ARTIFICIAL EUKARYOTIC EXPRESSION SYSTEM WITH ENHANCED PERFORMANCES

Abstract

The present invention concerns a method for expressing a recombinant DNA molecule in a eukaryotic host cell, comprising the steps of: (a) expressing or introducing at least one chimeric protein, in said host cell, wherein said chimeric protein comprises: (i) at least one catalytic domain of a capping enzyme, in particular selected in the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-1 capping enzymes and cap-2 capping enzymes; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase, (b) constitutively or transiently downregulating the phosphorylation level of subunit a of translation initiation factor eIF2 (eIF2α) in said host cell.

The invention also concerns an isolated nucleic acid molecule or a set of nucleic acid molecules, comprising or consisting of (1) at least one nucleic acid sequence encoding a chimeric protein comprising at least one catalytic domain of a capping enzyme; and at least one catalytic domain of a DNA-dependent RNA polymerase; and (2) at least one nucleic acid sequence downregulating the phosphorylation level of eIF2α in a eukaryotic host cell or encoding a polypeptide downregulating said phosphorylation level; and (3) optionally, at least one nucleic acid sequence encoding a poly(A) polymerase, as well as vectors, kits and cells comprising said nucleic acid molecule or set, and different uses and applications thereof.

Claims

1. An ex vivo, in vitro or in cellulo method for expressing a recombinant DNA molecule in a eukaryotic host cell, comprising the steps of: (a) expressing or introducing at least one chimeric protein, in said host cell, wherein said chimeric protein comprises: at least one catalytic domain of a capping enzyme, in particular selected in the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-1 capping enzymes and cap-2 capping enzymes; and at least one catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase, (b) constitutively or transiently downregulating the phosphorylation level of subunit a of translation initiation factor eIF2 (eIF2α) in said host cell.

2. The method according to claim 1, wherein step (b) comprises introducing, into said host cell, at least one polypeptide or a nucleic acid molecule encoding said polypeptide, wherein the polypeptide modulates the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α, preferably of a target host cell protein selected from EIF2AK2, EIF2AK3, DDX58, IFIH1, MAVS, IFNAR1, IFNAR2, IRF3, IRF7, IFNB1, TBK1, TRAF2, TRAF3, IFIT1, JAK1, TYK2, STAT1, STAT2, IRF9, or protein phosphatase 1 PP1 or a subunit thereof, in particular PPP1CA or PPP1R15.

3. The method according to claim 2, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is selected from: (a) a viral protein selected from E3L of vaccinia virus, NSs from Rift Valley fever virus, NPRO from Bovine Viral Diarrhea Virus, V protein from parainfluenza virus type 5, ICP34.5 from human Herpes-simplex virus-1, NS1 from Influenza A virus, NS1 protein from human respiratory syncytial virus, K3L of vaccinia virus, DP71L from African swine fever virus, in particular the DP71(s) and DP71L(1) isoforms, VP35 from Zaire Ebolavirus, VP40 from the Marburg virus, LMP-1 from Epstein-Barr virus, μ2 from reovirus, B18R of vaccinia virus, and ORF4a from Middle East respiratory syndrome coronavirus, a protein with at least 40% amino acid sequence identity with one of E3L of vaccinia virus, NSs from Rift Valley fever virus, NPRO from Bovine Viral Diarrhea Virus, V protein from parainfluenza virus type 5, ICP34.5 from human Herpes-simplex virus-1, NS1 from Influenza A virus, NS1 protein from human respiratory syncytial virus, K3L of vaccinia virus, DP71L from African swine fever virus, in particular the DP71(s) and DP71L(1) isoforms, VP35 from Zaire Ebolavirus, VP40 from the Marburg virus from the Marburg virus, LMP-1 from Epstein-Barr virus, μ2 from reovirus, B18R of vaccinia virus and ORF4a from Middle East respiratory syndrome coronavirus, or a biologically active fragment thereof; (b) PPP1CA catalytic subunit and its regulatory proteins, in particular its host-cell regulatory proteins such as the eukaryotic protein PPP1R15, or a protein with at least 40% amino acid sequence identity with PPP1CA or PPP1R15, or a biologically active fragment thereof; (c) an inactive mutant of a host cell protein involved in the regulation of the phosphorylation level of eIF2α, in particular selected from EIF2AK2 or EIF2AK3, or a biologically active fragment thereof, in particular the K296R mutant of the human EIF2AK2, or the dsRNA binding domain from EIF2AK2 deleted of its carboxy-terminal kinase domain, or a biologically active fragment thereof.

4. The method according to claim 2, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is an eIF2AK2 inhibitor comprising at least one Zα domain, in particular a Zα domain from E3L of vaccinia virus or mammalian ADAR1 operably linked to at least one dsRNA-binding domain, in particular a dsRNA-binding domain from Influenza A virus NS1 protein, mammalian EIF2AK2, Flock House virus B2 protein, orthoreovirus σ3 protein, preferably selected from Influenza A virus NS1 and mammalian EIF2AK2 proteins.

5. The method according to claim 2, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is an eIF2AK2 inhibitor comprising: (a) the amino acid sequence set forth in SEQ ID NO. 16; or (b) an amino acid sequence with at least 40% amino acid sequence identity with SEQ ID NO. 16; or (c) a biologically active fragment of (a) or (b).

6. The method according to claim 2, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is a chimeric protein comprising: a. a polypeptide capable of selectively binding to EIF2AK2, preferably selected from dsRNA-binding region from EIF2AK2 protein deleted of its carboxyl-terminal kinase domain; or orthologous dsRNA binding domains such as the dsRNA-binding domain of E3L protein from vaccinia virus; or single-chain antibodies, such as nanobodies or ScFv, raised against EIF2AK2; and b. a specific domain from multimeric E3 ligases, preferably selected from: Skp1-interacting domains from BTRCP, FBW7, SPK2; or Elongin BC-interacting domains from VHL; or Cullin3-interacting domains from SPOP; or DDB1-interacting domains from CRBN or DDB2; or Elongin BC-interacting domains from SOCS2; or U-box interacting domain and coiled-coil dimerization domain from STUB1; or CUL1-interacting domain from Skp1.

7. The method according to claim 1, wherein step (b) comprises introducing, into said host cell, at least two polypeptides, or one or more nucleic acid molecules encoding said polypeptides, wherein said polypeptides modulate the activity or the expression of at least two different target host cell proteins involved in the regulation of the phosphorylation level of eIF2α, preferably wherein the modulation by said polypeptides has a supra-additive effect on the expression of said recombinant DNA by said host cell.

8. The method according to claim 7, wherein one of said at least two polypeptides inhibits the phosphorylation of eIF2α, preferably is an EIF2AK2 inhibitor such as the dsRNA binding domain from EIF2AK2 deleted of its carboxy-terminal kinase domain, or a biologically active fragment thereof, and wherein another of said at least two polypeptides activates the dephosphorylation of eIF2α, preferably is selected from PPP1CA or its viral and host-cell regulatory proteins, in particular PPP1R15, DP71L from African swine fever virus, such as its isoforms DP71L(s) or DP71L(1) and ICP34.5 from human Herpes-simplex virus-1 or a biologically active fragment thereof.

9. The method according to claim 1, wherein step (b) comprises introducing, into said host cell, a polypeptide comprising, the sequence of SEQ ID NO. 20 or SEQ ID NO. 36 or a sequence with at least 40% identity to SEQ ID NO. 20 or SEQ ID NO. 36, or a nucleic acid sequence encoding said polypeptide, wherein said polypeptide is capable of downregulating the phosphorylation level of eIF2α.

10. The method according to claim 1, wherein step (a) further comprises expressing at least one catalytic domain of a poly(A) polymerase, potentially tethered through a lambdoid N-peptide, in said host cell.

11. A eukaryotic host cell for the expression of a recombinant protein, characterized in that the phosphorylation level of eIF2α is constitutively or transiently downregulated in said cell, and wherein said cell comprises at least one nucleic acid molecule encoding at least one chimeric protein comprising: (i) at least one catalytic domain of a capping enzyme, in particular selected in the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-1 capping enzymes and cap-2 capping enzymes; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase.

12. A eukaryotic host cell according to claim 11, further comprising a heterologous nucleic acid sequence encoding at least one polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α by introducing, into said host cell, at least one polypeptide or a nucleic acid molecule encoding said polypeptide, wherein the polypeptide modulates the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α, preferably of a target host cell protein selected from EIF2AK2, EIF2AK3, DDX58, IFIH1, MAVS, IFNAR1, IFNAR2, IRF3, IRF7, IFNB1, TBK1, TRAF2, TRAF3, IFIT1, JAK1, TYK2, STAT1, STAT2, IRF9, or protein phosphatase 1 PP1 or a subunit thereof, in particular PPP1CA or PPP1R15.

13. The eukaryotic host cell according to claim 12, further comprising a nucleic acid molecule comprising: at least one nucleic acid sequence encoding a chimeric protein comprising: (i) at least one catalytic domain of a capping enzyme; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase; at least one nucleic acid sequence encoding at least one polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α by introducing, into said host cell, at least one polypeptide or a nucleic acid molecule encoding said polypeptide, wherein the polypeptide modulates the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α, preferably of a target host cell protein selected from EIF2AK2, EIF2AK3, DDX58, IFIH1, MAVS, IFNAR1, IFNAR2, IRF3, IRF7, IFNB1, TBK1, TRAF2, TRAF3, IFIT1, JAK1, TYK2, STAT1, STAT2, IRF9, or protein phosphatase 1 PP1 or a subunit thereof, in particular PPP1CA or PPP1R15; and optionally, at least one nucleic acid sequence encoding a poly(A) polymerase, potentially tethered through a lambdoid N-peptide.

14. An isolated nucleic acid molecule or a set of nucleic acid molecules, comprising: (a) at least one nucleic acid sequence encoding a chimeric protein comprising: (i) at least one catalytic domain of a capping enzyme, in particular selected in the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-1 capping enzymes and cap-2 capping enzymes; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase; and (b) at least one nucleic acid sequence downregulating the phosphorylation level of eIF2α in a eukaryotic host cell, or encoding a polypeptide downregulating said phosphorylation level.

15. The isolated nucleic acid molecule or set of nucleic acid molecules according to claim 14, wherein said nucleic acid sequence downregulating the phosphorylation level of eIF2α encodes at least one polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α in a eukaryotic host cell, preferably modulating the activity or the expression of a target host cell protein selected from EIF2AK2, EIF2AK3, DDX58, IFIH1, MAVS, IFNAR1, IFNAR2, IRF3, IRF7, IFNB1, TBK1, TRAF2, TRAF3, IFIT1, a type-I interferon protein, JAK1, TYK2, STAT1, STAT2, IRF9, or protein phosphatase 1 PP1 or a subunit thereof, in particular PPP1CA or PPP1R15.

16. The isolated nucleic acid molecule or set of nucleic acid molecules according to claim 14, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is selected from: (a) a viral protein selected from E3L from vaccinia virus, NSs from Rift Valley fever virus, NPRO from Bovine Viral Diarrhea Virus, V protein from parainfluenza virus type 5, ICP34.5 from human Herpes-simplex virus-1, NS1 from influenza A virus, K3L from vaccinia virus, DP71L from African swine fever virus, in particular the DP71(s) and DP71L(1) isoforms, VP35 from Zaire Ebolavirus, VP40 from Marburg virus, LMP-1 from Epstein-Barr virus, μ2 from reovirus, B18R from vaccinia virus and ORF4a from Middle East respiratory syndrome coronavirus, a protein with at least 40% amino acid sequence identity with one of E3L from vaccinia virus, NSs from Rift Valley fever virus, NPRO from Bovine Viral Diarrhea Virus, V protein from parainfluenza virus type 5, ICP34.5 from human Herpes-simplex virus-1, NS1 from influenza A virus, K3L from vaccinia virus, DP71L from African swine fever virus, in particular the DP71(s) and DP71L(1) isoforms, VP35 from Zaire Ebolavirus, VP40 from Marburg virus, LMP-1 from Epstein-Barr virus, μ2 from reovirus, B18R from vaccinia virus and ORF4a from Middle East respiratory syndrome coronavirus, or a biologically active fragment thereof; (b) PPP1CA catalytic subunit and its regulatory proteins, in particular host-cell regulatory proteins such as the eukaryotic protein PPP1R15, or a protein with at least 40% amino acid sequence identity with PPP1CA or PPP1R15, or a biologically active fragment thereof; (c) an inactive mutant of a host cell protein involved in the regulation of the phosphorylation level of eIF2α, in particular selected from EIF2AK2 or EIF2AK3 or a biologically active fragment thereof, in particular the K296R mutant of the human EIF2AK2 or a biologically active fragment thereof.

17. The isolated nucleic acid molecule or set of nucleic acid molecules according to claim 15, wherein said polypeptide modulating the activity or the expression of a target host cell protein involved in the regulation of the phosphorylation level of eIF2α is an eIF2AK2 inhibitor comprising at least one Zα domain, in particular a Zα domain from E3L of vaccinia virus or mammalian ADAR1 operably linked to at least one dsRNA-binding domain, in particular a dsRNA-binding domain from Influenza A virus NS1 protein, mammalian EIF2AK2, Flock House virus B2 protein, orthoreovirus σ3 protein, preferably selected from Influenza A virus NS1 and mammalian EIF2AK2 proteins.

18. The isolated nucleic acid molecule or set of nucleic acid molecules according to claim 14, further comprising at least one nucleic acid sequence encoding a poly(A) polymerase, potentially tethered through a lambdoid N-peptide and, from the 5′-terminus to the 3′-terminus: said at least one nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase potentially tethered through a lambdoid N-peptide; said at least one nucleic acid sequence encoding said polypeptide downregulating the phosphorylation level of eIF2α; and said at least one nucleic acid sequence encoding a chimeric protein comprising: (i) at least one catalytic domain of a capping enzyme; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase.

19. The isolated nucleic acid molecule or set of nucleic acid molecules according to any one of claim 14, further comprising at least one nucleic acid sequence encoding a poly(A) polymerase, potentially tethered through a lambdoid N-peptide and, from the 5′-terminus to the 3′-terminus: said at least one nucleic acid sequence encoding said polypeptide downregulating the phosphorylation level of eIF2α; said at least one nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase potentially tethered through a lambdoid N-peptide; and said at least one nucleic acid sequence encoding a chimeric protein comprising: (iii) at least one catalytic domain of a capping enzyme; and (iv) at least one catalytic domain of a DNA-dependent RNA polymerase.

20.-24. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0502] FIG. 1: shows the generic map of pC3P3 plasmids. The open-readings frames of the different generations of the C3P3 enzyme are subcloned into the pCMVScript plasmid backbone, downstream to the standard CpG-rich RNA polymerase II-dependent promoter IE1 promoter/enhancer from the human cytomegalovirus (CMV).

[0503] FIG. 2: shows the map of the pK1Ep-Luciferase-4xλBoxBr plasmid, which consists of a K1E phage RNA polymerase promoter, 5′-UTR from the human β-globin gene, Kozak consensus sequence followed by the ORF of Firefly luciferase gene, four BoxBL RNA tethering repeats in tandem from the λ bacteriophage, artificial poly[A] track of 40 adenosine residues, followed by a self-cleaving genomic ribozyme from of the hepatitis D virus, and terminated by the bacteriophage T7 φ10 transcription stop.

[0504] FIG. 3: illustrates the map of the pCMVScript-Luciferase, which consists of the ORF of Firefly luciferase gene subcloned into the pCMVScript plasmid backbone, downstream to the standard CpG-rich RNA polymerase II-dependent promoter IE1 promoter/enhancer from the human cytomegalovirus (CMV).

[0505] FIG. 4: illustrates the regulation of translation initiation by the eIF2, which is a heterotrimer consisting of eIF2α, eIF2β, and eIF2γ subunits. The conserved serine 52 residue the α subunit can be phosphorylated by several kinases, which increase the affinity of eIF2 for eIF2B. Since eIF2B can only exchange GDP for GTP if eIF2α is in its unphosphorylated state, the resulting decrease the activation of unphosphorylated eIF2 to its active GTP-bound state and concomitant decrease in translation initiation rates. Conversely, eIF2α can be dephosphorylated by the catalytic Protein Phosphatase 1 (PP1) subunit (PPP1CA) and its regulatory subunit PPP1R15. The unphosphorylated eIF2 complex can load the methionine-charged initiator tRNA Met-tRNA.sub.i.sup.Met and GTP though eIF2B guanosine nucleotide exchange factor, then assembles with other initiation factors to form the 43S preinitiation complex.

[0506] FIG. 5: shows the polysome profile observed in human HEK-293 transfected with the C3P3-G1, C3P3-G2 and C3P3-G3a plasmids, together with the pK1Ep-Luciferase-4xλBoxBr plasmid (solid lines). The profile obtained with the standard RNA polymerase II-dependent pCMVScript-Luciferase plasmid is shown for comparison (dotted line). From left to right, each profile shows the 40S, 60S, and 80S peaks, followed by the polysomes

[0507] FIG. 6: Western-blot analysis of eIF2α phosphorylation rate. The top blot is obtained with an anti-human eIF2α antibody which specifically recognizes its phosphorylated form on the Ser52 residue. The bottom blot assays the total human eIF2α using an anti-human eIF2α antibody that binds to the protein whether or not it is phosphorylated.

[0508] FIG. 7: illustrates the structure of the test plasmids encoding engineered proteins tested in Example 3. In the first series, the Z domain at the amino terminus extremity of the E3L protein is substituted by other domain with similar function. In the second series, the dsRNA-binding domain at the carboxyl-terminus end of the E3L protein is substituted by other domain with similar function. In the third series, leucine-zippers are added to the carboxyl terminus of the chimeric protein pE3L-Zα/NS1-dsDNA through flexible (G4S)2 linkers for its di/multimerization.

[0509] FIG. 8: depicts the structure of the plasmids encoding protein assemblies tested as C3P3-G3 systems as shown in Example 6. The ORF of the artificial protein E3L-Zα/NS1-dsDNA/(G4S)2/SZIP is inserted into the scaffold of the C3P3-G2 enzyme, either at its start of immediately before the Nλ-mPAPOLA block (C3P3-G3d and C3P3-G3e), or within its coding sequence between the Nλ-mPAPOLA and NP868R-(G4S)2-K1ERNAP blocks (C3P3-G3a, C3P3-G3b, and C3P3-G3c).

[0510] FIG. 9: depicts the structure of the plasmids encoding protein assemblies tested as further C3P3-G3 systems. The dsRNA-binding domain from the human EIF2AK2 and the DP71L(I) ORF were inserted into the scaffold of the C3P3-G2 enzyme, either at its start of immediately before the Nλ-mPAPOLA block (C3P3-G3f and C3P3-G3g), or within its coding sequence between the Nλ-mPAPOLA and NP868R-(G4S)2-K1ERNAP blocks (C3P3-G3h and C3P3-G3i). In addition, two types of intervening sequences were used, either the flexible (Gly4Ser)2 linker (C3P3-G3f and C3P3-G3h), or the 2A ribosome skipping sequence (C3P3-G3g and C3P3-G3i).

EXAMPLES

[0511] The examples describe different improvements of the C3P3 artificial expression system previously developed by the inventor.

Example 1: Polysome Profiling LED to the Discovery of Translation Initiation Defect in Human Cells by the C3P3 Artificial Expression System

[0512] Objectives

[0513] The aim of the present series of experiments was to assess by polysomal profiling whether translation by C3P3 expression system was normal or altered.

[0514] Polysome profiling is a method of global analysis of cell translation that separates mRNAs on a sucrose gradient according to the number of bound ribosomes. Due to strong reproducibility and sensitivity, polysome profiling is regarded as the reference method to study the translation process in cells (Chasse, Boulben et al. 2017). This technique is particularly sensitive to any alteration in the initiation of translation. More specifically, this method has been used successfully for analyzing eIF2α kinases effects on translation (Dey, Baird et al. 2010, Teske, Baird et al. 2011, Baird, Palam et al. 2014, Andreev, O'Connor et al. 2015, Knutsen, Rødland et al. 2015).

[0515] The profiling of the polysomes obtained in human cultured cells with the 1St generation (C3P3-G1) system was reported (Jais, Decroly et al. 2019). Its absence of detectable anomaly suggested that the translation was normal using this system.

[0516] In addition to C3P3-G1, the present inventor has assayed by polysome profiling the 2.sup.nd generation (C3P3-G2) and the 3.sup.rd generation system CP3-G3a.

[0517] Methods

[0518] Plasmids

[0519] Artificial gene sequences were synthesized and assembled from stepwise PCR using oligonucleotides, cloned and fully sequence verified by GeneArt AG (Regensburg, Germany). The coding sequences of all constructions were optimized for expression in human cells with respect to codon adaptation index (Raab, Graf et al. 2010).

[0520] C3P3 enzyme sequences were subcloned into the pCMVScript plasmid backbone (Stratagene, La Jolla, Calif.), downstream to the standard CpG-rich RNA polymerase II-dependent promoter IE1 promoter/enhancer from the human cytomegalovirus (CMV), as shown FIG. 1. The structure of different generations of the C3P3 enzyme were as follows: [0521] C3P3-G1, which allows the synthesis of mRNA with cap-0 modification at 5′-end, was described in WO2011/128444 and elsewhere (Jais 2011, Jais 2011, Jais, Decroly et al. 2019). The C3P3 enzyme consists of the following fusion from the N- to C-terminus (pC3P3-G1 plasmid; SEQ ID NO. 1 and SEQ ID NO. 2; FIG. 8A): African Swine Fever Virus capping enzyme NP868R (UniProtKB/Uniprot accession number P32094.1), flexible (G4S)2 linker, and mutant R551S K1E DNA-dependent RNA polymerase from bacteriophage K1E (UniProtKB/Uniprot accession number Q2WC24). [0522] C3P3-G2, which allows the extension of a polyadenylation mRNA at 3′-end of the target mRNA in addition to cap-0 modification, has been described elsewhere with slight modifications in WO2019/020811 (Jais 2017). The C3P3 enzyme consists of the following fusion from the N- to C-terminus (pC3P3-G2 plasmid; SEQ ID NO. 3 and SEQ ID NO. 4; FIG. 8B): N-peptide from the lambda bacteriophage that binds at high affinity to the BoxBL sequences from the lambda bacteriophage inserted in the 3′UTR of the target mRNA (Genbank AAA32249.1), mutant S605A/S48A/S654A/KK656-657RR mouse poly(A) polymerase a isoform 1 (PAPOLA, UniProtKB/Uniprot accession number Q61183-1), ribosome skipping F2A sequence from the Foot-and-mouth disease virus that allows ribosome skipping (Genbank AAT01770.1), African Swine Fever Virus capping enzyme NP868R (UniProtKB/Uniprot accession number P32094.1), flexible (G4S)2 linker, and mutant R551S K1E DNA-dependent RNA polymerase from bacteriophage K1E (UniProtKB/Uniprot accession number Q2WC24). [0523] A third generation C3P3-G3 enzyme (C3P3-G3a) is described below in Example 6 (pC3P3-G3a plasmid; SEQ ID NO. 19 and SEQ ID NO. 20).

[0524] The C3P3 system was used to express the Firefly luciferase test gene (FIG. 2, i.e. pK1Ep-Luciferase-4xλBoxBr), which consists of a K1E phage RNA polymerase promoter transcribed by the C3P3 enzyme, 5′-UTR from the human β-globin gene (Genbank NM_000518.4), Kozak consensus sequence for initiation of translation followed by the ORF of the Photinus pyralis gene (i.e. Firefly luciferase; UniProtKB/Uniprot accession number Q27758) and stop codon, four BoxBL RNA tethering repeats in tandem from the lambda bacteriophage that binds at high affinity to the N-peptide of the C3P3-G2 and C3P3-G3a enzyme (nucleotides 38312-38298 of genomic sequence of Enterobacteria phage lambda KT232076.1), an artificial poly[A] track of 40 adenosine residues, followed by a self-cleaving genomic ribozyme from of the hepatitis D virus, and terminated by the bacteriophage T7 φ10 transcription stop. As a control the Firefly luciferase was expressed by standard nuclear expression system using RNA polymerase II-dependent CMV promoter (FIG. 3). The corresponding pCMVScript-Luciferase plasmid therefore contained the IE1 human CMV promoter/enhancer, Kozak consensus sequence followed by the ORF from Photinus pyralis gene (UniProtKB/Uniprot accession number Q27758), and late SV40 polyadenylation signal.

[0525] For standard experiments, the Human Embryonic Kidney 293 (HEK-293, ATCC CRL 1573) were routinely grown at 37° C. in 5% CO.sub.2 atmosphere at 100% relative humidity. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine, 10% fetal bovine serum (FBS), 1% non-essential amino-acids, 1% sodium pyruvate, 1% penicillin and streptomycin, and 0.25% fungizone.

[0526] Cells were routinely plated in 24-well plates at 1×10.sup.5 cells per well the day before transfection and transfected at 80% cell confluence. Transient transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) according to manufacturer's recommendations. Except otherwise stated, cells were transfected with 2 μl of Lipofectamine 2000 plus 0.8 pg of total plasmid DNA, and were assayed two days after transfection.

[0527] Polysome Fractionation

[0528] Polysome fractionation of HEK-293 transfected cells was performed as described elsewhere with minor modifications (Verrier and Jean-Jean 2000). A single 75 cm.sup.2 tissue culture flask of HEK-293 transfected cells was used for each sucrose gradient. The culture medium was removed 24 hours after transfection and replaced with fresh medium. After overnight incubation, the medium was changed again and 2 hours later, cycloheximide at 100 pg/ml was added for 10 min. Cells were washed with PBS, collected by trypsinization, and pelleted. The dry cell pellet was resuspended in 500 μl of lysis buffer (50 mM Tris-HCl at pH 7.4, 300 mM KCl, 10 mM Mg-Acetate, 1 mM DTT, 0.05% Nonidet P40) containing 200 units/ml of SUPERaseln RNAse inhibitor (Invitrogen) and 100 pg/ml of cycloheximide, and lyzed by incubation on ice for 10 min with occasional shaking. Cycloheximide blocks the movement of peptidyl-tRNA from acceptor (aminoacyl) site to the donor (peptidyl) site on ribosomes and locks them onto the mRNA. Nuclei and cell debris were removed by centrifugation at 1,000×g for 10 min and 400 μl of supernatant was layered directly onto a 12 ml 15-50% (w/v) sucrose gradient in 50 mM Tris-Acetate (pH 7.5), 50 mM NH.sub.4Cl, 12 mM MgCl.sub.2 and 1 mM DTT. The gradient was centrifuged at 39,000 rpm in a SW41 Beckman rotor for 2.75 hours at 4° C. After centrifugation, optical density (O. D.) at 254 nm was monitored by pumping the gradient through a Retriever 500 (Teledyne Isco) fraction collector.

[0529] Results

[0530] Polysome profiling allows global analysis of host-cell translation by separating translated mRNAs on a sucrose gradient according to the number of bound ribosomes. HEK-293 cells expressing the Firefly

[0531] Luciferase gene under control of the C3P3 systems (i.e. C3P3-G1, C3P3-G2 and C3P3-G3a) or standard CMV-promoter-based nuclear expression plasmid were compared. Cell lysates are loaded of a 15-50% sucrose gradient. After ultracentrifugation, the gradient is monitored at A254 using a flow cell coupled to a spectrophotometer and then fractionated into equal fractions.

[0532] As previously described (Jais 2017), virtually no difference in ribosome distribution patterns was observed between HEK-293 cells transfected with pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr and pCMVScript-Luciferase (FIG. 5a). This finding therefore shows that expression by C3P3-G1 expression system has no detectable effect on global translation of human HEK-293 cells in comparison to standard nuclear expression system as assessed by polysome profile analysis.

[0533] Surprisingly, the expression by C3P3-G2 of the Firefly Luciferase has major impact on global translation of human HEK-293 cells (FIG. 5B). Cells transfected pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr showed very reduced 40S and 60S ribosome peaks in comparison of those of control cells transfected with pCMVScript-Luciferase. Moreover, the 80S monosome peak, which is formed during protein synthesis by assembly of the 40S and 60S subunits, was considerably increased, while polysome peaks were reduced. Such patterns are suggestive of a translation initiation defect, and more specifically by eIF2α hyperphosphorylation (Dey, Baird et al. 2010, Baird, Palam et al. 2014, Andreev, O'Connor et al. 2015, Knutsen, Rødland et al. 2015). As described above, several kinases can phosphorylate eIF2α and are activated by various stress signals, including oxidative stress [heme-regulated inhibitor (HRI) or EIF2AK1], dsRNA generated by viral infection [protein kinase double-stranded RNA-dependent (PKR) or EIF2AK2], unfolded protein response activated by endoplasmic reticulum overload [PKR-like ER kinase (PERK) or EIF2AK3], and ROS accumulation or amino acid starvation [general control non-derepressible-2 (GCN2) or EIF2AK4].

[0534] The ribosome distribution patterns were also investigated in HEK-293 cells expressing the Firefly Luciferase gene under control of the C3P3-G3a system. As detailed in Example 6, the C3P3-G3a enzyme contains an artificial interferon-inhibitory protein consisting of the Z-α domain from E3L of the vaccinia virus fused the dsRNA-binding domain from NS1 protein of the influenza A virus and terminated by the leucine sZip leucine zipper for homodimerization. The ORF of this artificial protein is inserted in frame into the open-reading of C3P3-G2 enzyme and is separated by 2A ribosome skipping motifs (FIG. 8C). Cells were transfected pC3P3-G3a/pK1Ep-Luciferase-4xλBoxBr as described above. Polysome profile of transfected cells was very similar, although not strictly identical, to that of cells transfected with pCMVScript-Luciferase plasmid (FIG. 5C). These results suggest that blocking the phosphorylation of eIF2α, returns translation to an almost normal state in the cell expressing Firefly Luciferase reporter gene under the control of the C3P3-G3a system.

[0535] Conclusions

[0536] These experiments on cultured human cells show that the second generation of the C3P3 system, induces a strong defect in the initiation of translation, which can be largely corrected by the C3P3-G3a system. This defect was not visible on the polysome profile of the first-generation C3P3 system. The mechanism of this finding is investigated in the following experiments.

Example 2 Characterization of the Mechanisms Involved in the Translation Initiation Defect

Example 2(a) RNA Interference Show that the Translation Initiation Defect Observed with the C3P3-G2 Artificial Expression System is Induced by Type I Interferon and Unfolded Protein Responses

[0537] Objectives

[0538] The objectives of these experiments were to investigate the mechanisms involved in the translation initiation defect observed in human cultured cells with the C3P3-G2 system. The C3P3-G1 system was also investigated. In addition, key candidate genes that could be involved in such translation initiation defect and could be targeted by viral proteins as selected in Examples 3 and 7, were sought. This was investigated using small interfering RNA (siRNA) to target key cell genes and with Firefly Luciferase reporter gene driven by the artificial C3P3 system expression as a readout.

[0539] Methods

[0540] Plasmids

[0541] The pC3P3-G1, pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously.

[0542] siRNA

[0543] Small interfering RNA (siRNA) is a class of non-coding dsRNA molecules of 20-25 base pairs in length with 3′-overhangs that operate within the RNA interference (RNAi) pathway to target RNA transcript for destruction.

[0544] RNA interference was performed using pools of four siRNA (Dharmacon, Lafayette, Colo., USA). Each pool consists of four chemically siRNA, which were designed in order to reduce off-targets by modifications of the sense strand to prevent interaction with RISC and favor antisense strand uptake, as well as modifications of antisense strand seed region to destabilize off-target activity and enhance target specificity (Birmingham, Anderson et al. 2006, Jackson, Burchard et al. 2006, Anderson, Birmingham et al. 2008).

[0545] The following pools of siRNA were targeting the following human genes: EIF2AK2 (NCBI GenBank accession number NM_002759), EIF2AK3 (NCBI GenBank accession number NM_004836), IRF3 (NCBI GenBank accession number NM_001571), IRF7 (NCBI GenBank accession number NM_004030), IRF9 (NCBI GenBank accession number NM_006084), JAK1 (NCBI GenBank accession number NM_002227), STAT1 (NCBI GenBank accession number NM_139266), STAT2 (NCBI GenBank accession number NM_005419), TYK2 (NCBI GenBank accession number NM_003331), DDX58 (NCBI GenBank accession number NM_014314), IFIH1 (NCBI GenBank accession number NM_022168), MAVS (NCBI GenBank accession number NM_020746), IFNAR1 (NCBI GenBank accession number NM_000629), IFNAR2 (NCBI GenBank accession number NM_207584) and IFNB1 (NCBI GenBank accession number NM_002176). A pool of four siRNAs designed and microarray tested for minimal targeting of human genome was used as negative control.

[0546] Cell Culture and Transfection

[0547] Human cells were cultured and cotransfected with the pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr or pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr plasmids as previously described. For standard luciferase and hSEAP gene reporter expression assays, cells were analyzed 48 hours after transfection. Pools of test and control siRNA were added to transfection reagent and used at a final concentration of 100 nM.

[0548] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0549] Luciferase luminescence was assayed by the Luciferase Assay System (Promega, Madison, Wis.) according to the manufacturer's recommendations. In brief, cells were lysed in Cell Culture Lysis Reagent buffer (CLR), and then centrifuged at 12,000×g for two minutes at 4° C. Luciferase Assay Reagent (Promega; 100 μl/well) diluted at 1:10 was added to supernatant (20 μl/well). Luminescence readout was taken on a Tristar 2 microplate reader (Berthold, Bad Wildbad, Germany) with a read time of one second per well.

[0550] In order to normalize for transfection efficacy, cells were transfected with the pORF-eSEAP plasmid (InvivoGen, San Diego, Calif.), which encodes for the human secreted embryonic alkaline phosphatase (hSEAP) driven by the EF-1α/HTLV composite promoter. Enzymatic activity was assayed in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen). Gene reporter expression was expressed as the ratio of luciferase luminescence (RLU, relative light units) to eSEAP absorbance (OD, optic density).

[0551] Statistical Analysis

[0552] Student t-test was used for single comparison. For multiple comparisons, one-way ANOVA with

[0553] Dunnett's Post Hoc Test was used for testing the between means of the test groups with the control group. All data are presented as are means (n≥4)±standard deviation (SD). Statistical significance was set at P<0.05.

[0554] Results

[0555] In a first series of experiments, the effects of gene expression inhibition in human HEK-293 cells were investigated with the C3P3-G2 system.

[0556] Strong increase of Firefly Luciferase expression was found in cells transfected with EIF2AK2 (relative ratio of 2.68-fold vs. negative control cells, P<0.0001), and at lesser extend EIF2AK3 siRNA (relative ratio of 1.94-fold vs. negative control cells, P<0.0001). Both kinases phosphorylate eIF2α at serine 52 and thereby inhibit translation initiation. EIF2AK2 is activated by dsRNA, which is central trigger of type I-interferon response, while EIF2AK3 is activated by unfolded protein response, which is initiated by ER overload.

[0557] The effects of inhibition of key genes involved in type I-interferon response were also investigated using pools of siRNA. Inhibition of DDX58 and IFIH1 by siRNA increases by 2.17-fold and 1.38-fold the expression of Firefly Luciferase, respectively (P<0.0001 for both comparisons). Both factors are cytosolic RNA helicases that sense short and long dsRNA, respectively and trigger type-I interferon production, which in turn induces the expression of EIF2AK2 (Saito and Gale 2008). IRF3 and IFR7 siRNA also increases by 1.70 and 1.36-fold expression of Firefly Luciferase. IRFs are transcription factors that dimerize and translocate to the nucleus following DDX58/RIG-I or IFIH1/MDA5 activation, therefore resulting in type-I interferon response (Honda, Takaoka et al. 2006). The inhibition of interferon β by siRNA also increases the expression of Firefly Luciferase by 1.82-fold. Upon their release, interferons bind their ubiquitous heterodimeric membrane receptor which is composed of two subunits, referred to as the low affinity subunit, IFNAR1, and the high affinity subunit, IFNAR2 (Piehler, Thomas et al. 2012). The inhibition of expression of these subunits by specific siRNA increases by 1.21- and 1.23-fold the expression of Firefly Luciferase. IFN receptors transduce signals via the JAK-STAT pathway, which consists of the Janus tyrosine kinases such as JAK1 and TYK2, which in turn phosphorylate the transcription proteins STAT1 and STAT2 that associate with IRF9 to form a homo- or heterotrimeric transcription complex (Au-Yeung, Mandhana et al. 2013, Platanitis, Demiroz et al. 2019). Noticeably, inhibition of all factors in the JAK-STAT pathway increases the expression of Firefly Luciferase of 1.62-(JAK1), 1.33-(TYK2), 1.21-(STAT1), 1.14-(STAT1), and 1.19-fold (IRF9), respectively.

TABLE-US-00001 Firefly Luciferase siRNA (mean ± SD); Conditions reference relative ratio P-value pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:EIF2AK2 L-003527-00 1 456 721 ± 20 541; 2.68 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:EIF2AK3 L-004883-00 1 053 783 ± 21 830; 1.94 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IRF3 L-006875-00 923 733 ± 40 675; 1.70 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IRF7 L-011810-00 739 675 ± 24 448; 1.36 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IRF9 L-020858-00 648 538 ± 11 900; 1.19 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:JAK1 L-003145-00 879 285 ± 30 622; 1.62 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:STAT1 L-003543-00 658 428 ± 11 998; 1.21 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:STAT2 L-012064-00 618 861 ± 9 585; 1.14 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:TYK2 L-003182-00 720 392 ± 18 927; 1.33 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:DDX58 L-012511-00 1 180 432 ± 25 457; 2.17 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IFIH1 L-013041-00 750 167 ± 7 974; 1.38 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:MAVS L-024237-00 669 935 ± 23 610; 1.23 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IFNAR1 L-020209-00 651 619 ± 11 100; 1.20 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IFNAR2 L-015411-00 665 850 ± 17 500; 1.23 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, siRNA:IFNB1 L-019656-00 988 203 ± 27 864; 1.82 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL, ON-TARGETplus LP_104738 543 084 ± 13 675; 1.00 Non-targeting Pool pC3P3-G2, empty plasmid, ON-TARGETplus Non-targeting Pool 37 847 ± 821; 0.07 Transfection reagent only 26 511 ± 821; 0.05

[0558] In a second series of experiments, the effects of gene expression inhibition in human HEK-293 cells were investigated with the C3P3-G1 system with the same methodology as previously. Similar findings were obtained although less marked than with the C3P3-G2 system. Both EIF2AK2 and EIF2AK3 siRNAs increased the expression of Firefly Luciferase, thereby confirming the activation of the type !-interferon, and at lesser extend the unfolded protein responses. Moreover, inhibition of most of the key genes involved in type I-interferon response previously tested significantly increased Firefly Luciferase expression level, the greatest effect being observed with DDX58 siRNA, a key cytoplasmic sensor for dsRNA.

TABLE-US-00002 Firefly Luciferase siRNA (mean ± SD); Conditions reference relative ratio P-value pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:EIF2AK2 L-003527-00 1 019 065 ± 14 370; 1.88 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:EIF2AK3 L-004883-00 818 408 ± 16 954; 1.51 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IRF3 L-006875-00 714 264 ± 31 451; 1.32 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IRF7 L-011810-00 655 279 ± 21 658; 1.21 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IRF9 L-020858-00 600 286 ± 10 350; 1.11 <0.001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:JAK1 L-003145-00 679 860 ± 23 677; 1.25 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:STAT1 L-003543-00 603 668 ± 11 000; 1.11 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:STAT2 L-012064-00 533 121 ± 8 257; 0.98 NS pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:TYK2 L-003182-00 651 240 ± 17 110; 1.20 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:DDX58 L-012511-00 908 279 ± 19 588; 1.67 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IFIH1 L-013041-00 594 715 ± 6 322; 1.10 <0.001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:MAVS L-024237-00 598 848 ± 21 105; 1.10 <0.001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IFNAR1 L-020209-00 585 131 ± 9 967; 1.08 <0.001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IFNAR2 L-015411-00 599 730 ± 15 763; 1.10 <0.001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL, siRNA:IFNB1 L-019656-00 755 455 ± 21 301; 1.39 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL LP_104738 543 084 ± 13 675; 1.00 <0.0001 pC3P3-G1, empty plasmid 37 847 ± 821; 0.00 Transfection reagent only 26 511 ± 822; 0.00

[0559] Conclusions

[0560] These RNA interference studies confirm that EIF2AK2, an effector of the type I interferon response, and EIF2AK3, a key effector of the unfolded protein response, are both activated by expression of the C3P3-G2, and at lesser extend C3P3-G1 system. Key genes involved in their pathway can be also identified.

Example 2(b): Repression of EK2AK2 Activity by Small Molecule Increases Expression Levels of Reporter Gene by the C3P3-G2 and C3P3-G1 Systems

[0561] Objectives

[0562] The present study aims to confirm the activation of EIF2AK2 effectively repress expression levels by the C3P3 system, as suggested by the previous experiments. EIF2AK2 kinase was inhibited by small molecules acting as selective competitive inhibitors. As in the previous experiments, the C3P3-G1 and C3P3-G2 generations of the system were tested.

[0563] Methods

[0564] Plasmids

[0565] The pCMVScript-Luciferase, pC3P3-G1, pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously.

[0566] Chemicals

[0567] The EIF2AK2/PKR inhibitor CAS 608512-97-6 (Sigma-Aldrich) is an imidazolo-oxindole compound, which inhibits RNA-induced EIF2AK2 autophosphorylation (Jammi, Whitby et al. 2003). CAS 608512-97-6 binds ATP-binding site directed human with an IC50 of 210 nM.

[0568] Cell Culture and Transfection

[0569] Human cells were cultured and cotransfected with the pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr or pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr plasmids as previously described. For standard luciferase and hSEAP gene reporter expression assays, cells were analyzed 48 hours after transfection. The EIF2AK2 inhibitor was added to culture medium at the time of transfection and tested at concentrations ranging from 10 nM to 10 μM.

[0570] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0571] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0572] Statistical Analysis

[0573] Statistical analyses were performed as previously described.

[0574] Results

[0575] The effects of EIF2AK2 inhibition by CAS 608512-97-6 were investigated in human HEK-293 cells expressing the Firefly Luciferase reporter gene under control of the C3P3-G1 and C3P3-G2 system. A dose-dependent increase of Firefly Luciferase expression was found with the C3P3-G1 (pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr plasmids) and C3P3-G2 (pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr plasmids) expression systems, when cells were treated with the CAS 608512-97-6 EIF2AK2 inhibitor. Maximum efficacy was found at 1 μM with the C3P3-G1 (relative ratio of 1.31-fold vs. negative control cells, P<0.0001) and C3P3-G2 (relative ratio of 1.67-fold vs. negative control cells, P<0.0001) expression systems.

TABLE-US-00003 Firefly Luciferase (mean ± SD); P-value vs. no CAS 608512-97-6 (relative ratio) pC3P3-G1, pK1Ep(G)- pC3P3-G2, pK1Ep(G)- Luciferase-4xλBoxBl Luciferase-4xλBoxBl no CAS 608512-97-6 2 576 402 ± 6 231 980 ± 172 961; NA (1.00) 786 566; NA (1.00) 10 nM CAS 608512-97-6 2 708 078 ± 7 148 341 ± 159 871; NS (1.05) 497 010; NS (1.15) 100 nM CAS 608512-97-6 3 095 106 ± 8 113 967 ± 424 730; <0.01 (1.20) 451 546; <0.0001 (1.30) 1 μM CAS 608512-97-6 3 380 978 ± 10 387 086 ± 367 210; <0.0001 (1.31) 180 533; <0.0001 (1.67) 10 μM CAS 608512-97-6 3 228 759 ± 9 317 870 ± 235 564; <0.0001 (1.25) 488 998; <0.0001 (1.50) Lipofectamine only 829 ± 274 1 0427 ± 140

[0576] The effects of the inhibition of EIK2AK2 by the specific inhibitor CAS 608512-97-6 were also tested with the plasmid pCMVScript-Luciferase which allows conventional nuclear expression of the Firefly Luciferase reporter gene. As shown in the table below, no detectable effect was observed at any concentration ranging from 10 nM to 10 μM. These results suggest that phosphorylation of eIF2α by EIF2AK2 is not a limiting factor for expression by conventional nuclear expression systems.

TABLE-US-00004 P-value vs. no CAS 608512-97-6 Firefly Luciferase CAS 608512-97-6 concentrations (mean ± SD) (relative ratio) pCMVScript-Luciferase, no CAS 608512-97-6 2 576 402 ± 37 323 NS (1) pCMVScript-Luciferase, 10 nM CAS 608512-97-6 2 490 730 ± 158 348 NS (0.97) pCMVScript-Luciferase, 100 nM CAS 608512-97-6 2 542 067 ± 164 518 NS (0.99) pCMVScript-Luciferase, 1 μM CAS 608512-97-6 2 639 581 ± 64 324 NS (1.02) pCMVScript-Luciferase, 10 μM CAS 608512-97-6 2 640 768 ± 120 057 NS (1.02) Lipofectamine only 32 093 ± 8 421 NA

[0577] Conclusions

[0578] Inhibition of EIF2AK2 by the specific antagonist CAS 608512-97-6 increases the expression of a reporter gene under control of first and second generation of the C3P3 artificial expression systems. These findings suggest that the phosphorylation of eIF2α by EIF2AK2 is a limiting factor for expression by the C3P3-G1 and C3P3-G2 systems, but not for conventional nuclear expression systems.

Example 2(c): Phosphorylation of eIF2α Assessed by Western-Blot

[0579] Objectives

[0580] The objectives of this experiment were to assay by means of a direct method the level of eIF2 phosphorylation induced by the first- and second-generation expression systems. This was assessed by Western blotting, which makes it possible to quantify the rate of phosphorylated eIF2α protein and the total eIF2α protein.

[0581] Methods

[0582] HEK-293 cells were transfected as described above with plasmids allowing the expression of the reporter gene Firefly Luciferase by the artificial expression system of the first generation (pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr), second generation (pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr), second generation with dsRNA-binding domain from human EIF2AK2 (pC3P3-G2/phEIF2AK2:DRB/pK1Ep-Luciferase-4xλBoxBr), co-expression of C3P3-G2 and E3L:Zα-NS1:dsDNA-(G4S)2-sZIP as described in Example 5 (pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr/pE3L:Zα-NS1:dsDNA-(G4S)2-sZIP), co-expression of C3P3-G2 and hEIF2AK2:DRB as described in Example 7 (pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr/phEIF2AK2:DRB), and co-expression of C3P3-G2, hEIF2AK2:DRB and DP71L(I) as described in Example 8 (pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr/phEIF2AK2:DRB/pDP71L(I)).

[0583] As a positive control, HEK-293 cells were transfected with pools of siRNA against the protein phosphatase 1 catalytic subunit alpha (PPP1CA, Dharmacon LQ-008927-00, NCBI GenBank accession number NM_002708.4) or its regulatory subunit PPP1R15A (GADD34, Dharmacon LQ-004442-02, NCBI GenBank accession number NM_014330.5). Cells treated with non-targeting pool of siRNA was used to assess off-target effects. Pools of test siRNA were added to transfection reagent and used at a final concentration of 100 nM.

[0584] Cells were lysed in 200 μl of CLR buffer and lysate was clarified by spinning for 15 sec at 12,000×g at room temperature. Twenty milligrams of total protein were resolved on 4-12% NuPAGE SDS-polyacrylamide gradient gel (Life Technologies, Carlsbad, Calif.), and subjected to western blotting onto nitrocellulose Hybond membrane (GE Healthcare, Pittsburgh, Pa.) overnight at +4° C.

[0585] To assess eIF2α phosphorylation, membranes with transferred proteins were blocked with 5% skim milk powder in PBS, then incubated with the rabbit IgG phospho-EIF2S1 (Ser52) polyclonal antibody 44-728G (1:1000; ThermoFisher) raised against human Ser52 phosphorylated eIF2α, then with anti-rabbit IgG-conjugated horseradish peroxidase NA9340V antibody (1:10000; GE Healthcare). Bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Scientific) and scanned with the Fusion XPRESS gel imager (Vilber Lourmat, Marne-la-Vallée, France). Molecular weights were determined using the Novex Sharp pre-stained Protein Standard color markers (Thermo Fisher).

[0586] For total eIF2α assay, the membranes were then dehybridized, blocked with 5% skim milk powder in PBS, and reprobed with the rabbit IgG EIF2S1 polyclonal antibody AHO1182 raised against total human eIF2α protein (1:500; ThermoFisher), then analyzed by Western blotting as previously described.

[0587] Results

[0588] An increased rate of phosphorylation of eIF2α was found in cells transfected with the plasmids of the artificial expression system of the first generation (FIG. 6, track 1 vs. 6) and second generation C3P3 system (track 2 vs. 6), as compared to cells treated with the transfection agent only.

[0589] Conversely, the rate of phosphorylation was reduced with the C3P3-G2 expression system when the E3L:Zα-NS1:dsDNA-(G4S)2-sZIP artificial protein was co-expressed (FIG. 6, track 3 vs. 6). Likewise, decrease in the rate of phosphorylation of eIF2α was observed when dsRNA-binding domain from human EIF2AK2 was co-expressed alone (FIG. 6, track 4 vs. 6) or in combination with the DP71L(I) (FIG. 6, track 5 vs. 6).

[0590] Finally, pools of siRNA of the catalytic subunit of the phosphatase PPP1CA (FIG. 6, track 7 vs. 9) or of its regulatory subunit PPP1R15A (FIG. 6, track 8 vs. 9), was associated to an increase in the rate of phosphorylation of eIF2α compared to a non-targeting pool of siRNA.

[0591] Conclusions

[0592] This experiment shows an increase of phosphorylation of eIF2α induced by first- and second-generation artificial expression systems, which can be reserved by protein inhibitors.

Example 3: Expression of Viral and Host-Cell Proteins and RNA Sequence Triggering the Type-I Interferon Pathway can Increase Expression Levels by the C3P3-G2 and C3P3-G1 Systems

[0593] Objectives

[0594] The objective of these experiments was to screen for viral proteins and RNA sequences able to inhibit type-I interferon response, which could increase expression of Firefly Luciferase reporter gene driven by the artificial C3P3-G1 and C3P3-G2 systems. This screening phase was secondarily extended to host-cell proteins also involved type-I interferon response.

[0595] Plasmids

[0596] The pC3P3-G1, pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously. Viral genes known or anticipated to interfere with the host-cell interferon response pathway or other related biological activities were subcloned in the pCMVScript plasmid backbone (Stratagene), following the removal of the T7 φ10 promoter sequence. These corresponding plasmids, designated as p-followed by the name of the ORF, have the following design: 1E1 promoter/enhancer from the human cytomegalovirus (CMV), 5′-untranslated region (5′-UTR), Kozak consensus sequence, selected open-reading frames, 3′-untranslated region (3′-UTR), and SV40 polyadenylation signal.

[0597] A first series test plasmids was synthetized, which consists of viral genes known to inhibit host-cell interferon response pathway. These genes have been selected to target host cellular proteins whose inhibition by siRNA has shown the most important effects: [0598] EIF2AK2 was targeted by the long isoform of E3L protein from vaccinia virus (UniProtKB/Uniprot accession number P21081-1; pE3L-1NV) and its short (UniProtKB/Uniprot accession number P21081-2; pE3L-2NV) isoforms (Davies, Chang et al. 1993), NS1 protein (UniProtKB/Uniprot accession number P03496; pNS1/IAV) from Influenza A virus (Bergmann, Garcia-Sastre et al. 2000), K3L protein (UniProtKB/Uniprot accession number P18378; pK3L/VV) from vaccinia virus (Davies, Chang et al. 1993), NSs protein (UniProtKB/Uniprot accession number P21698; pNSs/RVFV) from Rift Valley fever virus, which promotes EIF2AK2 proteasomal degradation (Habjan, Pichlmair et al. 2009), and the dominant-negative mutant K296R of the human EIF2AK2 (UniProtKB/Uniprot accession number P19525; pEIF2AK2:K296R), which is inactive as a result of a mutation in the ATP-binding/phosphotransfer site (Katze, Wambach et al. 1991). [0599] DDX58 was targeted by the VP35 from Zaire Ebolavirus (UniProtKB/Uniprot accession number Q05127; pVP35/EBOV), which can also caps the ends of dsRNA (Kimberlin, Bornholdt et al. 2010, Leung, Prins et al. 2010, Jiang, Ramanathan et al. 2011, Kowalinski, Lunardi et al. 2011), [0600] IRF3 was targeted by the N(pro) (N-terminal autoprotease; UniProtKB/Uniprot accession number Q6Y4U2; pN(pro)/BVDV) from Bovine Viral Diarrhea Virus (Seago, Hilton et al. 2007, Peterhans and Schweizer 2013), [0601] IFNAR1/IFNAR2 heterodimeric receptor was targeted by the B18R secreted protein from vaccinia virus (UniProtKB/Uniprot accession number P25213; pB18R/VV) that binds to type I interferons (Alcami, Symons et al. 2000), [0602] JAK1 was targeted by the VP40 protein (UniProtKB/Uniprot accession number P35260; VP40/MBV) from the Marburg virus (Valmas and Basler 2011), [0603] TYK2 was targeted by the LMP-1 protein (UniProtKB/Uniprot accession number P03230; LMP-1/EBV) from Epstein-Barr virus (Geiger and Martin 2006), [0604] STAT1 was targeted by the V protein (UniProtKB/Uniprot accession number P11207; pV/PIV5) from parainfluenza virus type 5 (Didcock, Young et al. 1999, Precious, Carlos et al. 2007) [0605] STAT2 was targeted by NS1 protein from human respiratory syncytial virus (UniProtKB/Uniprot accession number O42083; NS1/RSV) that mediates its proteasomal degradation (Elliott, Lynch et al. 2007), [0606] IRF9 was targeted by μ2 protein (UniProtKB/Uniprot accession number Q00335; μ2/REOV) from reovirus that induces its nuclear accumulation (Zurney, Kobayashi et al. 2009).

[0607] In a second series, viral and host-cell proteins which recruit human PPP1CA (UniProtKB/Uniprot accession number P62136, pPPP1CA), the serine/threonine-protein Phosphatase 1 catalytic subunit, to dephosphorylate eIF2α were tested, including the human PPP1R15A (also known as GADD34, UniProtKB/Uniprot accession number O75807; pPPP1R15A), which the regulatory subunit of PPP1CA (Novoa, Zeng et al. 2001), DP71L(s) from African swine fever virus (UniProtKB/Uniprot accession number Q65212; pDP71L(s)/ASFV) (Afonso, Zsak et al. 1998, Zhang, Moon et al. 2010), and ICP34.5 from human Herpes-simplex virus-1 (UniProtKB/Uniprot accession number P36313; pICP34.5/HVS1) (Goatley, Marron et al. 1999).

[0608] In addition, the effects of 5′UTR RNA sequence from alphavirus Sindbis virus (strain ArB7761, Genbank ID MH212167.1) on expression of Firefly Luciferase was investigated by substituting 5′UTR of the pK1Ep-Luciferase-4xλBoxBr plasmid by the 5′UTR sequence from Sindbis virus (strain ArB7761, Genbank ID MH212167.1; pK1Ep-5′UTR/SINV-Luciferase-4xλBoxBr) (Hyde, Gardner et al. 2014, Reynaud, Kim et al. 2015).

[0609] Cell Culture and Transfection

[0610] Human cells were cultured as previously described. Cells were cotransfected with the pC3P3-G1/pK1Ep-Luciferase-4xλBoxBr or pC3P3-G2/pK1Ep-Luciferase-4xλBoxBr plasmids, together with the test plasmid listed above or an empty dummy plasmid to transfect the same amount of DNA to all conditions.

[0611] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0612] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0613] Statistical Analysis

[0614] Statistical analyses were performed as previously described.

[0615] Results

[0616] In this first series of experiments, the level of expression of the Firefly Luciferase reporter gene expressed by the C3P3-G2 plasmid was investigated in presence several test plasmids encoding for viral proteins known to interfere directly or indirectly with the type-I interferon response.

[0617] All test plasmids encoding proteins that target EIF2AK2 statistically significantly increased the level of expression of the Firefly luciferase reporter gene. Greatest increase of nearly 3-fold was observed with the long isoform of E3L from vaccinia virus and at much lesser extend of 1.49-fold with its short isoform (P<0.0001 for both comparisons). This protein is well-characterized competitive inhibitor for the binding of dsRNA to EIF2AK2. NSs protein from Rift Valley fever virus increased by 2.63-fold the expression of the Firefly Luciferase reporter gene (P<0.0001). NS1 protein from Influenza A virus and K3L from vaccinia virus also statistically significantly increased the expression of Firefly Luciferase reporter gene (relative ratio of 2.63-fold and 1.68-fold, P<0.0001). In addition, the dominant-negative mutant K296R of the human EIF2AK2, which is inactive as a result of a mutation in the ATP-binding/phosphotransfer site increased substantially the expression level of Firefly Luciferase reporter gene (relative ratio of 2.63-fold, P<0.0001). This body of evidence confirms the crucial role of eIF2AK2 activation for expression by the artificial C3P3 expression system.

[0618] Other viral genes involved in type-I interferon pathway also statistically significantly increased the level of Firefly Luciferase reporter gene expression. They ranged in the following order: pVP35 (RIG-I inhibitor), N.sup.PRO (IRF3 inhibitor), VP40 (JAK1 inhibitor), V protein (STAT1 inhibitor), LMP-1 (TYK2 inhibitor), μ2 (IRF9 inhibitor), NS1 from human respiratory syncytial virus (STAT2 inhibitor) and B18R (type I interferons inhibitor).

[0619] A second series of candidate genes was tested, which recruit the Protein Phosphatase 1 (PPP1CA) catalytic subunit that dephosphorylate eIF2α at Ser52, thereby reversing the shut-off of protein synthesis induced by eIF2 kinases. The expression of human PPP1R15, a host-cell protein that directs the catalytic PPP1CA subunit to its specific substrate, significantly increased Firefly Luciferase expression by 2.25-fold (P<0.0001). The herpes virus simplex 1 ICP34.5 protein (Mossman and Smiley 2002), which also recruit PPP1CA had similar efficacy to that of PPP1R15 with 2.16-fold increase of Firefly Luciferase expression, whereas DP71L(s) which also recruit PPP1CA has much reduced efficacy of only 1.24-fold (Barber, Netherton et al. 2017).

[0620] Finally, we tested the effects of the 5′-UTR from the genomic RNA of the Sindbis alphavirus, which antagonize IFIT1 (Hyde, Gardner et al. 2014, Reynaud, Kim et al. 2015). IFIT1 are effectors induced in response to type-I interferon, which sensors viral RNA that carries a triphosphate group on its 5′-terminus or 5′-cap lacking 2′-O methylation and induces type-I-interferon response (Abbas, Laudenbach et al. 2017). This genomic RNA sequence from the Sindbis alphavirus inserted in the 5′-UTR of the Firefly Luciferase gene reporter plasmid also increased its expression by 2.7-fold (P<0.0001).

TABLE-US-00005 Firefly Luciferase (mean ± SD); Plasmids relative ratio P-value pC3P3-G2, pE3L-1/VV, pK1E-Luciferase-4xλBoxBL 3 312 609 ± 54 621; 2.98 <0.0001 pC3P3-G2, pK1Ep-5′UTR/SINV-Luciferase-4xλBoxBL 2 947 342 ± 83 023; 2.66 <0.0001 pC3P3-G2, pNSs/RVFV, pK1E-Luciferase-4xλBoxBL 2 924 273 ± 100 916; 2.63 <0.0001 pC3P3-G2, pEIF2AK2:K296R, pK1E-Luciferase-4xλBoxBL 2 923 612 ± 91 169; 2.63 <0.0001 pC3P3-G2, pPPP1R15A, pK1E-Luciferase-4xλBoxBL 2 498 553 ± 35 329; 2.25 <0.0001 pC3P3-G2, pICP34.5/HVS1, pK1E-Luciferase-4xλBoxBL 2 394 434 ± 97 118; 2.16 <0.0001 pC3P3-G2, pNS1/IAV, pK1E-Luciferase-4xλBoxBL 2 311 610 ± 74 121; 2.08 <0.0001 pC3P3-G2, pVP35/EBOV, pK1E-Luciferase-4xλBoxBL 2 039 576 ± 68 123; 1.84 <0.0001 pC3P3-G2, pNPRO/BVDV, pK1E-Luciferase-4xλBoxBL 1 901 080 ± 59 654; 1.71 <0.0001 pC3P3-G2, pK3L/VV, pK1E-Luciferase-4xλBoxBL 1 859 375 ± 62 784; 1.68 <0.0001 pC3P3-G2, VP40/MBV, pK1E-Luciferase-4xλBoxBL 1 782 322 ± 38 538; 1.61 <0.0001 pC3P3-G2, pV/PIV5, pK1E-Luciferase-4xλBoxBL 1 750 866 ± 37 434; 1.58 <0.0001 pC3P3-G2, LMP-1/EBV, pK1E-Luciferase-4xλBoxBL 1 721 541 ± 56 310; 1.55 <0.0001 pC3P3-G2, pE3L-2/VV, pK1E-Luciferase-4xλBoxBL 1 648 821 ± 59 950; 1.49 <0.0001 pC3P3-G2, μ2/REOV, pK1E-Luciferase-4xλBoxBL 1 402 716 ± 48 746; 1.26 <0.0001 pC3P3-G2, NS1/RSV, pK1E-Luciferase-4xλBoxBL 1 396 909 ± 50 590; 1.26 <0.0001 pC3P3-G2, DP71L(s)/ASFV, pK1E-Luciferase-4xλBoxBL 1 379 443 ± 57 682; 1.24 <0.0001 pC3P3-G2, pB18R/VV, pK1E-Luciferase-4xλBoxBL 1 363 247 ± 30 232; 1.23 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL 1 109 854 ± 8 750; 1.00 <0.0001 pC3P3-G2, empty plasmid 13 499 ± 228; 0.01 <0.0001 Transfection reagent only 8 890 ± 150; 0.01 <0.0001

[0621] The effects of test plasmids driven by the C3P3-G1 system were investigated with the same methodology as above. Similar findings were observed although less marked than with the C3P3-G2 system. All test plasmids encoding proteins that target EIF2AK2, except the short isoform of E3L and K3L, statistically significantly increased the level of expression of the Firefly luciferase reporter gene, with the greatest increase observed with the long isoform of E3L from vaccinia virus (relative ratio of 0.69-fold; P<0.0001).

[0622] Most of the previous test genes that target the type-I interferon pathway also statistically significantly increased the expression of the Firefly Luciferase reporter gene, except DP71L(s), LMP-1, NS1, μ2 and B18R. Finally, the 5′-UTR from the genomic RNA of the Sindbis alphaviruses also statistically significantly increased the expression of the Firefly Luciferase reporter gene.

TABLE-US-00006 Firefly Luciferase (mean ± SD); Plasmids relative ratio P-value pC3P3-G1, pE3L-1/VV, pK1E-Luciferase-4xλBoxBL 1 938 320 ± 80 126; 1.69 <0.0001 pC3P3-G1, pK1Ep-5′UTR/SINV-Luciferase-4xλBoxBL 1 810 266 ± 22 395; 1.58 <0.0001 pC3P3-G1, pNSs/RVFV, pK1E-Luciferase-4xλBoxBL 1 646 400 ± 39 468; 1.44 <0.0001 pC3P3-G1, pN(pro)/BVDV, pK1E-Luciferase-4xλBoxBL 1 371 893 ± 23 469; 1.20 <0.0001 pC3P3-G1, pEIF2AK2:K296R, pK1E-Luciferase-4xλBoxBL 1 520 092 ± 32 013; 1.33 <0.0001 pC3P3-G1, pE3L-2/VV, pK1E-Luciferase-4xλBoxBL 1 209 281 ± 30 214; 1.06 NS pC3P3-G1, pV/PIV5, pK1E-Luciferase-4xλBoxBL 1 252 842 ± 42 091; 1.09 <0.01  pC3P3-G1, pPPP1R15A, pK1E-Luciferase-4xλBoxBL 1 500 449 ± 56 022; 1.31 <0.0001 pC3P3-G1, pICP34.5/HVS1, pK1E-Luciferase-4xλBoxBL 1 423 032 ± 54 867; 1.24 <0.0001 pC3P3-G1, pNS1/IAV, pK1E-Luciferase-4xλBoxBL 1 520 613 ± 37 328; 1.33 <0.0001 pC3P3-G1, pK3L/VV, pK1E-Luciferase-4xλBoxBL 1 166 878 ± 24 155; 1.02 NS pC3P3-G1, DP71L(s)/ASFV, pK1E-Luciferase-4xλBoxBL 1 072 925 ± 14 178; 0.94 NS pC3P3-G1, pVP35/EBOV, pK1E-Luciferase-4xλBoxBL 1 418 038 ± 28 761; 1.24 <0.0001 pC3P3-G1, VP40/MBV, pK1E-Luciferase-4xλBoxBL 1 339 210 ± 26 474; 1.17 <0.0001 pC3P3-G1, LMP-1/EBV, pK1E-Luciferase-4xλBoxBL 1 203 105 ± 32 203; 1.05 NS pC3P3-G1, NS1/RSV, pK1E-Luciferase-4xλBoxBL 954 663 ± 20 205; 0.83 <0.0001 pC3P3-G1, μ2/REOV, pK1E-Luciferase-4xλBoxBL 1 049 444 ± 40 645; 0.92 <0.01 pC3P3-G1, pB18R/VV, pK1E-Luciferase-4xλBoxBL 1 005 633 ± 35 402; 0.88 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL 1 145 467 ± 47 573; 1.00 pC3P3-G1, empty plasmid 10 685 ± 5 716; 0.01 Transfection reagent only 10 935 ± 3 918; 0.01

[0623] Noticeably, some differences in the activity of these proteins were observed depending on the types of mammalian cell lines tested. For example, a statistically significant efficacy has been observed with K3L from vaccinia virus using the C3P3-G1 and C3P3-G2 systems in monkey kidney cells COS-1 (increased expression of the Firefly Luciferase reporter gene of 1.15 and 1.28-fold with C3P3-G1 and C3P3-G2, respectively; P<0.01 and P<0.0001) and human hepatocellular carcinoma cells HepG2 (increased expression of the Firefly Luciferase reporter gene of 1.29- and 1.43-fold with C3P3-G1 and C3P3-G2, respectively, P<0.0001 for both comparisons). Similarly, a significant increase in expression was observed with DP71L (s) from African swine fever virus in human HeLa cells (increased expression of the Firefly Luciferase reporter gene of 1.12 and 1.26-fold with C3P3-G1 and C3P3-G2, respectively; P<0.05 and P<0.01) and monkey kidney cells COS-1 (increased expression of the Firefly Luciferase reporter gene of 1.30 and 1.42-fold with C3P3-G1 and C3P3-G2 systems, respectively; P<0.0001 for both comparisons). A statistically significant effect of LMP-1 from Epstein-Barr virus was also found with C3P3-G1 and C3P3-G2 systems in the human B myelomonocytic leukemia cell line MV-4-11 (increased expression of the Firefly Luciferase reporter gene of 1.32 and 1.58-fold with C3P3-G1 and C3P3-G2, respectively; P<0.0001 for both comparisons). These results are consistent with previous findings of other investigators, showing that the degree of biological activity of certain viral proteins interacting with the interferon response are dependent on that of the host-cell and therefore differ from one cell type and more generally from one species to another (Langland and Jacobs 2002, Park, Peng et al. 2020).

[0624] Conclusions

[0625] Experiments show that various viral or cellular proteins, as well as certain viral RNA sequences, involved in the type-I interferon response can significantly increase expression with the artificial C3P3 expression system. The best results were obtained with the long isoform of the E3L protein of vaccinia virus, which was selected for the following protein engineering shown in Example 4.

Example 4: An Artificial Protein Generated from E3L Protein Scaffold can Increase Expression Levels by the C3P3 System

[0626] Objectives

[0627] The aim of this series of experiments is to develop an artificial protein using the E3L protein of vaccinia virus as a scaffold, in order to further increase the levels of expression by the C3P3 system.

[0628] Methods

[0629] Plasmids

[0630] The pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously.

[0631] The vaccinia virus E3L protein contains two distinct domains: one Zα binding domain (also named Zα domain) at its amino-terminal extremity and a single dsRNA-binding domain at its carboxy-terminal end (FIG. 7A). These two domains, which are separated by a protein region with no distinct structure or function. In a first series of protein engineering, each of the domains of E3L were substituted by other domains having similar functional activities: [0632] The amino-terminal Zα binding domain was substituted by the Zα binding domain at amino-terminal end of human ADAR1 protein (UniProtKB/Uniprot accession number P55265; Zα domain from human ADAR1, fused to the dsRNA-binding domain from E3L of vaccinia virus, through a linker: pADAR1-Zα/(G4S)2/E3L-dsDNA (SEQ ID NO. 5 and SEQ ID NO. 6; pADAR1-Zα/(G4S)2/E3L-dsDNA plasmid; FIG. 7B), which contains two Zα binding domain in tandem (Schwartz, Rould et al. 1999) though a flexible (G4S)2 linker, [0633] The carboxyl-terminal dsRNA was substituted by several other dsRNA-binding domains from Influenza A virus NS1 protein (UniProtKB/Uniprot accession number P03496; SEQ ID NO. 7 and SEQ ID NO. 8; pE3L-Zα/NS1-dsDNA plasmid; FIG. 7C) (Bergmann, Garcia-Sastre et al. 2000), Flock House virus B2 protein (UniProtKB/Uniprot accession number P68831; SEQ ID NO. 9 and SEQ ID NO. 10; pE3L-Zα/B2-dsDNA plasmid; FIG. 7D) (Lingel, Simon et al. 2005), the amino-terminal region of human EIF2AK2 (UniProtKB/Uniprot accession number P19525; SEQ ID NO. 11 and SEQ ID NO. 12; pE3L-Zα/hEIF2AK2-dsDNA plasmid; FIG. 7E), which contains two dsRNA binding motifs separated by a short spacer (Patel and Sen 1992), and the orthoreovirus structural σ3 protein (UniProtKB/Uniprot accession number P07939; SEQ ID NO. 13 and SEQ ID NO. 14; pE3L-Zα/σ3-dsDNA plasmid; FIG. 7F) (Olland, Jane-Valbuena et al. 2001).

[0634] The E3L-Zα/NS1-dsDNA was selected from previous series of experiments and further optimized in a second series of protein engineering. Unlike the wild-type E3L protein, which can dimerize through its carboxyl-terminal region or even to form high order multimers at low ionic strength (Ho and Shuman 1996), the artificial protein E3L-Zα/NS1-dsDNA lacks known dimerization domain. To generate dimerization or multimerization of this protein, two different leucine zippers were introduced at the carboxy-terminal extremity of the artificial E3L-Zα/NS1-dsDNA protein: [0635] Super leucine zipper (sLZ), which can homodimerize in parallel orientation through its super long coiled coil helix (SEQ ID NO. 15 and SEQ ID NO. 16; pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP; FIG. 7G) (Harbury, Zhang et al. 1993, Harbury, Kim et al. 1994), [0636] GCN4-pVg leucine zipper, which can homotetramerize in antiparallel orientation (SEQ ID NO. 17 and SEQ ID NO. 18; pE3L-Zα/NS1-dsDNA/(G4S)2/GCN4; FIG. 7H) (Pack, Kujau et al. 1993, Pluckthun and Pack 1997).

[0637] Cell Culture and Transfection

[0638] Human cells were cultured and transfected as previously described.

[0639] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0640] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0641] Statistical Analysis

[0642] Statistical analyses were performed as previously described.

[0643] Results

[0644] The vaccinia virus E3L protein contains two distinct domains (FIG. 7A), which are both necessary to inhibit the interferon response (White and Jacobs 2012). First, its amino-terminal region (residues 5-70) contains a Z-DNA-binding domain. Second, the carboxyl-terminal region (residues 117-185) of E3L has typical dsRNA-binding domain, which binds to and sequesters as a homodimer dsRNA synthesized during viral infection (Ho and Shuman 1996). Such binding mask the dsRNA thereby preventing recognition and subsequent activation of EIF2AK2. Finally, a region with no distinct structure or function separates these two functional domains (residues 71-116).

[0645] The substitution of the amino-terminal Zα-binding domain of the wild-type E3L protein by the amino-terminal end of human ADAR1 protein, which contains two Zα binding domains, results in a functional protein that increased the expression of the Firefly Luciferase reporter, although at statistically lower level than that of the wild-type E3L protein (relative ratio of 1.74-fold vs. no test plasmid; P<0.0001).

[0646] This result is consistent with that of other authors who have shown that the existence of the Zα functional domain is essential for its biological activity of the E3L protein involved in the virulence of the vaccinia virus and could be substituted by the Zα binding domain from ADAR1 (Kim, Muralinath et al. 2003). The dsRNA-binding domain of E3L was then substituted by others from other human and viral proteins. Greatest increased expression levels of the Firefly Luciferase reporter gene were observed with two dsRNA-binding domain substitutions, i.e. NS1 protein from Influenza A virus and human EIF2AK2 (relative ratio of 3.11-fold and 3.02-fold vs. no test plasmid, respectively; P<0.0001 for both comparisons). In addition, the expression levels with these two test plasmids were statistically greater than with the wild-type E3L plasmid (relative ratio of 3.11-fold and 3.02-fold vs. 2.73, respectively; P<0.0001 for both comparisons). The two other substitutions by the B2 and σ3 dsRNA-binding domain were also functional, but to a lesser extent than the wild-type E3L plasmid.

[0647] To further optimize the E3L-Zα/NS1-dsDNA, this artificial protein was engineered by grafting leucine zippers at its carboxyl-terminal end through a flexible (G4S)2 linker. Leucine zippers are coiled-coil protein structures composed of two amphipathic α-helices that interact with each other and are commonly used to homo- or hetero-di/multimerize proteins (O'Shea, Klemm et al. 1991). Each helix consists of repeats of seven amino acids, in which the first amino-acid (residue a) is hydrophobic, the fourth (residue d) is usually a Leucine, while the other residues are polar. The super leucine zipper (sZIP), which can form homodimers in parallel orientation, increased significantly the expression of Firefly Luciferase in comparison to the E3L-Zα/NS1-dsDNA protein (relative ratio of 3.62-fold vs. 3.11-fold; P<0.0001). In contrast, the addition of the GCN4-pVg leucine zipper, which forms homotetramers in antiparallel orientation, had no detectable effects in comparison to the E3L-Zα/NS1-dsDNA protein (relative ratio of 3.01-fold vs. 3.11-fold; P=NS).

TABLE-US-00007 Firefly Luciferase (mean ± SD); P-value vs. Plasmids ratio pE3L-1 pC3P3-G2, pE3L-1, pK1E- 3 940 065 ± 76 802; 2.73 — Luciferase-4xλBoxBL pC3P3-G2, pADAR1-Zα/ 2 382 402 ± 71 147; 1.65 <0.0001 (G4S)2/E3L-dsDNA, pK1E- Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 4 494 430 ± 90 526; 3.11 <0.0001 NS1-dsDNA, pK1E- Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 2 518 407 ± 34 326; 1.74 <0.0001 B2-dsDNA, pK1E- Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 4 358 392 ± 89 250; 3.02 <0.0001 hEIF2AK2-dsDNA, pK1E- Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 3 132 092 ± 57 352; 2.17 <0.0001 σ3-dsDNA, pK1E- Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 5 236 071 ± 191 878; 3.62 <0.0001 NS1-dsDNA/(G4S)2/sZIP, pK1E-Luciferase-4xλBoxBL pC3P3-G2, pE3L-Zα/ 4 346 079 ± 179 900; 3.01 <0.0001 NS1-dsDNA/(G4S)2/GCN4, pK1E-Luciferase-4xλBoxBL pC3P3-G2, pK1E-Luciferase- 1 444 957 ± 49 361; 1.00 <0.0001 4xλBoxBL pC3P3-G2, empty plasmid 28 979 ± 527; 0.02 Transfection reagent only 17 326 ± 385; 0.01

[0648] Conclusions

[0649] These experiments shown that an artificial protein with greater activity than that of the long isoform of the E3L protein can be engineered. The artificial E3L-Zα/NS1-dsDNA/(G4S)2/sZIP protein was selected for further development.

Example 5: The Co-Expression of the Artificial E3L-Zα/NS1-dsDNA/(G4S)2/sZIP, Together with Other Proteins or RNA Sequences Tested in Example 3, can Even Increase Expression Levels by the C3P3 Expression System

[0650] Objectives

[0651] The objective of this series of experiments is to test the additivity of the coexpression of pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP with plasmids encoding other proteins or RNA sequences previously tested.

[0652] Methods

[0653] Plasmids

[0654] All plasmids were described in the above examples.

[0655] Cell Culture and Transfection

[0656] Human cells were cultured and transfected as previously described.

[0657] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0658] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0659] Statistical Analysis

[0660] Statistical analyses were performed as previously described.

[0661] Results

[0662] A possible additive effect on the expression of the reporter gene Firefly Luciferase expressed by the C3P3-G2 system was tested by co-transfection of the previous test plasmids, together with the plasmid pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP.

[0663] The results of the table below show successively the effect of each of the two plasmids transfected separately, then of the two plasmids co-transfected simultaneously. A supra-additive effect (SA) is defined as being at a statistically higher level of expression than by the simple addition of the effects tested separately, i.e. pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP on one hand and test plasmid on the other hand. The infra-additive effect (IA) and the strict additive effects (SA) were defined respectively as a statistically lower or non-different expression compared to the simple addition of the effects tested separately.

[0664] Supra-additive effect, was observed with plasmids encoding for the N(pro) from Bovine Viral Diarrhea Virus that target IRF3 (P<0.05), NSs from Rift Valley fever virus that promotes EIF2AK2 proteasomal degradation (P<0.001) and 5′UTR from Sindbis viral genome (P<0.001), together with the pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP plasmid.

[0665] Infra-additive (IA) or strict additive (SA) effects, which were defined as on a statistically lower or not different levels of expression than the addition of the individual effects, were observed with all other test plasmids.

TABLE-US-00008 Firefly Luciferase (mean ± SD); Plasmids relative ratio 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, VP40/MBV, pK1E-Luciferase-4xλBoxBL 2 001 557 ± 43 278; 1.80 1 + 2) pC3P3-G2, VP40/MBV, pE3L-Zα/ 3 472 301 ± 75 079; 3.13 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pEIF2AK2:K296R, pK1E-Luciferase-4xλBoxBL 2 924 887 ± 97 692; 2.64 1 + 2) pC3P3-G2, pEIF2AK2:K296R, pE3L-Zα/ 5 202 600 ± 173 769; 4.69 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pVP35/EBOV, pK1E-Luciferase-4xλBoxBL 2 120 069 ± 70 811; 1.91 1 + 2) pC3P3-G2, pVP35/EBOV, pE3L-Zα/ 3 679 190 ± 122 886; 3.32 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pV/PIV5, pK1E-Luciferase-4xλBoxBL 2 031 546 ± 43 435; 1.83 1 + 2) pC3P3-G2, pV/PIV5, pE3L-Zα/ 4 000 943 ± 85 541; 3.60 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pPPP1R15A, pK1E-Luciferase-4xλBoxBL 2 642 205 ± 37 360; 2.38 1 + 2) pC3P3-G2, pPPP1R15A, pE3L-Zα/ 5 670 692 ± 80 182; 5.11 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pNSs/RVFV, pK1E-Luciferase-4xλBoxBL 3 333 228 ± 115 029; 3.00 1 + 2) pC3P3-G2, pNSs/RVFV, pE3L-Zα/ 9 000 800 ± 310 617; 8.11 (SA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pN(pro)/BVDV, pK1E-Luciferase-4xλBoxBL 2 208 016 ± 69 286; 1.99 1 + 2) pC3P3-G2, pN(pro)/BVDV, pE3L-Zα/ 6 649 423 ± 208 654; 5.99 (SA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pK3L/VV, pK1E-Luciferase-4xλBoxBL 1 976 221 ± 66 730; 1.78 1 + 2) pC3P3-G2, pK3L/VV, pE3L-Zα/ 2 885 041 ± 97 417; 2.60 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pK1Ep-5′UTR/SINV-Luciferase-4xλBoxBL 3 257 540 ± 91 760; 2.94 1 + 2) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/LZ, 9 017 711 ± 254 016; 8.13 (SA) pK1Ep-5′UTR/SINV-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pICP34.5/HVS1, pK1E-Luciferase-4xλBoxBL 2 568 071 ± 104 161; 2.31 1 + 2) pC3P3-G2, pICP34.5/HVS1, pE3L-Zα/ 6 875 963 ± 278 889; 6.20 (SA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pDP71L(s)/ASFV, pK1E-Luciferase-4xλBoxBL 1 438 249 ± 60 141; 1.30 1 + 2) pC3P3-G2, pDP71L(s)/ASFV, pE3L-Zα/ 4 253 506 ± 177 863; 3.83 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, pB18R/VV, pK1E-Luciferase-4xλBoxBL 1 600 590 ± 35 496; 1.44 1 + 2) pC3P3-G2, pB18R/VV, pE3L-Zα/ 4 245 040 ± 94 141; 3.82 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, NS1/RSV, pK1E-Luciferase-4xλBoxBL 1 531 635 ± 55 469; 1.38 1 + 2) pC3P3-G2, NS1/RSV, pE3L-Zα/ 4 142 101 ± 150 009; 3.73 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, LMP-1/EBV, pK1E-Luciferase-4xλBoxBL 1 818 683 ± 59 488; 1.64 1 + 2) pC3P3-G2, LMP-1/EBV, pE3L-Zα/ 4 209 742 ± 137 698; 3.79 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL 1) pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 4 085 116 ± 149 701; 3.68 pK1E-Luciferase-4xλBoxBL 2) pC3P3-G2, μ2/REOV, pK1E-Luciferase-4xλBoxBL 1 669 720 ± 58 025; 1.50 1 + 2) pC3P3-G2, μ2/REOV, pE3L-Zα/ 3 317 346 ± 115 282; 2.99 (IA) NS1-dsDNA/(G4S)2/LZ, pK1E-Luciferase-4xλBoxBL pC3P3-G2, pK1E-Luciferase-4xλBoxBL 1 109 854 ± 8 750; 1.00 pC3P3-G2, empty plasmid 13 499 ± 228; 0.01

[0666] Conclusions

[0667] These results show synergistic effects between the E3L-Zα/NS1-dsDNA/(G4S)2/sZIP protein and the Nss and N(pro) proteins, as well as with the sequence 5′UTR of the Sindbis virus genome, on the expression of the Firefly Luciferase reporter gene expressed with the C3P3-G2 system.

Example 6: Assembly of the Artificial E3L-Zα/NS1-dsDNA/(G4S)2/sZIP within the C3P3 Enzyme Results in Active Polyproteins

[0668] Objectives

[0669] The objective of the following experiment was to assemble in frame the artificial E3L-Zα/NS1-dsDNA/(G4S)2/sZIP within the open-reading frame of the C3P3-G2 enzyme.

[0670] Methods

[0671] Plasmids

[0672] The assemblies tested hereinafter are designed according to two protein scaffolds: [0673] either Nλ-mPAPOLA-[X1]-E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-[X2]-NP868R-(G4S)2-K1ERNAP, [0674] or E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-[X3]-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP,
where [X1], [X2] and [X3] are variable. Each of the [X] positions correspond either to a (G4S)2 flexible linker or to an F2A ribosomal skipping motif.

[0675] The resulting proteins were named C3P3-G3x, where is the numbering of the construction: [0676] C3P3-G3a, where [X1]=F2A and [X2]=F2A (SEQ ID NO. 19 and SEQ ID NO. 20; FIG. 8C), i.e. Nλ-mPAPOLA-F2A-E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-F2A-NP868R-(G4S)2-K1ERNAP, [0677] C3P3-G3b, where [X1]=(G4S)2 and [X2]=F2A (SEQ ID NO. 21 and SEQ ID NO. 22; FIG. 8D), i.e. Nλ-mPAPOLA-(G4S)2-E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-F2A-NP868R-(G4S)2-K1ERNAP, [0678] C3P3-G3c, where [X1]=F2A and [X2]=(G4S)2 (SEQ ID NO. 23 and SEQ ID NO. 24; FIG. 8E), i.e. Nλ-mPAPOLA-F2A-E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-(G4S)2-NP868R-(G4S)2-K1ERNAP [0679] C3P3-G3d, where [X3]=F2A (SEQ ID NO. 25 and SEQ ID NO. 26; FIG. 8F), i.e. E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-F2A-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP), [0680] C3P3-G3e, where [X3]=(G4S)2 (SEQ ID NO. 27 and SEQ ID NO. 28; FIG. 8G), i.e. E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-(G4S)2-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP.

[0681] Cell Culture and Transfection

[0682] Human cells were cultured and transfected as previously described.

[0683] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0684] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0685] Statistical Analysis

[0686] Statistical analyses were performed as previously described.

[0687] Results

[0688] The E3L-Zα/NS1-dsDNA/(G4S)2/SZIP coding sequence was inserted in-frame into the open-reading frame of the C3P3-G2 enzyme. The E3L-Zα/NS1-dsDNA/(G4S)2/SZIP coding sequence could be readily inserted at only two positions: either within the coding sequence of C3P3 between the Nλ-mPAPOLA and NP868R-(G4S)2-K1ERNAP blocks (C3P3-G3a, C3P3-G3b, and C3P3-G3c), or at the start of the ORF immediately before the Nλ-mPAPOLA block (C3P3-G3d and C3P3-G3e). In contrast, in-frame insertions of E3L-Zα/NS1-dsDNA/(G4S)2/SZIP at the end of the coding sequence of the C3P3 enzyme was not tested because phage RNA polymerases such as K1ERNAP do not tolerate carboxyl-terminal extensions (Mookhtiar, Peluso et al. 1991, Gardner, Mookhtiar et al. 1997).

[0689] Two types of intervening sequences have been used, i.e. (G4S)2 and F2A. The (Gly4Ser)n linkers (where n indicates the number of repeats) are prototypes of flexible protein linkers for appropriate separation of the functional domains (Huston, Levinson et al. 1988). The 2A are protein sequence of viral origin, which causes ribosomal skipping during translation, which thereby result in apparent co-translational cleavage of the protein (Donnelly, Luke et al. 2001). The F2A sequence, which was used for present constructions, is from the Foot-and-mouth disease aphtovirus (UniProtKB/Uniprot accession number AAT01756, residues 934-955).

[0690] The greatest effect was observed with the CP3P-G3a plasmid (Nλ-mPAPOLA-F2A-E3L-Zα/NS1-dsDNA/2A/sZIP-F2A-NP868R-(G4S)2-K1ERNAP). This construction is characterized by an in-frame insertion of E3L-Zα/NS1-dsDNA coding sequence in the open-reading frame of C3P3-G2, flanked by two F2A motifs, thereby producing a polyprotein consisting of three distinct subunits. The expression levels observed in cells transfected with pC3P3a were statistically significantly higher than those obtained by co-transfection of pC3P3-G2 and pE3L-Zα/NS1-dsDNA (relative ratio of 3.73-fold vs. 3.50-fold; P<0.0001).

[0691] The C3P3-G3d construction (E3L-Zα/NS1-dsDNA/(G4S)2/sZIP-F2A-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP), where the coding sequence of E3L-Zα/NS1-dsDNA/(G4S)2/sZIP is inserted at the start of the C3P3 ORF and flanked by a F2A motif gave similar expression level to that of cells co-transfection of pC3P3-G2 and pE3L-Zα/NS1-dsDNA (relative ratio of 3.51-fold vs. 3.49-fold; P=NS). Other constructions were still functional but to a lesser extent than the E3L-Zα/NS1-dsDNA/(G4S)2/sZIP coding sequence not inserted in the C3P3a open-reading frame. Finally, the performances of these constructions ranged in the following order: C3P3-G3a>C3P3-G3d>C3P3-G3c≈C3P3-G3b>C3P3-G3e.

TABLE-US-00009 Firefly Luciferase (mean ± SD); Plasmids relative ratio pC3P3-G2, pE3L-Zα/NS1-dsDNA/(G4S)2/sZIP, 5 058 433 ± 180 056; 3.49 pK1E-Luciferase-4xλBoxBL pC3P3-G3a, pK1E-Luciferase-4xλBoxBL 5 399 055 ± 142 014; 3.73 pC3P3-G3b, pK1E-Luciferase-4xλBoxBL 4 391 463 ± 176 880; 3.03 pC3P3-G3c, pK1E-Luciferase-4xλBoxBL 4 398 126 ± 156 526; 3.04 pC3P3-G3d, pK1E-Luciferase-4xλBoxBL 5 074 674 ± 133 124; 3.51 pC3P3-G3e, pK1E-Luciferase-4xλBoxBL 4 192 816 ± 61 479; 2.90 pC3P3-G2, pK1E-Luciferase-4xλBoxBL 1 447 470 ± 32 976; 1.00 pC3P3-G2, empty plasmid 28 820 ± 1 143; 0.02 Transfection reagent only 17 050 ± 171; 0.01

[0692] Conclusions

[0693] The E3L-Zα/NS1-dsDNA/(G4S)2/SZIP coding sequence could be efficiently inserted in-frame into the scaffold of the C3P3-G2 enzyme, the C3P3-G3a construction having the best performance.

Example 7: The dsRNA-Binding Domain of EIF2AK2 from Different Species can Increase Expression Levels by the C3P3-G2 and C3P3-G1 Systems

[0694] Objectives

[0695] This experiment has a goal of developing new artificial protein inhibitors of EIF2αphosphorylation. We thought that the dsRNA binding domain of EIF2AK2 deleted from its carboxy-terminal kinase domain could act as an inhibitor by dimerizing with the full-length wild-type EIF2AK2 protein. The resulting dimer is therefore possibly capable of trapping dsRNA, which in turn activates wild-type EIF2AK2. Moreover, due to the absence of a kinase domain, this dimer is likely to have reduced or no phosphorylation activity of its molecular target eIF2α.

[0696] Methods

[0697] Plasmids

[0698] The pC3P3-G1, pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously. EIF2AK2 (eukaryotic translation initiation factor 2-alpha kinase 2 also known as Protein kinase RNA-activated (PKR); human protein UniProtKB/Uniprot accession number P19525) has two functional domains which are separated by a region with no distinct structure or function: [0699] a. N-terminal dsRNA binding domain (dsRBD), which consists of two tandem copies of a conserved double stranded RNA binding motif, dsRBM1 and dsRBM2 (residues 100-167). This domain is capable of homodimerizing and binding to dsRNA (Zhang, Romano et al. 2001), [0700] b. C-terminal serine/threonine kinase domain, capable of autophosphorylating, then phosphorylating eIF2α, after its homodimerization (Zhang, Romano et al. 2001, Dey, Mann et al. 2014).

[0701] We hypothesized that the isolated dsRNA domain of EIF2AK2 without a kinase domain can act as an efficient competitive inhibitor. Also note that the relatively small size of this domain is well suited for the construction of C3P3 enzymes as shown in Example 9

[0702] The following mutant EIF2AK2 proteins consisting only of the dsRNA-binding domain were tested: [0703] Human EIF2AK2 dsRNA-binding domain (UniProtKB/Uniprot accession number P19525, residues 2-167; phEIF2AK2:DRB), [0704] Mouse EIF2AK2 dsRNA-binding domain (UniProtKB/Uniprot accession number Q03963, residues 2-162; pmEIF2AK2:DRB), [0705] Bovine EIF2AK2 dsRNA-binding domain (UniProtKB/Uniprot accession number A0A4W2CP11 residues 2-167; pbEIF2AK2:DRB).

[0706] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0707] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0708] Statistical Analysis

[0709] Statistical analyses were performed as previously described.

[0710] Results

[0711] The dsRNA binding domains of EIF2AK2 from different species, deleted of their carboxyl-terminal domains, were tested. Coexpression of all these proteins increased the level of expression of the Firefly Luciferase reporter gene expressed with the C3P3-G1 system. The effects observed were in the following order: human>mouse≈bovine.

TABLE-US-00010 Firefly Luciferase (mean ± SD); Plasmids relative ratio P-value pC3P3-G1, K1Ep(G)-Luciferase, phEIF2AK2:DRB 1 575 067 ± 41 527; 1.37 <0.0001 pC3P3-G1, K1Ep(G)-Luciferase, pmEIF2AK2:DRB 1 373 402 ± 43 643; 1.20 <0.0001 pC3P3-G1, K1Ep(G)-Luciferase, pbEIF2AK2:DRB 1 363 178 ± 78 703; 1.19 <0.0001 pC3P3-G1, pK1E-Luciferase-4xλBoxBL 1 147 342 ± 53 282; 1.00 NA pC3P3-G1, empty plasmid 9 247 ± 239; 0.01 Transfection reagent only 5 581 ± 188; 0.01

[0712] A similar effect was observed by the coexpression of these dsRNA binding domains of EIF2AK2 from several species, but more marked with the second generation of the artificial expression system. The dsRNA binding domains from the human EIF2AK2 has the greatest effects.

TABLE-US-00011 Firefly Luciferase (mean ± SD); Plasmids relative ratio P-value pC3P3-G2, K1Ep(G)-Luciferase, phEIF2AK2:DRB 3 788 243 ± 99 879; 1.78 <0.0001 pC3P3-G2, K1Ep(G)-Luciferase, pmEIF2AK2:DRB 3 188 517 ± 101 322; 1.50 <0.0001 pC3P3-G2, K1Ep(G)-Luciferase, pbEIF2AK2:DRB 3 164 781 ± 182 719; 1.49 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL 2 130 953 ± 98 961; 1.00 NA pC3P3-G2, empty plasmid 12 245 ± 239; 0.01 Transfection reagent only 8 721 ± 188; 0.01

[0713] Conclusions

[0714] dsRNA binding domains of EIF2AK2 can increase the expression of the reporter gene under the control of both the first and the second generation of the artificial expression system C3P3, which supports a dominant negative effect by competitive inhibition. The strongest effect was observed with human protein which has been selected for the development of a new artificial protein capable of inhibiting the phosphorylation of eIF2α.

[0715] Such dsRNA binding domains of EIF2AK2 can also be used to drive specifically its ubiquitination by E3 ligases and thereby their degradation by the 28S proteasome. To implement this mechanism of action, the present inventor has designed chimeric proteins resulting of the fusion of wild-type and mutant dsRNA binding domains of EIF2AK2 with specific subunit domains of multimeric E3 ligases, especially Skp1-interacting domains from F-box proteins (e.g. BTRCP, FBW7 or SPK2), elongin BC-interacting domains (e.g. VHL or SOCS2), Cullin3-interacting domains from SPOP, DDB1-interacting domains from CRBN, dimerization domain from STUB1 (also named CHIP) or CUL1-interacting domain from Skp1.

Example 8: The Co-Expression of the dsRNA-Binding Domain of EIF2AK2 with Proteins Involved in eIF2α Dephosphorylation, can Even Increase Expression Levels by the C3P3-G1 and C3P3-G2 Expression Systems

[0716] Objectives

[0717] The aim of this experiment is to test whether a potentiation of the effect of the dsRNA-binding domain of EIF2AK2 with other protein factors. Due to the mechanism of action of dsRNA-binding domain of EIF2AK2 which makes it possible to repress the phosphorylation of eIF2α, we were particularly interested to the reverse modification pathway, namely proteins involved in dephosphorylation of eIF2α.

[0718] Methods

[0719] Plasmids

[0720] The pC3P3-G1, pC3P3-G2 and pK1Ep-Luciferase-4xλBoxBr plasmids were described previously. Viral and host-cell gene involved in eIF2α dephosphorylation were subcloned in the pCMVScript plasmid backbone (Stratagene, La Jolla, Calif.), as previously described, most of which were previously described: [0721] pPPP1CA plasmid, which encodes the human serine/threonine-protein phosphatase PP1-alpha catalytic subunit (UniProtKB/Uniprot accession number P62136) that dephosphorylates eIF2α, [0722] pPPP1R15A plasmid, which encodes a regulatory subunit that recruits the serine/threonine-protein phosphatase PPP1CA to dephosphorylate eIF2α (UniProtKB/Uniprot accession number O75807), [0723] pICP34.5/HVS1 plasmid, which encodes the Herpes Simplex Virus ICP34.5 protein that serves as a regulatory subunit of protein phosphatase PPP1CA (UniProtKB/Uniprot accession number P03496), [0724] Plasmids pDP71L(s)/ASFV and pDP71L(I)/ASFV, which respectively encode the short (UniProtKB/Uniprot accession number Q65212) and long (UniProtKB/Uniprot accession number P0C755) isoforms of African swine fever virus DP71L protein, both of which regulatory subunit of protein phosphatase PPP1CA.

[0725] Cell Culture and Transfection

[0726] Human cells were cultured and transfected as previously described.

[0727] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0728] Firefly luciferase luminescence was assayed from cell lysate as previously described.

[0729] Statistical Analysis

[0730] Statistical analyses were performed as previously described.

[0731] Results

[0732] A possible additive effect on the expression of the reporter gene Firefly Luciferase expressed by the

[0733] C3P3 system was tested by co-transfection of the previous test plasmids, together with the plasmid phEIF2AK2:DRB.

[0734] The results of the table below show successively the effect of each of the two plasmids transfected separately, then of the two plasmids co-transfected simultaneously. Supra-additive effect (SA), infra-additive (IA) or purely additive (PA) effects were statistically defined as previously described in Example 5.

[0735] Surprisingly, we discovered supra-additive effect with all the plasmids coding for proteins involved in the eIF2α dephosphorylation pathway listed above. The efficacy on the expression of the reporter gene Firefly Luciferase was observed in the following order: pDP71L(I)/ASFV (ratio 4.54; P<0.001)>pPPP1CA (ratio 4.46; P<0.001)>pPPP1R15A (ratio 4.39; P<0.001) pICP34.5/HVS1 (ratio 4.36; P<0.05)>>pDP71L(s)/ASFV (ratio 3.51; P<0.001).

[0736] DP71L(I) encoding the long isoform of African swine fever virus DP71L protein was therefore used for construction of the new generation of C3P3-G3 enzymes shown in Example 9.

TABLE-US-00012 Firefly Luciferase (mean ± SD); relative ratio Plasmids (additivity, P-value) 1) pC3P3-G2, phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL 3 831 026 ± 102 718; 1.75 2) pC3P3-G2, pPPP1CA, pK1E-Luciferase-4xλBoxBL 5 040 224 ± 137 764; 2.30 1 + 2) pC3P3-G2, phEIF2AK2:DRB, pPPPICA, 9 796 322 ± 241 884; 4.46 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL (SA, P < 0.001) 1) pC3P3-G2, phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL 3 831 026 ± 102 718; 1.75 2) pC3P3-G2, pPPP1R15A, pK1E-Luciferase-4xλBoxBL 4 990 197 ± 94 700; 2.27 1 + 2) pC3P3-G2, phEIF2AK2:DRB, pPPP1R15A, 9 627 003 ± 330 477; 4.39 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL (SA; P < 0.001) 1) pC3P3-G2, phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL 3 831 026 ± 102 718; 1.75 2) pC3P3-G2, pICP34.5/HVS1, pK1E-Luciferase-4xλBoxBL 4 893 675 ± 172 443; 2.23 1 + 2) pC3P3-G2, phEIF2AK2:DRB, pICP34.5/HVS1, 9 573 417 ± 413 280; 4.36 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL (SA; P < 0.05) 1) pC3P3-G2, phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL 3 831 026 ± 102 718; 1.75 2) pC3P3-G2, pDP71L(s)/ASFV, pK1E-Luciferase-4xλBoxBL 2 719 144 ± 68 774; 1.24 1 + 2) pC3P3-G2, phEIF2AK2:DRB, pDP71L(s)/ASFV, 7 694 041 ± 281 447; 3.51 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL (SA; P < 0.001) 1) pC3P3-G2, phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL 3 831 026 ± 102 718; 1.75 2) pC3P3-G2, pDP71L(I)/ASFV, pK1E-Luciferase-4xλBoxBL 4 837 262 ± 279 279; 2.20 1 + 2) pC3P3-G2, phEIF2AK2:DRB, pDP71L(I)/ASFV, 9 954 686 ± 292 508; 4.54 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL (SA; P < 0.0001) pC3P3-G2, pK1E-Luciferase-4xλBoxBL 2 194 881 ± 52 197 pC3P3-G2, empty plasmid 17 431 ± 403 Transfection reagent only 21 096 ± 870

[0737] Conclusions

[0738] These results therefore demonstrate the supra-additivity effect between the dsRNA-binding domain from hEIF2AK2 and all genes involved the eIF2α dephosphorylation pathway.

Example 9: Assembly of New Generation C3P3 Enzymes

[0739] Objectives

[0740] The objective of the following experiment was to assemble in frame the genes identified from Example 8 within the open-reading frame of the C3P3-G2 enzyme. The resulting C3P3 genes are numbered C3P3-G3f to C3P3-G3i.

[0741] Methods

[0742] Plasmids

[0743] The assemblies tested hereinafter are designed according to two scaffolds: [0744] hEIF2AK2:DRB-[X1]DP71L(I)-F2A-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP, where [X1] is either a (G4S)2 flexible linker (C3P3-G3f, SEQ ID NO. 35 and SEQ ID NO. 36; FIG. 9A) or an F2A ribosomal skipping motif (C3P3-G3g, SEQ ID NO. 37 and SEQ ID NO. 38; FIG. 9B), [0745] Nλ-mPAPOLA-F2A-hEIF2AK2:DRB-[X2]-DP71L(I)]-F2A-NP868R-(G4S)2-K1ERNAP, where [X2] is either a (G4S)2 flexible linker (C3P3-G3h, SEQ ID NO. 39 and SEQ ID NO. 40; FIG. 9C) or an F2A ribosomal skipping motif (C3P3-G3i, SEQ ID NO. 41 and SEQ ID NO. 42; FIG. 9D).

[0746] Firefly Luciferase Luminescence and SEAP Colorimetric Assays

[0747] Results

[0748] The coding sequence hEIF2AK2:DRB-X-DP71L(I) was inserted in-frame into the open-reading frame of the C3P3-G2 enzyme, where X is an intervening sequence (i.e. either the flexible (Gly4Ser)2 linker, or the 2A are protein sequence, which causes ribosomal skipping). The hEIF2AK2:DRB-X-DP71L(I) coding sequence could be readily inserted at only two positions: either within the coding sequence of C3P3 either at the start of the ORF immediately before the Nλ-mPAPOLA block (C3P3-G3f and C3P3-G3g), or between the Nλ-mPAPOLA and NP868R-(G4S)2-K1ERNAP blocks (C3P3-G3h, C3P3-G3i). As previously stated in Example 6, it was not possible to position the hEIF2AK2:DRB-X-DP71L(I) block at the carboxyl-terminal end of the C3P3 protein because phage RNA polymerases such as K1ERNAP do not tolerate carboxyl-terminal extensions (Mookhtiar, Peluso et al. 1991, Gardner, Mookhtiar et al. 1997). As shown in the table below, all constructions were functional. The efficacy of the C3P3 enzymes on the expression of the reporter gene Firefly Luciferase was observed in the following order: C3P3-G2f (ratio 5.47 vs. C3P3-G2 expression system)>C3P3-G2g (ratio 5.22)>C3P3-G2h (ratio 4.77)>C3P3-G2i (ratio 4.52).

[0749] The best results were therefore obtained when the hEIF2AK2:DRB-X-DP71L(I) block was inserted at the start of the protein before the Nλ-mPAPOLA block (C3P3-G3f and C3P3-G3g), rather than between the Nλ-mPAPOLA and NP868R-(G4S)2-K1ERNAP blocks (C3P3-G3h and C3P3-G3i). In addition, better results were apparently obtained when flexible (Gly4Ser)2 linker used as intervening sequences between hEIF2AK2:DRB and DP71L(I) sequences rather than the 2A ribosome skipping sequences. Finally, the C3P3-G3f construction which gave the best performance was selected that has the following design: EIF2AK2:dsDNA-(G4S)2-DP71L(I)-F2A-Nλ-mPAPOLA-F2A-NP868R-(G4S)2-K1ERNAP.

TABLE-US-00013 Firefly Luciferase (mean ± SD); Plasmids relative ratio P-value pC3P3-G2, phEIF2AK2:DRB, pDP71L(I)/ASFV, 9 229 953 ± 291 877; 4.64 <0.0001 phEIF2AK2:DRB, pK1E-Luciferase-4xλBoxBL C3P3-G3f, pK1E-Luciferase-4xλBoxBL 10 885 745 ± 352 233; 5.47 <0.0001 C3P3-G3g, pK1E-Luciferase-4xλBoxBL 10 389 482 ± 318 248; 5.22 <0.0001 C3P3-G3h, pK1E-Luciferase-4xλBoxBL 9 491 167 ± 307 109; 4.77 <0.0001 C3P3-G3i, pK1E-Luciferase-4xλBoxBL 8 996 018 ± 275 564; 4.52 <0.0001 pC3P3-G2, pK1E-Luciferase-4xλBoxBL 1 988 881 ± 47 298; 1.00 NA pC3P3-G2, empty plasmid 19 116 ± 788 Transfection reagent only 15 795 ± 365

[0750] Conclusions

[0751] The hEIF2AK2:DRB-X-DP71L(I) coding sequence could be efficiently inserted in-frame into the scaffold of the C3P3-G2 enzyme, the C3P3-G3f construction having the best performance.

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