacturer’s instructions.

[0150] Statistical analysis

[0151] The statistical details of all experiments are reported in the figure including statistical analysis performed, error bars, statistical significance and exact N numbers. Statistical tests used in this study were two-sided. Data normality was assessed using the Shapiro-Wilk test. In the case of multiple sample comparisons, we employed the "two-stage" Benjamini, Krieger, and Yekutieli procedure to control the false discovery rate (FDR). Statistics were performed using GraphPad Prism 6 or 9 software.

[0152] Results

[0153] SMYD2 localizes at the midbody and methylates the CHMP2B protein

[0154] The SMYD2 enzymes was first described as a histone methyltransferase, but subsequent literature suggests that its natural substrates are non-histone proteins. To investigate the cellular functions of the SMYD2 lysine methyltransferase, we monitored the localization of GFP- tagged SMYD2 in HeLa cells using live-cell imaging. We observed that GFP SMYD2 primarily localized to the cytoplasm, with a portion of SMYD2 recruited to the intercellular bridge during cytokinesis (data not shown). This localization pattern raised the potential involvement of SMYD2 in cytokinesis by methylation of midbody proteins. To identify potential substrates of SMYD2 at the midbody, we explored two proteomic datasets: (1) data from a methyl-lysine proteomic screen to identify SMYD2-dependent methylation sites in KYSE-150 cells, and (2) a Flemmingsome dataset from a midbody remnant proteome of 489 proteins enriched in post-abscission midbodies from HeLa cells. By comparing these two datasets, we identified 16 common proteins, three of which are known to be involved in cytokinesis: the kinesin KIF14, the p50RhoGAP, and the ESCRT-III component CHMP2B (data not shown). We focused on the CHMP2B ESCRT-III protein as a potential SMYD2 target. To explore the potential interaction between SMYD2 and CHMP2B, we performed coimmunoprecipitation experiments in HeLa cells, which revealed the formation of a complex between these two proteins (data not shown). Subsequently, we tested whether SMYD2 could directly methylate CHMP2B using in vitro H3 -radiolabeled S-adenosyl-m ethionine (SAM) methylation assays. We demonstrated the direct methylation of CHMP2B by recombinant SMYD2 enzyme, but not by the catalytic-dead (CD) mutant SMYD2 Y240A (data not shown). To identify the specific methylated lysine residue in CHMP2B, we performed mass spectrometry (MS)-based proteomic analysis of endogenous CHMP2B in HeLa cells. We detected mono-methylation at lysine 6 of endogenous CHMP2B (CHMP2B K6) in HeLa cell extracts (data not shown). To quantify the level of methylated CHMP2B K6, we performed label-free quantification using CHMP2B transiently transfected with either Flag-tagged SMYD2 or the catalytic-dead mutant SMYD2 Y240A, followed by MS analysis of immunoprecipitated CHMP2B GFP. The results showed a significant increase in K6 monomethylation (K6mel) upon expression of SMYD2 compared to the SMYD2 Y240A mutant (data not shown), demonstrating that SMYD2 catalytic activity is required for monomethylation of CHMP2B K6 in HeLa cells. We conducted mutagenesis experiments to confirm that CHMP2B K6 is the main lysine target of SMYD2. In vitro methylation assay using a lysine- to-alanine CHMP2B K6A recombinant mutant protein as substrate, revealed the abrogation of CHMP2B methylation, thus confirming the direct and exclusive methylation of CHMP2B K6 by SMYD2 (data not shown). This result was further validated using GFP CHMP2B and the CHMP2B K6A mutant, transiently transfected and immunoprecipitated from HeLa cells (data not shown). Furthermore, we used fluorescently-coupled CHMP2B peptides containing K6 to demonstrate efficient in vitro methylation of the unmodified peptide by SMYD2. In contrast, the K6mel peptide could not undergo further methylation (data not shown). This indicates that SMYD2 specifically mono-methylates CHMP2B and does not efficiently proceed to di- or trimethylation. Additionally, two specific SMYD2 inhibitors, BAY-598 or LLY-507, completely abolished methylation of the CHMP2B peptide (data not shown), demonstrating the druggability of this methylation. Taken together, these biochemical results establish CHMP2B as a novel cytoplasmic substrate of SMYD2.

[0155] SMYD2 methylates CHMP2B K6 at the intercellular bridge of dividing cells

[0156] To evaluate the role of CHMP2B K6 methylation in cells, we first generated an antibody that can specifically recognize CHMP2B K6 mel in vivo. The anti-CHMP2B K6 mel antibody successfully detected CHMP2B GFP in the presence of Flag_SMYD2, but not in the presence of the catalytic-dead Flag_SMYD2 Y240A mutant enzyme in total protein extracts (Data not shown). Furthermore, the CHMP2B K6 mel antibody did not detect the K6mel signal in protein extracts from cells expressing the GFP CHMP2B K6A mutant, demonstrating the high specificity for K6mel (data not shown). These findings were further validated using recombinant proteins (Fig. S2A) and the methylation signal was lost upon treatment with pharmacological inhibitors of SMYD2 (data not shown). This antibody detected endogenous CHMP2B K6mel by Western blot analysis following CHMP2B immunoprecipitation and the signal diminished upon SMYD2 inhibition (data not shown). Collectively, these results provide evidence that CHMP2B K6 methylation is specifically recognized by the CHMP2B K6 mel antibody in protein extracts. Additionally, we observed a distinct endogenous CHMP2B K6 methylation signal at the ICB of dividing HeLa cells by immunofluorescence analysis. The ICB signal was reduced in experiments using CHMP2B siRNA, supporting the antibody’s specificity for CHMP2B (data not shown). The signal intensity significantly decreased with either SMYD2 silencing or pharmacological inhibition (data not shown), demonstrating its specificity for methylation. Moreover, while the signal at the ICB increased upon SMYD2 expression in wild-type (WT) CHMP2 GFP expressing cells, this effect was not observed in CHMP2 K6A or CHMP2B K6R mutants expressing cells (data not shown). Thus, CHMP2B K6 methylation at the midbody is dependent on SMYD2 catalytic activity and could play a role during cytokinesis. Importantly, the K6 lysine residue is highly conserved in CHMP2 across evolution, including in yeast, which suggests its functional importance (data not shown). The CHMP2B K6 residue is positioned at the interface between a hydrophobic and hydrophilic region. The addition of a methyl group to this region could thus locally enhance the hydrophobicity potentially leading to a conformational change and CHMP2B activation (data not shown). Collectively, these findings demonstrate the presence of in cellulo methylation of endogenous CHMP2B at the intercellular bridge of cells undergoing cytokinesis and suggest an unexplored contribution of SMYD2 to the late cytokinetic steps.

[0157] CHMP2B K6 is involved in the timely progression of CHMP2B to the abscission site

[0158] To evaluate the impact of SMYD2-mediated methylation on CHMP2B function during cytokinesis, we established stable HeLa cell lines expressing RNAi-resistant (siR) forms of CHMP2B GFP or an un-methylatable mutant, CHMP2B K6A GFP (data not shown). These cell lines exhibited comparable expression levels of CHMP2B to endogenous levels and displayed similar localization patterns at the midbody (data not shown). To assess the effect on abscission kinetics, we performed time-lapse phase contrast microscopy to monitor ICB scission upon CHMP2B silencing. Our time-lapse phase contrast microscopy analysis revealed a delay in abscission upon depletion of endogenous CHMP2B (data not shown). Reintroduction of WT CHMP2B rescued the abscission timing, while reintroduction of the un-methylatable CHMP2B K6A mutant failed to rescue the cytokinetic defect (data not shown). Indeed, the CHMP2B K6A cells exhibited slower resolution of their intercellular bridges compared to control cells, underscoring the crucial role of CHMP2B lysine 6 in regulating the timing of abscission during cell division (data not shown). We observed an increased proportion of ICBs with CHMP2B K6A GFP localized at ring structures at the midbody and a comparatively reduced proportion of ICBs with CHMP2B K6A GFP localized at the midbody arms (compared to wild-type CHMP2B) (data not shown). We confirmed this observation in independent clones (data not shown). To investigate whether the delayed abscission observed in cells expressing CHMP2B K6A GFP was due to defective recruitment at the intercellular bridge, we employed time-lapse spinning-disk confocal microscopy. We categorized three stages of abscission based on CHMP2B localization: early abscission stage with no CHMP2B at the ICB, CHMP2B recruitment as midbody rings, and CHMP2B at midbody arms (data not shown). Time-lapse microscopy confirmed the delayed abscission timing in cells expressing CHMP2B K6A GFP compared to CHMP2B GFP, monitored by the microtubule cut (MT cut) (Fig. 1 A), which was consistent with results observed by phase-contrast microscopy (data not shown). This delay was associated with slower recruitment of CHMP2B K6A GFP at the midbody and reduced progression to the abscission site compared to CHMP2B GFP (Fig. IB). To confirm these results, we generated stable HeLa cell lines expressing a CHMP2B K6R GFP mutant, where lysine 6 is substituted with arginine, a mutation that conserves the residue's charge. Using time-lapse spinning-disk confocal microscopy, we show that the CHMP2B K6R mutant also resulted in delayed abscission, slower recruitment, and reduced progression to the abscission site (Fig. 1C-D). Taken together, these results demonstrate the significance of CHMP2B lysine 6 in both the recruitment of CHMP2B at the midbody and its transition to the abscission site during cytokinesis.

[0159] SMYD2 regulates the localization of CHMP2B and the timing of abscission

[0160] We next investigated whether SMYD2-mediated methylation of CHMP2B altered ESCRT dynamics at the intercellular bridge. Silencing SMYD2 or the chemical inhibition of SMYD2 (BAY-598 drug) markedly decreased CHMP2B methylation (data not shown) and resulted in a significant delay in the timing of abscission, observed by monitoring the microtubule cut (data not shown). In addition, SMYD2 loss-of-function (genetically or pharmacologically) increased the retention of CHMP2B GFP at ring structures at the midbody without impacting the CHMP2B GFP initial recruitment at the ICB (data not shown). These results partially phenocopied the observations with the methyl-resistant CHMP2B K6A (Fig. IB) and CHMP2B K6R mutants (Fig. 1C). The difference in amplitude observed between the use of CHMP2B K6 mutants and SYMD2 depletion / inhibition suggests that mutating the K6 residue has a stronger physicochemical impact than inhibiting its methylation. To explore the potential mechanism responsible for the in cellulo effect observed with the methylated CHMP2B, we turned to in vitro reconstitution using purified ESCRT-III proteins and model membranes. CHMP2B lacking the C-terminal region (CHMP2BAC) was previously shown to increase the efficiency of CHMP3 binding to membranes. We analyzed the effect of CHMP2BAC methylation on the recruitment of CHMP3. We compared unmethylated CHMP2B-AC with CHMP2B-AC that was methylated in vitro by SMYD2 (70% methylation of K6 residue) (data not shown). To monitor CHMP2B-AC / CHMP3 recruitment on curved membranes, we chemically labelled CHMP3 to circumvent the aggregation issues encountered with CHMP2B-AC during the labeling process (data not shown). To experimentally obtain curved membranes, we pulled membrane nanotubes from giant unilamellar vesicles (GUVs) dopped with Atto-647 fluorochrome. This allowed us to study the effect of CHMP2B methylation on CHMP3 recruitment and polymerization on curved membranes, as previously shown for unmethylated CHMP2A-AC / CHMP3 complex. We measured the fluorescence intensity of CHMP3 normalized to the membrane fluorescence intensity on both the tube (curved) and the GUV (flat), and calculated the sorting coefficient as the ratio between them. CHMP2B-AC / CHMP3 complex showed stronger preference for positively curved membranes as compared with the CHMP3 protein alone that was not able to bind by itself to the curved membrane. Interestingly, we found that a higher sorting coefficient value was obtained with the methylated CHMP2B-AC / CHMP3 complex (Fig.4K), showing that methylation may promote the formation of curvature-sensing filaments, either because they a have a smaller preferred curvature, or because they are more rigid. These results suggest that CHMP2B-AC K6 methylation facilitates the formation of ESCRT-III filaments on curved membrane.

[0161] CHMP2B K6 and its methylation contribute to HIV particles release

[0162] ESCRT-III proteins play a crucial role in diverse membrane remodelling processes, including HIV-1 budding, particularly involving the CHMP2 proteins. To extend our understanding on the function of the CHMP2B K6 methylation in broader processes, such as HIV budding, we monitored the impact of the CHMP2B K6A and CHMP2B K6R mutants in a single round of HIV-1 infection. HeLa cells expressing either the WT or the un-methylatable (K6A / R) CHMP2B GFP were transfected with HIV-1 WT provirus. After allowing viral proteins to accumulate for 48 hours, we measured the viral titer and levels of cell-associated and released CAp24. Cells expressing the un-methylatable CHMP2B K6A / R exhibited a reduction in the release of HIV-1 WT compared to cells expressing GFP alone (Fig. 2A). This effect was the result of a decrease of released CAp24 in the supernatant (Fig. 2B). Moreover, CHMP2B K6A and CHMP2B K6R inhibited the production of infectious virions (Fig. 2C). A previous study showed that simultaneous silencing of both CHMP2B and CHMP2A is necessary to interrupt virus budding44, highlighting the functional redundancy between these two members of the CHMP2 family. The substantial decrease in virus release that we observed with the CHMP2B K6A / R mutants could be the result of a dominant-negative effect via copolymerization with endogenous ESCRT-III members, including CHMP2A. To explore the impact of K6 methylation on HIV-1 release, we investigated the silencing of SMYD2. The depletion of SMYD2 significantly decreased the release of HIV-1 WT (Fig. 2D-E) and inhibited the production of infectious virions (Fig. 2F). These results collectively suggest that the function of CHMP2B K6 and its methylation are not limited to ESCRT-III functions in abscission, but also play an important role in ESCRT -Ill-dependent HIV budding and membrane remodelling processes in general.

[0163] To develop innovative pan-antiviral strategies based on alteration of CHMP2B, we designed minimal ESCRT-III N-terminal protein (CHMP2B) with antiviral activity. We first generated a C-terminal truncated CHMP2B construct (that conserves only the first 154 amino acid of CHMP2B) and evaluated their inhibitory activity on HIV-1 particle release

[0164] Our results showed that a truncated form limited to the first 154 residues of CHMP2B has an inhibitory effect on the release of infectious HIV-1 particles (Fig. 2G-H). This effect is similar to the one described previously for truncated mutant of the same length of CHMP3.

[0165] Furthermore, it is very interesting to note that the mutation of the lysine residue in position 6 in alanine (K6A) increases the inhibitory effect on the release of HIV-1. This result shows that we can design more efficient negative transdominant ESCRT-III antiviral mutant by deleting CHMP2B ESCRT-III proteins and mutated CHMP2B K6 residue.

[0166] REFERENCES:

[0167] 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.

[0168] 1. Vietri, M., Radulovic, M. & Stenmark, H. The many functions of ESCRTs. Nat. Rev. Mol. Cell Biol. 21, 25-42 (2020).

[0169] 2. Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. & Stenmark, H. Cellular Functions and Molecular Mechanisms of the ESCRT Membrane-Scission Machinery. Trends Biochem. Sci. 42, 42-56 (2017). Olmos, Y. The ESCRT Machinery: Remodeling, Repairing, and Sealing Membranes. Membranes (Basel). 12, (2022). Samson, R. Y., Obita, T., Freund, S. M., Williams, R. L. & Bell, S. D. A role for the ESCRT system in cell division in archaea. Science 322, 1710-1713 (2008). Mierzwa, B. & Gerlich, D. W. Cytokinetic abscission: molecular mechanisms and temporal control. Dev. Cell 31, 525-538 (2014). Stoten, C. L. & Carlton, J. G. ESCRT-dependent control of membrane remodelling during cell division. Semin. Cell Dev. Biol. 74, 50-65 (2018). Andrade, V. & Echard, A. Mechanics and regulation of cytokinetic abscission. Front, cell Dev. Biol. 10, 1046617 (2022). Carlton, J. G. Parallels Between Cytokinesis and the ESCRT Machinery. Current 1908, (2007). Morita, E. el al. Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215-4227 (2007). Pfitzner, A. et al. An ESCRT -III Polymerization Sequence Drives Membrane Deformation and Fission 11 11 An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission. Cell 182, 1140-1155 (2020). Pfitzner, A.-K., Moser von Filseck, J. & Roux, A. Principles of membrane remodeling by dynamic ESCRT-III polymers. Trends Cell Biol. 31, 856-868 (2021). Mierzwa, B. E. et al. Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat. Cell Biol. 19, 787- 798 (2017). Adell, M. A. Y. et al. Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding. Elife 6, (2017). Elia, N., Sougrat, R., Spurlin, T. A., Hurley, J. H. & Lippincott-Schwartz, J. Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc. Natl. Acad. Sci. U. S. A. 108, 4846-4851 (2011). Remec Pavlin, M. & Hurley, J. H. The ESCRTs - converging on mechanism. J. Cell Sci. 133, (2020). Caballe, A. et al. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT- III proteins. Elife 4, e06547 (2015). Carlton, J. G., Caballe, A., Agromayor, M. & Kloc, M. ESCRT-III Governs the Aurora B-Mediated Abscission Checkpoint Through CHMP4C. Science. 336, 220-225 (2012). Mathieu, J., Michel-Hissier, P., Boucherit, V. & Huynh, J.-R. The deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division. Science 3 6, 818-823 (2022). Votteler J, Sundquist WI. Virus budding and the ESCRT pathway. Cell Host Microbe. 2013 Sep 11;14(3):232-41. doi: 10.1016 / j.chom.2013.08.012 Morita E, Sandrin V, McCullough J, Katsuyama A, Baci Hamilton I, Sundquist WI. ESCRT-III protein requirements for HIV-1 budding. Cell Host Microbe. 2011 Mar 17;9(3):235-242. Zamborlini A, Usami Y, Radoshitzky SR, Popova E, Palu G, Gbttlinger H. Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding. Proc Natl Acad Sci U S A. 2006 Dec 12; 103(50): 19140-5. Yamano S, Dai J, Moursi AM. Comparison of transfection efficiency of nonviral gene transfer reagents. Mol Biotechnol. 2010 Nov;46(3):287-300. Kumar P, Nagarajan A, Uchil PD. DNA Transfection Mediated by Cationic Lipid Reagents. Cold Spring Harb Protoc. 2019 Mar 1 ;2019(3). Feigner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413-7. Rahimi P, Mobarakeh VI, Kamalzare S, SajadianFard F, Vahabpour R, Zabihollahi R. Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents. Bratisl Lek Listy. 2018; 119(11):701-705. Pardi N, Tuyishime S, Muramatsu H, Kariko K, Mui BL, Tam YK, Madden TD, Hope MJ, Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J Control Release. 2015 Nov 10;217:345-51. Khairkhah N, Namvar A, Bolhassani A. Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections. Mol Biotechnol. 2023 Sep;65(9): 1387-1402. Kardani K, Bolhassani A. Exploring novel and potent cell penetrating peptides in the proteome of SARS-COV-2 using bioinformatics approaches. PLoS One. 2021 Feb 19;16(2):e0247396. Rivera-Cuevas Y, Carruthers VB. The multifaceted interactions between pathogens and host ESCRT machinery. PLoS Pathog. 2023 May 4;19(5):el011344.

Claims

CLAIMS:

1. An isolated polypeptide derived from a Charged multivesicular body protein 2b (CHMP2B), wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 3 to the amino acid residue at position 10 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO:1 is substituted by another amino acid residue.

2. The isolated polypeptide of claim 1, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 54 in SEQ ID NO: 1 and wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 mutant”).

3. The isolated polypeptide of claim 1, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 117 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO:1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 2 mutant”).

4. The isolated polypeptide of claim 1, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 136 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO:1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 4 mutant”).

5. The isolated polypeptide of claim 1, wherein said polypeptide comprises the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 154 in SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO:1 is substituted by another amino acid residue (“CHMP2B Helix alpha 1 to 4 mutant bis”).

6. The isolated polypeptide of claim 1, wherein said polypeptide comprises the sequence as set forth as SEQ ID NO: 1, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by another amino acid residue (“CHMP2B mutant”).

7. The isolated polypeptide of claim 1 to 6, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by an amino acid residue selected from the group consistingof a alanine residue (A), a arginine residue (R), a glutamic acid residue (E) and a phenylalanine (F).

8. The isolated polypeptide of claim 7, wherein the lysine residue (K) at position 6 in SEQ ID NO: 1 is substituted by a alanine residue (A).

9. The isolated polypeptide of claim 1 to 8, wherein the polypeptide further comprises at 1, 2, 3, 4 or 5 mutations selected from the group consisting of F5L, K59T, K81P, K90R, Q110R.

10. A nucleic acid that encodes for a polypeptide according to claim 1 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. A method of treating an infection induced by a pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of i) the isolated polypeptide of claim 1 to 9, ii) the nucleic acid of claim 10, or iii) the vector of claim 11.

13. The method of claim 12, wherein the subject from a viral infection, and in particular an infection selected from the group consisting of human immunodeficiency virus (HIV) infection, ebola virus infection, dengue virus infection, chikungunya virus infection and SARS-Cov 2 infection.

14. The method of claim 12, wherein the pathogen is toxoplasma gondii or an intracellular bacteria selected from the group consisting of anaplasma phagocytophilum, brucella abortus, uropathogenic Escherichia coli (UPEC), mycobacterium tuberculosis, salmonella enterica, Coxiella bumetti.

15. A pharmaceutical composition comprising i) the polypeptide of claim 1 to 9, ii) the nucleic acid of claim 10, or iii) the vector of claim 11.