Self-cleaving Sequence Research Articles

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

Immunogenicity of Varicella Zoster Virus DNA Vaccines Encoding Glycoprotein E and Immediate Early Protein 63 in Mice.

Herpes zoster (HZ) is caused by the reactivation of latent varicella-zoster virus (VZV) from the sensory ganglia due to aging or immunosuppression. Glycoprotein E (gE) is a widely used vaccine antigen for specific humoral and cellular immune responses. Immediate early protein 63 (IE63) is expressed during latency, suggesting that it is a potential antigen against HZ reactivation. In this study, HZ DNA vaccines encoding gE, IE63, IE63-2A-gE (where 2A is a self-cleaving sequence), or IE63-linker-gE were developed and investigated for immunogenicity in mice. The results showed that each HZ DNA vaccine induced VZV-specific antibody production. The neutralizing antibody titer elicited by IE63-2A-gE was comparable to that elicited by gE or live attenuated HZ vaccine (LAV). IE63-2A-gE-induced gE or IE63-specific INF-γ+ T cell frequencies in splenocytes were comparable to those of LAV. Furthermore, IE63-2A-gE, gE, or IE63 led to a significant increase in IFN-γ (IE63 stimulation) and IL-2 (gE stimulation) secretion compared to LAV, showing a Th1-biased immune response. Moreover, IE63-2A-gE and gE induced cytotoxic activity of CD8+ T cells compared to that of LAV. This study elucidates that the IE63-2A-gE DNA vaccine can induce both humoral and cell-mediated immune responses, which provides a candidate for the development of an HZ vaccine.

Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga-Euglena gracilis.

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A–gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.

Development of an experimental method of systematically estimating protein expression limits in HEK293 cells

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins’ expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.

Development of a “deconstructed” vector based on the genome of grapevine virus A

The development of new vectors based on plant viruses creates more efficient vectors driving high expression levels of heterologous proteins. Currently, deconstructed vectors are more suitable for the expression of target genes. We attempted to develop a deconstructed vector based on the genome of grapevine virus A (GVA) by replacing the coat protein gene of GVA to the coat protein gene (aCP) of ACLSV (apple chlorotic leaf spot virus). This is the first work on creation of deconstructed vector based on GVA genome. The heterologous gene was placed under the control of the CP subgenomic promoter of GVA and fused with ORF5. The 2A self-cleaving sequence was inserted between the target protein and the protein encoded by ORF5 to enable the expression of separate proteins. The transient expression of coat protein of ACLSV from this vector was assessed in both transgenic plants carrying the CP of GVA and in non-transgenic plants. The level of aCP expression in transgenic plants was higher than the expression level in non-transgenic plants. Transgenic plants were developed in this study to increase target protein yield, because GVA cannot move between cells in its non-encapsidated form. The developed vector based on the grapevine virus A genome can be used in transient expression of different heterologous proteins utilizing magnifection method or via co-agroinfiltration with native genome of GVA.

3170 - Variation in HoxA9 Expression in the NUP98-HOXD13 Myelodysplasia Model, Using a Novel HoxA9-eGFP Reporter Mouse

HoxA9 is normally expressed in haematopoietic stem and progenitor cells of humans and of mice, but expression ceases in differentiated cells. Many patients with a variety of myeloid and lymphoid malignancies overexpress HoxA9 in their disease cells, a fact reflected in many mouse models of these diseases. HoxA9 overexpression is thought to drive a differentiation block or abnormal self-renewal in these cells, promoting the oncogenic phenotype. We sought to produce a transgenic mouse that reports expression of the HoxA9 gene from the endogenous locus via co-expression of a fluorescent marker, in hopes that this might facilitate the better understanding of the origins of disease. We designed a targeting strategy using CRISPR-Cas9 technology, inserting an eGFP cassette immediately before the endogenous HoxA9 stop codon, separated from the HoxA9 coding sequence by a P2A self-cleaving sequence. We used this strategy to generate transgenic mice and found that the resultant mice contained the expected sequence and were developmentally normal and fertile. We characterised the expression of eGFP in the haematopoietic system of 6 week old mice using flow cytometry and found that, as expected, mature blood cells were eGFP negative while within the stem and progenitor compartments there were consistent, discrete HoxA9-positive and -negative compartments. We sorted these cells and confirmed by RQ-PCR analysis that eGFP expression did indeed correlate with expression of HoxA9. We subsequently crossed these mice with NUP98-HOXD13 transgenic mice, an established model of MDS characterised by HoxA9 overexpression in progenitor and mature cells, and found a markedly higher percentage of eGFP positive stem and progenitor cells, and that the positive cells were also markedly brighter. eGFP expression levels within the stem and progenitor compartments were more variable in the NHD13 mice, perhaps suggesting variable growth of a clonal population. As expected, we also observed expression of eGFP in certain mature cell compartments. These mice represent a valuable tool for studying the role of HoxA9 in haematopoiesis.

Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG.

Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii

Fluorescent proteins (FPs) have become essential tools for a growing number of fields in biology. However, such tools have not been widely adopted for use in microalgal research. The aim of this study was to express and compare six FPs (blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato and red mCherry) in the popular model microalga Chlamydomonas reinhardtii. To circumvent the transgene silencing that often occurs in C.reinhardtii, the FPs were expressed from the nuclear genome as transcriptional fusions with the sh-ble antibiotic resistance gene, with the foot and mouth disease virus 2A self-cleaving sequence placed between the coding sequences. All ble-2A-FPs tested are well-expressed and efficiently processed to yield mature, unfused FPs that localize throughout the cytoplasm. The fluorescence signals of each FP were detectable in whole cells by fluorescence microplate reader analysis, live-cell fluorescence microscopy, and flow cytometry. Furthermore, we report a comparative analysis of fluorescence levels relative to auto-fluorescence for the chosen FPs. Finally, we demonstrate that the ble-2A expression vector may be used to fluorescently label an endogenous protein (α-tubulin). We show that the mCerulean-α-tubulin fusion protein localizes to the cytoskeleton and flagella, as expected, and that cells containing this fusion protein had normal cellular function. Overall, our results indicate that, by use of the ble-2A nuclear expression construct, a wide array of FP tools and technologies may be applied to microalgal research, opening up many possibilities for microalgal biology and biotechnology.

Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice

Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-1,4-xylanase activities) from mollusca, Ampullaria crossean and a β-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.

Coexpression of double or triple copies of the rabies virus glycoprotein gene using a ‘self-cleaving’ 2A peptide-based replication-defective human adenovirus serotype 5 vector

Rabies virus glycoprotein (RVG) is a major structural protein and antigen of rabies virus that induces a highly immunogenic response. In the present study, we have used 2A self-cleaving sequence of the foot-and-mouth disease virus (FMDV) to express double or triple copies of the RVG from a single open reading frame derived from human adenovirus 5 (AdHu5). The recombinant adenoviruses produce similar virus titers, indicating that the insertion of double or triple copies of the RVG gene linked with the FMDV 2A sequence does not affect virus replication. The RVG was efficiently expressed by constructs containing the 2A sequence and retained its antigenic property. The 2A self-cleaving peptide mediated efficient generation of individual glycoprotein in transient expression assay and did not lead to an altered surface distribution of RVG. Flow cytometry demonstrated that the expression levels of RVG were improved in recombinant Ads carrying multiple RVG gene copies. We conclude that ribosome skipping induced by the FMDV 2A sequence is an effective strategy to express multiple glycoprotein genes of rabies virus in adenoviruses and 2A-containing recombinant Ads may represent an attractive alternative to other coexpression strategies for multiple gene expression.

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-beta1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. In pTriEx2-transfected LX-2 cells, TGF-beta1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-beta1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. Endogenous CCN2 is a mediator of basal or TGF-beta1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode.

A comparative analysis of 40 Trypanosoma cruzi L1Tc elements showed that the 2A self-cleaving sequence described in viruses is present in them. Of these elements, 72% maintain the canonical 2A motif (DxExNPGP). A high percentage has a conserved point mutation within the motif that has not been previously described. In vitro and in vivo expression of reporter polyproteins showed that the L1Tc2A sequence is functional. Mutations within certain L1Tc2A sequences affect the efficiency of the cleavage. The data indicate that the L1Tc2A sequence may be influencing the L1Tc enzymatic machinery determining the composition and level of the translated products. The residues located immediately upstream of the 2A consensus sequence increase the cleaving efficiency and appear to stabilize the relative amount of translated products.

Self-cleaving ribozymes of hepatitis delta virus RNA.

Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The self-cleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysis has not yet been proven. A minimal natural ribozyme sequence with proficient in vitro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (P1-P4). The two pairings that define the 5' and 3' boundaries of the ribozyme, P1 and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of P1, is in close proximity to three single-stranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

Hepatitis delta virus ribozymes fold to generate a solvent-inaccessible core with essential nucleotides near the cleavage site phosphate.

Self-cleaving sequences or ribozymes from the hepatitis delta virus (HDV) genomic RNA and its complement form similar secondary structures that suggest a core region and potential active site composed of "single-stranded" sequences. However, there is little data on tertiary interactions in these ribozymes, therefore structural features were investigated using cross-linking and hydroxyl radical cleavage. Cross-links in cis and trans forms of the antigenomic RNA were generated using the photoactivatable azidophenacyl group tethered to the cleavage site phosphate. Specific cross-links formed to J4/2, and to the 3' sides of P3 and L3. Different sites were cross-linked in low salt or monovalent cations versus divalent cations, suggesting a metal ion-dependent conformational change near the cleavage site. The solvent-inaccessible regions of both the genomic and antigenomic ribozymes were revealed by cleavage in Fe(II)-EDTA. In Mg2+, backbone segments most strongly protected from solvent-based hydroxyl radicals were mapped to J4/2 and parts of L3. Similar patterns of protection were seen in trans-acting ribozymes bound to a product oligonucleotide. These data provide evidence for a common tertiary structure for the HDV ribozymes. They would be consistent with a model in which the end of P1, including the cleavage site phosphate and the nucleotide 5' to the cleavage site, is positioned in an active site pocket or cleft formed by the three single-stranded regions, L3, J4/2, and J1/4.

Nonmutant forms of the avirulent satellite D of turnip crinkle virus are produced following inoculation of plants with mutant forms synthesized in Vitro

The avirulent satellite RNA D (sat D) of turnip crinkle virus (TCV) has been cloned as a copy DNA. RNA transcripts synthesized in vitro from the cloned cDNA are infectious when coinoculated with RNA transcripts of the cloned TCV genome. Sat D is the smallest and most likely the progenitor of the other TCV satellites and, in contrast to the virulent satellite RNA C (sat C), does not intensify viral symptoms. Mutant forms of sat D including internal deletions up to 50 bases yielded sat D in infected plants. However, sat D mutants were not recovered in mutant form, but reverted to normal size and sequence in infected turnip plants. Mutations at a site with homology to the catalytic strand of self-cleaving sequences in certain viroids and satellites appeared to confer virulence on sat D in that test plants showed severe crinkling and stunting normally associated with sat C. However, sat C appeared along with a restored form of sat D in the progeny RNAs of these severely infected plants. Sat C was presumably generated by recombination between sat D and the TCV genome. In contrast, when plants were inoculated with transcripts containing the equivalent mutations in sat C, sat C was recovered from infected plants in mutant form. These findings demonstrate the tendency for mutant forms of the avirulent satellite, sat D, to revert, but raise questions about the source of information used in the reversion process.

A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA

Hepatitis delta virus genomic and antigenomic RNAs contain a self-cleavage site hypothesized to function in processing the viral RNA during replication. Self-cleavage requires only a divalent cation and is mediated at the genomic site by a sequence of less than 85 nucleotides. We propose that the genomic self-cleaving sequence element and a corresponding sequence from the anti-genomic RNA could generate related secondary structures. The region of the antigenomic sequence, predicted from the proposed structure, was synthesized and shown to be sufficient for self-cleavage. Evidence for two stems which form a tertiary interaction was obtained by site-specific mutagenesis of the antigenomic sequence. Efficient self-cleavage in 10 M formamide or 5 M urea, also a property of the genomic sequence, was dependent on base-pairing in both stems. But in the absence of denaturants, the stem distal to the site of cleavage was not required, suggesting that the tertiary interaction stabilizes the structure required for self-cleavage.

Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester.

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.