Patent Grant
10730920
This application claims priority of Taiwanese Patent Application No. 106113165, filed on Apr. 19, 2017.
The disclosure relates to recombinant polypeptides derived from FBP1 and FBP2. Such recombinant polypeptides are useful and effective in increasing viral yield.
Many RNA viruses utilize internal ribosome entry sites (IRESs) located in the 5′ untranslated region (UTR) of genomic RNA to translate viral proteins in a cap-independent manner. Several cellular host proteins that can bind to and stabilize IRES structures and regulate IRES-driven translation have been reported, and these proteins are known as IRES trans-acting factors (ITAFs). Far upstream element-binding protein 1 (FUBP1, also known as FBP1) and far upstream element-binding protein 2 (KHSRP, also known as KH-type splicing regulatory protein, KSRP, FUBP2, FBP2 or P75) are among the known ITAFs that are functionally important in modulating viral IRES-driven translation.
IRES elements have been shown to exist in all genera of Picornaviridae, a type of cytoplasmic RNA virus known for its infectability on both animals and humans. At present, at least five different types of IRESs have been identified in picornaviruses, and each type is characterized by a distinct secondary structure and a different eIF (eukaryotic initiation factor)/ITAF-requirement. The picornavirus type I IRES is only found in enteroviruses such as enterovirus A71 (EVA71 or EV71), coxsackievirus B3 (CVB3), and human rhinovirus type 2 (HRV2).
EV71 is a positive single-strand RNA virus containing type I IRES in the Picornaviridae family, and is emerging as a potent threat worldwide. EV71 infections normally cause mild diseases, such as hand-foot-and-mouth disease (HFMD) or herpangina. However, children under five years of age are particularly susceptible to the most severe forms of EV71-associated neurological complications, including aseptic meningitis, brainstem and/or cerebellar encephalitis, acute flaccid paralysis (AFP), myocarditis, and rapid fatal pulmonary edema and hemorrhage.
The applicants previously found that FBP2 is a negative regulator (Lin J. Y. et al. (2009), Nucleic Acids Res., 37:47-59), while FBP1 is a positive regulator of EV71 IRES-dependent translation (Huang P. N. et al. (2011), Nucleic Acids Res., 39:9633-9648). Moreover, when the C-terminal of FBP2 is cleaved upon EV71 infection, the cleaved form of FBP2 (FBP21-503) then becomes a positive regulator of EV71 IRES-driven translation. Ubiquitination, and proteasomal and lysosomal activities may be involved in EV71-induced FBP2 truncation (Chen L. L. et al. (2013), J Virol., 87:3828-3838).
By conducting research, the applicants surprisingly found that polypeptides derived from FBP1 and FBP2, respectively, can enhance viral IRES activity and increase viral yield. Therefore, these FBP1 and FBP2 polypeptides are expected to be useful in vaccine production.
Therefore, according to a first aspect, the disclosure provides a recombinant polypeptide having an amino acid sequence corresponding to that of a truncated mutant product of a wild-type FBP1 protein having 644 amino acids in length, the truncated mutant product lacking a C-terminal domain of the wild-type FBP1 protein.
According to a second aspect, the disclosure provides a recombinant polypeptide having an amino acid sequence corresponding to that of a mutant product of a wild-type FBP2 protein having 711 amino acids in length, each of amino acid residues at positions 109, 121 and 122 of the wild-type FBP2 protein being lysine, at least one of amino acid residues at positions 109, 121 and 122 of the mutant product being arginine instead of lysine.
According to a third aspect, the disclosure provides a recombinant nucleic acid encoding a recombinant polypeptide as described above.
According to a fourth aspect, the disclosure provides a recombinant expression vector comprising a recombinant nucleic acid as described above.
According to a fifth aspect, the disclosure provides a recombinant cell comprising a recombinant expression vector as described above.
According to a sixth aspect, the disclosure provides a method for producing a picornavirus with a type I internal ribosome entry site (IRES), the method comprising the steps of:
providing a recombinant cell as described above;
infecting the recombinant cell with the picornavirus;
incubating the infected recombinant cell; and
harvesting the picornavirus produced.
According to a seventh aspect, the disclosure provides a method for preparing a viral vaccine using a harvested picornavirus as obtained by the method as described above.
Other features and advantages of the present disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which:
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Taiwan or any other country.
For the purpose of this specification, it should be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprise” has a corresponding meaning.
Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described.
The terms “polypeptide”, “peptide”, and “protein” as used herein can be interchangeably used, and refer to a polymer formed of amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids or artificial chemical mimics. The term “recombinant polypeptide” or “recombinant protein” as used herein refers to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or the desired protein.
As used herein, an amino acid can be represented by the full name thereof, by the three-letter symbol corresponding thereto, or by the one-letter symbol corresponding thereto, as well-known in the art. In addition, the proteins are represented in accordance with the conventional way of describing peptides, that is, with the N-terminus (amino terminus) on the left side and the C-terminus (carboxyl terminus) on the right side.
A “wild-type” protein means that the protein will be active at a level of activity found in nature and typically will be the amino acid sequence found in nature. In an aspect, the term “wild type” or “parental sequence” can indicate a starting or reference sequence prior to a manipulation of this invention.
As used herein, the term “mutation” refers to a change introduced into a parental sequence, including, but not limited to, substitutions, insertions, and deletions (including truncations). The consequences of a mutation include, but are not limited to, the creation of a new character, property, function, phenotype or trait not found in the protein encoded by the parental sequence.
As used herein, the term “mutant” refers to a gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The mutant may be one that exists in nature, such as an allelic mutant, or one not yet identified in nature. The mutant may be conservatively altered, wherein substituted amino acid(s) retain structural or chemical characteristics similar to those of the original amino acid(s). Rarely, mutants may be substituted non-conservatively.
The term “truncated product” with reference to a protein, polypeptide or fragment thereof generally denotes such product that has a N-terminal and/or C-terminal deletion of one or more amino acid residues as compared to said protein, polypeptide or fragment thereof.
A “DNA coding sequence” is a double-stranded DNA sequence that is transcribed into an RNA (further translated into a polypeptide) in vivo under the control of appropriate regulatory sequences. The boundaries of the DNA coding sequence are determined by a start codon at the 5′ (amino) terminus and a stop codon at the 3′ (carboxyl) terminus. A coding sequence may include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. “cDNA” is defined as copy DNA or complementary DNA, and is a product of a reverse transcription reaction from an mRNA transcript.
The terms “nucleic acid”, “nucleic acid sequence”, and “nucleic acid fragment” as used herein refer to a deoxyribonucleotide or ribonucleotide sequence in single-stranded or double-stranded form, and comprise naturally occurring nucleotides or artificial chemical mimics. The term “nucleic acid” as used herein is interchangeable with the terms “gene”, “cDNA”, “mRNA”, “oligo-nucleotide”, and “polynucleotide” in use.
As used herein, the term “DNA fragment” refers to a DNA polymer, in the form of a separate segment or as a component of a larger DNA construct, which has been derived either from isolated DNA or synthesized chemically or enzymatically such as by methods disclosed elsewhere.
Unless otherwise indicated, a nucleic acid sequence, in addition to the specific sequences described herein, also covers its complementary sequence, and the conservative analogs, related naturally occurring structural variants and/or synthetic non-naturally occurring analogs thereof, for example, homologous sequences having degenerative codon substitution, and conservative deletion, insertion, substitution, or addition. Specifically, degenerative codon substitution may be produced by, for instance, a nucleotide residue substitution at the third position of one or more selected codons in a nucleic acid sequence with other nucleotide residue(s).
The terms “recombinant vector” and “expression vector” as used herein can be interchangeably used, and refer to any recombinant expression system capable of expressing a selected nucleic acid sequence, in any competent host cell in vitro or in vivo, constitutively or inducibly. The recombinant vector may be an expression system in linear or circular form, and covers expression systems that remain episomal or that integrate into the host cell genome. The recombinant expression system may or may not have the ability to self-replicate, and it may drive only transient expression in a host cell.
As used herein, the term “transformation” can be used interchangeably with the term “transfection” and refers to the introduction of an exogenous nucleic acid molecule into a selected host cell. According to techniques known in the art, a nucleic acid molecule (e.g., a recombinant DNA construct or a recombinant vector) can be introduced into a selected host cell in various ways, such as calcium phosphate- or calcium chloride-mediated transfection, electroporation, microinjection, particle bombardment, liposome-mediated transfection, transfection using bacteriophages, or other methods.
The terms “cell”, “host cell”, “transformed host cell”, and “recombinant host cell” as used herein can be interchangeably used, and not only refer to specific individual cells but also include sub-cultured offsprings or potential offsprings thereof. Sub-cultured offsprings formed in subsequent generations may include specific genetic modifications due to mutation or environmental influences and, therefore, may factually not be fully identical to the parent cells from which the sub-cultured offsprings were derived. However, sub-cultured cells still fall within the coverage of the terms used herein.
In order to increase viral yield for use in vaccine production, the applicants endeavored to develop improved methods and found that polypeptides derived from FBP1 and/or FBP2 can enhance viral IRES activity and increase viral yield.
Accordingly, the present disclosure provides a first recombinant polypeptide having an amino acid sequence corresponding to that of a truncated mutant product of a wild-type FBP1 protein having 644 amino acids in length. The truncated mutant product lacks a C-terminal domain of the wild-type FBP1 protein.
The term “corresponding to” is used herein to describe a polypeptide or nucleic acid of this disclosure which is similar or homologous to a corresponding mutant product modified from a parental polypeptide or nucleic acid, wherein the sequence of the polypeptide or nucleic acid of this disclosure differs from the sequence of the corresponding mutant product modified from the parental polypeptide or nucleic acid only by the presence of at least one amino acid residue or nucleotide residue variation.
Typically, the sequences of the polypeptide or nucleic acid of the disclosure and the corresponding mutant product modified from the parental polypeptide or nucleic acid exhibit a high percentage of identity, such as at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% identity, or at least about 99% identity.
In certain embodiments, the truncated mutant product of the wild-type FBP1 protein has amino acid deletions at positions 372-644 of the wild-type FBP1 protein. In an embodiment of the present disclosure, the truncated mutant product of the wild-type FBP1 protein has an amino acid sequence of SEQ ID NO: 4.
In certain embodiments, the truncated mutant product of the wild-type FBP1 protein has amino acid deletions at positions 63-78 and positions 372-644 of the wild-type FBP1 protein. In an embodiment of the present disclosure, the truncated mutant product of the wild-type FBP1 protein has an amino acid sequence of SEQ ID NO: 46.
The present disclosure also provides a first recombinant nucleic acid encoding the first recombinant polypeptide as described above. In certain embodiments, the first recombinant nucleic acid has a nucleotide sequence selected from SEQ ID NO: 3, SEQ ID NO: 44 and SEQ ID NO: 45.
In addition, the present disclosure also provides a second recombinant polypeptide having an amino acid sequence corresponding to that of a mutant product of a wild-type FBP2 protein having 711 amino acids in length. Each of amino acid residues at positions 109, 121 and 122 of the wild-type FBP2 protein is lysine, and at least one of amino acid residues at positions 109, 121 and 122 of the mutant product is not lysine.
In certain embodiments, the at least one amino acid residue at positions 109, 121 and 122 of the mutant product is arginine. In an exemplary embodiment, one amino acid residue at positions 109, 121 and 122 of the mutant product is arginine. In an exemplary embodiment, two of the amino acid residues at positions 109, 121 and 122 of the mutant product are arginine. In yet another exemplary embodiment, all of the amino acid residues at positions 109, 121 and 122 of the mutant product are arginine.
In an embodiment of the present disclosure, the amino acid sequence of the second recombinant polypeptide is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.
The present disclosure also provides a second recombinant nucleic acid encoding the aforesaid second recombinant polypeptide derived from FBP2. In certain embodiments, the second nucleotide sequence is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15.
The first and second recombinant nucleic acid sequences according to the present disclosure can be utilized to construct various recombinant expression vectors for expressing the aforementioned recombinant polypeptides using a standard technique known to one of ordinary skill in the art.
Therefore, the present disclosure provides a recombinant expression vector comprising the first recombinant nucleic acid and/or the second recombinant nucleic acid.
In certain embodiments, the first and/or second recombinant nucleic acids of the recombinant expression vector may be introduced into a genome of the recombinant cell. In an exemplary embodiment, an endogenous FBP2 gene in the genome of the recombinant cell is deleted, disrupted, or disabled.
As used herein, the term “delete” refers to partial or entire removal of a coding region of a gene.
As used herein, the term “disrupt” refers to removal, insertion, or mutation of a nucleotide of a gene.
As used herein, the term “disable” refers to inactivating a gene or the protein encoded by the gene so as to force the gene or protein to lose its activity or function.
The recombinant expression vector according to the present disclosure can be used to transform or transfect a desired host cell. Consequently, the disclosure also provides a recombinant cell comprising the aforementioned recombinant expression vector.
According to the disclosure, the host cell may be a mammalian cell. Examples of the mammalian cell suitable for used in the present disclosure include, but are not limited to, an RD cell, a 293T cell, a Vero cell, a MDCK cell, a PER.C6 cell, a MRC-5 cell, a WI-38 cell, etc.
Since the aforementioned recombinant polypeptides derived from FBP1 and/or FBP2 can promote picornavirus type I IRES-driven translation and increase viral yield, these polypeptides are expected to be useful in the production of viral vaccine. Therefore, the present disclosure provides a method for producing a picornavirus with a type I IRES, which includes the steps of:
providing the aforesaid recombinant cell;
infecting the recombinant cell with the picornavirus;
incubating the infected recombinant cell; and
harvesting the picornavirus produced.
According to the disclosure, the picornavirus with the type I IRES may be an enterovirus selected from the group consisting of enterovirus A (such as enterovirus A71 and coxsackieviruses A6 and A16), enterovirus B (such as coxsackievirus B3), enterovirus C (such as coxsackieviruses A21 and A24, and polioviruses 1, 2 and 3), enterovirus D (such as enterovirus D68), rhinovirus A, rhinovirus B, and rhinovirus C, and combinations thereof. In an embodiment of the disclosure, the enterovirus is enterovirus A71. In another embodiment of the disclosure, the enterovirus is coxsackievirus B3.
The disclosure also provides a method for preparing a viral vaccine using the harvested picornavirus obtained by the aforesaid method for producing the picornavirus.
The present disclosure will be further described by way of the following examples. However, it should be understood that the following examples are intended solely for the purpose of illustration and should not be construed as limiting the disclosure in practice.
General Experimental Materials:
1. Primers used in the polymerase chain reaction (PCR) experiments, infra, were synthesized by Mission Biotech, Taipei, Taiwan.
2. EV71 strain Tainan/4643/98 viruses (GenBank accession number AF304458) and CVB3 strain viruses were obtained from Chang Gung Memorial Hospital, Linkou, Taiwan.
3. Cell Cultures:
Human embryonal rhabdomyosarcoma (RD) cells (ATCC, CCL-136) and 293T cells (ATCC, CCL-3216) used in the following experiments were purchased from ATCC (American Type Culture Collection, Manassas, Va., USA). The cells of the respective type were incubated in a Petri dish containing Dulbecco's modified Eagle medium (DMEM; Gibco, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/mL of penicillin, 100 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B, followed by cultivation in an incubator with culture conditions set at 37° C. and 5% CO2. Medium change was performed every two days. Cell passage was performed when the cultured cells reached 90% of confluence.
4. FBP1-Related Recombinant Plasmids:
Note:
AG
5. FBP2-Related Recombinant Plasmids
6. 2A Protease (2Apro)-Related Recombinant Plasmids
Concerning the experimental methods and relevant techniques for DNA cloning as employed in this disclosure, such as DNA cleavage by restriction enzymes, polymerase chain reaction (PCR), DNA ligation with T4 DNA ligase, agarose gel electrophoresis, plasmid transformation, etc., reference may be made to the following textbook widely known in the art: Sambrook J. and Russell D. W. (2001), Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York. Site-directed mutagenesis PCR was performed substantially according to the manufacturer's instructions. These techniques can be readily performed by those skilled in the art based on their professional knowledge and experience.
1. Virus Infection:
When the cultured cells reached 90% of confluence, infection by a virus, such as EV71 strain Tainan/4643/98 or CVB3 strain, at a given multiplicity of infection (m.o.i.), was performed in serum-free DMEM. The viruses were allowed to adsorb at 37° C. for 1 hour, after which the infected cells were washed with phosphate-buffered saline (PBS) and incubated at 37° C. in DMEM containing 2% FBS.
2. Expression and Purification of Recombinant Proteins:
The 2Apro and 2Apro-C110S proteins fused to a GST-tag as respectively produced by pGEX-6P-1-EV71-2A and pGEX-6P-1-EV71-2A-C110S in BL21 (DE3) E. coli cells (Yeastern Biotech, Taiwan) were purified using a GSTrap FF column (GE Healthcare, Waukesha, Wis.) according to the manufacturer's instructions. The GST-tag of the respective puried fusion protein was removed with PreScission Protease (GE Healthcare), so as to obtain 2Apro and 2Apro-C110S proteins.
Experimental procedures for the expression and purification of recombinant His-tagged FBP1 was reported previously in Huang P. N. et al. (2011), supra.
3. Coupled Transcription/Translation of [35S] Methionine-Labeled FBP1:
To produce each of [35S] methionine-labeled wild-type or mutant FBP1 proteins, a DNA fragment containing the T7 promoter and the designated coding sequence was amplified using PCR and a respective primer pair shown in Table 5 from the corresponding plasmid obtained in above Items (a) and (d) of the section entitled “4. FBP1-related recombinant plasmids” of the General Experimental Materials, and the designated protein was produced with the TNT-coupled reticulocyte lysate system (Promega, Madison, Wis.) according to the manufacturer's instructions.
In addition, each of [35S] methionine-labeled truncated FBP1 proteins was generally produced as set forth above, except that the template to be applied was pFLAG-CMV2-FBP1 and the respective primer pair shown in Table 5 was used.
4. Immunoblot Analysis:
Protein samples were resolved in sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, and the separated proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare). The blotted PVDF membranes thus obtained were blocked with tris-buffered saline (TBS) and 0.1% (vol/vol) TWEEN 20 (polyethylene glycol sorbitan monolaurate) containing 5% non-fat dry milk, followed by washing with TBST 3 times, each time for 15 min, and then probing with the indicated primary antibodies. For staining with secondary antibodies, the probed membranes were washed with TBST 3 times, each time for 15 min, and were then incubated with an HRP-conjugated anti-mouse antibody or an HRP-conjugated anti-rabbit antibody for 60 minutes at room temperature. HRP was detected using the Western Lightning Chemiluminescence Kit (PerkinElmer Life Sciences, Boston, Mass.).
5. Viral Plaque Assay:
6×105 RD cells were seeded into each well of a 6-well plate and incubated for 24 h, followed by addition of 500 μL of virus diluent prepared in serum-free DMEM in duplicate to each well. The viruses were allowed to adsorb at 37° C. for 1 hour, after which the infected cells were washed with PBS. 2 mL of an agarose overlay medium (0.3% agarose in DMEM containing 2% FBS) was added to each well and the plates were kept at room temperature until the agarose overlay medium turned solid. The plates were moved to a 37° C. incubator for 2 to 4 days of incubation, and then the cells were fixed with a 3.6% formaldehyde solution at room temperature for 2 hours. The agarose overlay was removed, and then the fixed cells in each well were stained with 0.5% crystal violet for 2 min. After rinsing the stained cells with water, the viral plaques in each well were counted. The viral titer was determined by substituting the value of the viral plaques counted into the following formula (1):
A=B/(C×0.5) (1)
wherein: A=viral titer (plaque forming units (pfu)/mL)
To examine FBP1 expression and to analyze patterns of cleaved FBP1 in EV71-infected RD cell lysates, the following experiments were conducted.
RD cells were infected with EV71 strain Tainan/4643/98 at a m.o.i. of 40 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures. Mock-infected cells served as a control group.
At 2, 3, 4, 5, 6, 8 and 10 hours post-infection (h.p.i.), the infected cells were washed with PBS and lysed with IGEPAL CA630 (octylphenoxy poly(ethyleneoxy)ethanol) lysis buffer (150 mM NaCl, 1% IGEPAL CA630 (octylphenoxy poly(ethyleneoxy)ethanol), 50 mM Tris-base [pH 8.0]) for 30 minutes on ice. The resultant cell lysates were centrifuged at 10,000×g for 10 minutes at 4° C., and the supernatants were collected to serve as total protein samples.
Total protein samples in equal amount (determined by Bradford assay) at each designated time point of post-infection were subjected to immunoblot analysis according to the procedures as described in the preceding section, entitled “4. Immunoblot analysis,” of the General Experimental Procedures. For the purpose of comparison, the total protein sample from the mock-infected cells was subjected to the same analysis.
For analyzing patterns of cleaved FBP1 protein in EV71-infected RD cell lysates, the total protein samples obtained from the mock-infected and EV71-infected cell lysates at each designated time point of post-infection (i.e. 4, 6, 8 and 10 h.p.i.) were also subjected to immunoblot analysis using two specific FBP1 antibodies generated from the applicants' lab, i.e. Ab-N antibody and Ab-C antibody that respectively recognize the N-terminal region (amino acid positions 61-180) and C-terminal region (amino acid positions 293-644) of FBP1. In addition, viral 3Dpol protein was used as an indicator for virus infection, and actin was used as a loading control. The primary and secondary antibodies used for detecting the respective protein in this example are shown in Table 6 below.
Results:
As shown in
As shown in
Moreover, to further determine the subcellular localization of FBP1 in EV-71-infected cells, nuclear and cytoplasmic protein fractions were isolated from the total protein sample of the infected cells, and then subjected to immunoblot analysis. The results indicated that FBP1 remained primarily in the nucleus during mock infection, but appeared in the cytoplasm with an increasing level over 2 to 6 h.p.i. In contrast, the cleavage products of FBP1 were mostly present in the cytoplasm, and their expression levels increased throughout the course of infection (data not shown).
Taken together, these results demonstrate that the FBP1 protein is likely to be subjected to proteolytic cleavage during the middle stage of EV71 infection.
To determine whether FBP1 cleavage was caused by viral factors, [35S] methionine-labeled FBP1 generated according to the procedures as described in the section, entitled “3. Coupled transcription/translation of [35S] methionine-labeled FBP1,” of the General Experimental Procedures was used in the following experiment.
A. In Vitro Proteinase Cleavage Assay with [35S] Methionine-Labeled FBP1:
5 μL of the [35S] methionine-labeled FBP1 protein was incubated with various doses (0, 0.5, 1, 2, 5 and 10 μg) of EV71 viral wild-type 2A proteinase (2Apro) or incubated with 10 μg of a mutant 2A proteinase (2Apro-C110S) in a cleavage buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM DTT and 1 mM EDTA; pH 7.5) with a total volume of 15 μL at 37° C. for 4 hours. The reaction products were analyzed by SDS-PAGE. After the electrophoresis, gel was removed from the electrophoresis apparatus and dried by Model 583 gel dryers (Bio-Rad) for 4 hours, and then the dried gel was loaded into an X-ray cassette with an X-ray film (Amersham Hyperfilm MP) in a darkroom for overnight exposure. Following the exposure, and the X-ray film was removed from the cassette for developing.
The applicants further examined the cleavage kinetics of FBP1 by incubating 5 μg of 2Apro with [35S] methionine-labeled FBP1, and then assayed the cleavage result at various time points (i.e., 15 min, 30 min, 1 hour, 2 hours and 4 hours). [35S] methionine-labeled FBP1 incubated with 5 μg of 2AC110S and without 2Apro, and [35S] methionine-labeled FBP1 incubated without 5 μg of 2AC110S and without 2Apro, were used as negative control groups.
Results:
In addition, it was found that Cp-N and Cp-C were detected after 15 minutes of incubation with 2Apro, and the levels of Cp-N and Cp-C increased over the 4 hours of incubation as observed (data not shown). These results provide evidence that the EV71 viral 2Apro is capable of cleaving FBP1.
Previous studies on picornaviruses have revealed that 2Apro preferentially cuts at glycine residues (Blom N. et al. (1996), Protein Sci., 5:2203-2216; Hellen C. U. et al. (1992), J Virol., 66:3330-3338), and the result in
A. In Vitro Proteinase Cleavage Assay with [35S] Methionine-Labeled Wild-Type FBP1 and Mutant FBP1 Proteins:
[35S] methionine-labeled wild-type FBP1 protein and seven mutant FBP1 proteins labeled with [35S] methionine as shown in Table 7 were generated according to the procedures as described in the section, entitled “3. Coupled transcription/translation of [35S] methionine-labeled FBP1,” of the General Experimental Procedures.
These obtained [35S] methionine-labeled proteins were subjected to in vitro proteinase cleavage assay using 10 μg of 2Apro at 37° C. for 4 hours according to the procedures as described in Example 2.
Results:
It can be seen from
It should be noted that regarding FBP1-G345-362K, FBP1-G364-380K, and FBP1-G371K, there are two additional non-specific cleavage products with a higher molecular weight (indicated by asterisks) as compared to Cp-N and Cp-C fragments. These additional cleavage products were unlikely to represent the cleavage products or cleavage intermediates of 2Apro, since they were not observed in the 2Apro cleavage profile of FBP1, as shown in
B. In Vitro Proteinase Cleavage Assay with [35S] Methionine-Labeled Wild-Type FBP1 and Truncated FBP1 Proteins:
In order to confirm whether Gly-371 is the only cleavage site of 2Apro, [35S] methionine-labeled wild-type FBP1 protein and five truncated FBP1 proteins labelled with [35S] methionine (i.e., FBP11-371, FBP1372-644, FBP11-443, FBP1185-644 and FBP1185-443) were produced according to the procedures as described in the section, entitled “3. Coupled transcription/translation of [35S] methionine-labeled FBP1”, of the General Experimental Procedures, followed by conducting in vitro proteinase cleavage assay as described above in section A of this example.
Results:
As shown in
C. The Cleavage Profiles of Wild-Type FBP1 or Mutant FBP1-G371K in EV71-Infected RD Cells:
To further confirm that the cleavage of FBP1 occurs in vivo during EV71 infection, expression of FLAG-HA dual-tagged wild-type FBP1 and mutant FBP1-G371K (resistant to 2Apro cleavage) in RD cells was carried out, followed by EV71 infection.
To be specific, RD cells were transfected with pFLAG-FBP1-HA or pFLAG-FBP1-G371K-HA, and were then infected with EV71 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures.
At 4, 6, 8 and 10 h.p.i., the cell lysates from the mock-infected and EV71-infected cells were processed according to the procedures as described in Example 1. The total protein samples thus obtained were subjected to immunoblotting analysis according to the procedures as described in the preceding section, entitled “4. Immunoblot analysis,” of the General Experimental Procedures. The primary and secondary antibodies used for detecting the respective protein in this example are shown in Table 8 below.
Results:
To address the roles of cleaved FBP1 proteins in EV71 IRES activity, the following experiments were conducted.
Experimental Materials:
Plasmids pGL3-EV71 5′UTR-FLuc and pCRII-TOPO-EV71 5′UTR were constructed according to the methods described in Huang P. N. et al. (2011), supra and Lin J. Y. et al. (2009), supra.
A DNA fragment of T7-EV71 5′UTR containing T7 promoter and the EV71 5′UTR sequence (nucleotide positions 1-745 of EV71) was excised from recombinant plasmid pCRII-TOPO-EV71 5′UTR using a EcoRI restriction enzyme. In addition, a DNA fragment of T7-EV71 5′UTR linker region containing the T7 promoter and the EV71 5′UTR linker region sequence (nucleotide positions 636-745 of EV71) was amplified from pCRII-TOPO-EV71 5′UTR using a forward primer 5′-TAATACGACTCACTATAGGGCCATCCGGTGTGCAACAGGGCAAT-3′ (SEQ ID No: 79) and a reverse primer 5′-GTTTGATTGTGTTGAGGGTCA-3′ (SEQ ID NO: 80).
Each DNA fragment was transcribed into a respective RNA transcript (i.e., a respective one of EV71 5′ UTR and EV71 5′ UTR linker region RNA probes) using a MEGAscript T7 kit (ThermoFisher Scientific, San Jose, Calif.), according to the protocol recommended by the manufacturer. In addition, biotinylated RNA transcripts, i.e. biotinylated EV71 5′UTR and EV71 5′UTR linker region RNA probes, were synthesized by adding 1.25 μL of 10 mM biotin-16-UTP (Roche, Mannheim, Germany) in the transcription reaction. These RNA transcripts were purified using an RNeasy Mini kit (Qiagen, Chatsworth, Calif.).
A. Pull Down Assay for Biotinylated EV71 5′ UTR RNA Probe with FLAG-Fused FBP1 Protein
RD cells were transfected with a respective one of pFLAG-CMV2-FBP1, pFLAG-CMV2-FBP11-371 and pFLAG-CMV2-FBP1372-644. At 48 hours post-transfection, the transfected cells were washed with PBS and lysed with a lysis buffer (containing 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM EGTA, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 10% glycerol, 0.1 mM phenyl-methylsulfonyl fluoride (PMSF) and 5 mM β-mercaptoethanol) for 30 minutes on ice. Afterwards, the resultant cell lysate was centrifuged at 10,000×g for 10 min at 4° C., and the supernatant thus formed was collected (to serve as an RD cell extract) and stored at −80° C. for further analysis.
The obtained RD cell extract (200 μg) was mixed with 12.5 pmol of a respective one of the following: biotinylated EV71 5′UTR RNA probe, non-biotinylated EV71 5′UTR RNA probe, biotinylated EV71 5′ UTR linker region RNA probe and non-biotinylated EV71 5′ UTR linker region RNA probe, followed by adding an RNA mobility buffer (5 mM HEPES (pH 7.1), 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol (DTT), 1 U RNasin, and 0.25 mg/mL heparin) until a final volume of 100 μL was reached. The resultant mixture were incubated at 30° C. for 15 minutes, and then added with 400 μL of streptavidin MagneSphere Paramagnetic Particles (Promega), followed by incubation for 10 minutes at room temperature, so as to pull down the biotinylated RNA-protein complex. The obtained pulled-down complex was washed 5 times with a heparin-free RNA mobility buffer, and then 25 μL of a 2×SDS sample buffer was added to the washed complex, followed by conducting incubation at 95° C. for 10 minutes to dissociate the proteins from the complex. The eluted protein sample was subjected to immunoblotting analysis with an anti-FLAG antibody as shown in Table 8.
B. Enzymatic RNA Footprinting Assay of Wild-Type FBP1 and FBP11-371 Bound to EV71 5′ UTR Linker Region RNA
EV71 5′ UTR linker region RNA probe synthesized in this example was labeled at the 5′ end using T4 polynucleotide kinase and [γ-32P] ATP.
The obtained 32P-labeled EV71 5′ UTR linker region RNA probe was treated with 2 μg of wild-type FBP1 or 1.14 μg of FBP11-371 in a binding buffer containing 1 μL of 0.02 μg/μL RNAse A (a pyrimidine nucleotide-specific endonuclease, Ambion AM2274, Thermo Fisher Scientific) or 0.02 U/mL RNAse T1 (a ribonucleotide that specifically degrades single-stranded RNA at G residues, Ambion AM2283, Thermo Fisher Scientific) at 4° C. for 10 minutes, so as to degrade the RNA sequence without the protection of a bound protein. The RNA probe without incubation of an FBP1 protein served as a control group.
Reactions were terminated with 10 μL of an inactivation buffer (Ambion, Austin, Tex.), and the cleavage products were separated in 12% acrylamide/7M urea gels, after which the gels were dried and scanned with a phosphorimager (GE Typhoon Trio Imager Scanner). RNA nucleotide positions were determined through comparison with the Decade marker (Ambion), so as to identify the sequence protected by the wild-type FBP1 and FBP11-371.
Results:
A. Pull Down Assay
B. Enzymatic Footprinting Assay
Taken together, these results clearly demonstrate that wild-type FBP1 and FBP11-371 can bind to distinct sequences of the EV71 5′UTR linker region RNA.
To investigate whether FBP11-371 exhibits a comparable effect on in vitro EV71 IRES activity as wild-type FBP1, shFBP1-RD stable cells with sustained knockdown of endogeneous fbp1 gene expression were prepared and used in this example.
Experimental Materials:
Short hairpin RNA (shRNA) targeting nucleotide positions 847 to 871 of human FBP1 mRNA (shFBP1, 5′-CCAAGATTTGCTGTTGGCATTGTAA-3′, SEQ ID No: 81) and the scramble control (shNC, 5′-AATTTGCGCCCGCTTACCCAGTT-3′ SEQ ID No: 82) were respectively inserted into lentivirus vectors pLKO_TRC005 (obtained from Taiwan National RNAi Core Facility, Academia Sinica) according to the instructions of the Taiwan National RNAi Core Facility, Academia Sinica, so as to construct two pLKO_TRC005-shRNA vectors, i.e. pLKO_TRC005-shFBP1 and pLKO_TRC005-shNC.
For lentivirus preparation, 293T cells were co-transfected with pLKO_TRC005-shFBP1 or pLKO_TRC005-shNC, and the helper plasmids pMD.G and pCMVΔR8.91, using X-tremeGENE transfection reagent (Roche) and then cultured at 37° C. for 36 hr. The obtained cell culture was centrifugated under 300×g. The resultant supernatant containing viral particles was harvested.
RD cells were transduced with the obtained viral particles for 24 hours, and then subjected to selection with puromycin (5 μg/mL), so as to prepare shFBP1-RD stable cells with sustained knockdown of endogeneous fbp1 gene expression and shNC-RD stable cells.
The plasmid pGL3-EV71 5′UTR-FLuc was constructed according to the method described in Huang P. N. et al. (2011), supra and linearized using XhoI or XbaI restriction enzymes, so as to generate EV71 5′UTR-FLuc reporter RNA.
shFBP1-RD stable cells were grown in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B, followed by cultivation in an incubator with culture conditions set at 37° C. and 5% CO2. When reaching 90% of confluence, the cultured cells were washed and scraped with PBS, and then subjected to centrifugation at 300×g for 10 minutes at 4° C. After discarding the supernatant, the cell pellet thus obtained was resuspended in 1.5× pellet volume of a hypotonic lysis buffer (10 mM HEPES-KOH, pH 7.6, 10 mM KOAc, 0.5 mM Mg(OAc)2, 2 mM DTT, and 1× protease inhibitor cocktail [Roche]), was placed on ice for 30 minutes, and was then homogenized with a 27-gauge ½-inch needle. The thus formed cell extract was centrifuged at 10,000×g for 20 minutes at 4° C., and the supernatant (serving as an shFBP1-RD extract) was recovered and used in the following in vitro IRES-driven translation assay to determine the impact of wild-type FBP1 and FBP11371 on EV71 IRES-driven translation in vitro.
To be specific, a designated concentration (i.e., 0, 25, 50, 100 or 200 nM) of recombinant wild-type FBP1 or FBP11-371 obtained as described in Huang P. N. et al. (2011), supra was mixed with the following components to reach a final volume of 25 μL: 60% volume of the shFBP1-RD extract obtained above, 0.25 μg of EV71 5′ UTR-Fluc RNA, 10 mM creatine phosphate, 50 μg/mL creatine phosphokinase, 79 mM KOAc, 0.5 mM Mg(OAc)2, 2 mM DTT, 0.02 mM hemin, 0.5 mM spermidine, 20 mM HEPES-KOH (pH 7.6), 20 μM amino acid mixture (Promega), 0.4 mM ATP (Promega), and an RNase inhibitor. The mixture was incubated at 30° C. for 90 minutes, and firefly luciferase activity regarding each designated concentration of wild-type FBP1 or FBP11-371 was measured using luciferase assay system (Promega) and a luminometer (Promega). A mixture without recombinant wild-type FBP1 or FBP11-371 added was used as a buffer control and subjected to the same analysis, and the measured firefly luciferase activity was set as 100%.
The additive effect of wild-type FBP1 and FBP11-371 on EV71 IRES-driven translation in vitro was also generally examined using the same procedure as mentioned above, except that a designated concentration of recombinant wild-type FBP1 (50, 100 or 200 nM), FBP11-371 (50, 100 or 200 nM) or a combination of wild-type FBP1 and FBP11-371 (each at 25, 50 or 100 nM) was respectively used.
Results:
To address whether the truncated FBP1 assists in affecting picornavirus (such as EV71 and CVB3) yielded in infected cells, four recombinant plasmids, including pFLAG-Hr-FBP1WM (expressing shRNA-resistant FLAG-FBP1WM), pFLAG-Hr-FBP1-G371KWM (expressing shRNA-resistant and 2Apro cleavage-resistant FLAG-FBP1-G371KWM), pFLAG-Hr-FBP11-371-WM (expressing shRNA-resistant FBP11-371-WM) and pFLAG-Hr-FBP11-371-WM-delNLS (expressing shRNA-resistant FBP11-371-WM-delNLS with a deletion of nuclear localization signal (NLS) sequence from FBP11-371-WM), were used in the following experiment.
shFBP1-RD cells obtained in Example 5 were transiently transfected with pFLAG-CMV2 to serve as a vector control, or with the recombinant plasmid pFLAG-Hr-FBP1WM or pFLAG-Hr-FBP1-G371KWM to rescue FBP1 protein expression. At 48 hours post-transfection, the shFBP1-RD cells transfected with the respective plasmid as mentioned above and shNC-RD cells as obtained in Example 5 (as positive control) were infected with EV71 at a m.o.i. of 40 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures. EV71 viral titers during the course of infection, i.e. at 3, 6, 9 and 12 hours, were measured by plaque assays. In comparison, shNC-RD cells were subjected to the same analysis.
On the other hand, shFBP1-RD cells were co-transfected with: (1) pFLAG-Hr-FBP1-G371KWM and pFLAG-Hr-FBP11-371-WM, or (2) pFLAG-Hr-FBP1-G371KWM and pFLAG-Hr-FBP11-371-WM-delNLS. At 48 hours post-transfection, these two types of transfected cells, alone with the previously obtained shFBP1-RD cells transfected with pFLAG-CMV2 vector or pFLAG-Hr-FBP1-G371KWM, were infected with EV71 or CBV3 at a m.o.i. of 40 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures. The EV71 and CBV3 viruses thus produced were harvested at 9 hours post-infection for determination of virus titers according to the procedures as described in the preceding section, entitled “5. Viral plaque assay,” of the General Experimental Procedures. The viral titers in shFBP1-RD cells transfected with pFLAG-CMV2 vector was set as 100%.
Statistical significance of the experimental data was analyzed by performing one-way ANOVA using Prism 6 software (GraphPad Software, San Diego, Calif.), where p<0.05 was considered to be statistically significant.
Results:
The applicants found that the ubiquitination of FBP2 is promoted by the KLHL12-based CUL3 ubiquitin E3 ligase complex (data not shown). In order to identify the main ubiquitination sites on FBP2 from those predicted via proteomics analysis (see
A respective one of these wild-type FBP2 and mutant FBP2 recombinant plasmids was co-transfected with a plasmid pcDNA3-HA-Ub, which expressed HA tagged-ubiquitin (HA-Ub) and was provided by Dr. Rei-Lin Kuo (Chang Gung University, Taiwan) and Dr. Chen Zhao (University of Texas at Austin, USA), into 293T cells, using X-tremeGENE transfection reagent (Roche) according to the manufacturer's instructions. 293T cells transfected with pcDNA3-HA-Ub only served as a control group.
The transfected cells were treated with 20 μM MG132 (Sigma), followed by incubation for 4 hours. The cell culture was harvested and lysed with a lysis buffer containing 5 mM of N-ethylmaleimide (Sigma). The resultant cell lysate was immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitated product was subjected to immunoblotting according to the procedures as described in the preceding section, entitled “4. Immunoblot analysis,” of the General Experimental Procedures, so as to detect the ubiquitinated FBP2 protein. In addition, actin was used as a loading control. The primary and secondary antibodies used for detecting the respective protein in this example are shown in Table 9 below.
In addition, the immunoblotting results (i.e. ubiquitination levels) of wild-type FBP2 and FBP2-K109,121,122R, FBP2-K121,122R and FBP2-K109R were each normalized by that of wild-type FBP2, so as to determine the fold-changes in ubiquitin modification.
Results:
As shown in the left panel of
In addition, it can be seen from the right panel of
Therefore, the applicants contemplate that Lys109, Lys121 and Lys122 are the likely sites for KLHL12-mediated ubiquitination in FBP2.
To assess the impact of ubiquitinated FBP2 on EV71 5′ UTR RNA-driven translation and to investigate how FBP2 ubiquitination affects the viral protein synthesis, shFBP2-RD stable cells with sustained knockdown of endogeneous fbp2 gene expression were prepared and used in this example.
Experimental Materials:
shFBP2-RD stable cells with sustained knockdown of endogeneous fbp2 gene expression were prepared by the procedures similar to that of shFBP1-RD stable cells as described in Example 5, except that the short hairpin RNA (shRNA) used was shFBP2 targeting nucleotide positions 813 to 837 of human FBP2 mRNA (5′-CACATTCGTATTCTGAGATCCGTCC-3′, SEQ ID No: 83). In comparision, the pLKO.1-shLacZ control plasmid TRCN0000072224 (provided by Taiwan National RNAi Core Facility, Academia Sinica) was used to prepare shLacZ-RD stable cells without knockdown of endogenous fbp2 gene expression.
shFBP2-RD stable cells were grown in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B, followed by cultivation in an incubator with culture conditions set at 37° C. and 5% CO2. When reaching 90% of confluence, the cultured cells were washed and scraped with PBS, and were then subjected to centrifugation at 300×g for 10 minutes at 4° C. After discarding the supernatant, the cell pellet thus obtained was resuspended in 1.5× pellet volume of a hypotonic lysis buffer (10 mM HEPES-KOH, pH 7.6, 10 mM KOAc, 0.5 mM Mg(OAc)2, 2 mM DTT, and 1× protease inhibitor cocktail [Roche]), was placed on ice for 30 minutes, and was then homogenized with a 27-gauge ½-inch needle. The thus formed cell extract was centrifuged at 10,000×g for 20 minutes at 4° C., and the resultant supernatant (serving as an shFBP1-RD cell translation extract) was recovered and used in the following in vitro IRES-driven translation assay.
293T cells were co-transfected with (1) the recombinant plasmids pFLAG-CMV2-FBP2 and pcDNA3-HA-Ub or (2) pFLAG-CMV2-FBP2-K109,121,122R and pcDNA3-HA-Ub, using the X-tremeGENE transfection reagent. At 48 hours post-transfection, the transfected cells were harvested using a lysis buffer (50 mM Tris-HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% Triton-X-100) and placed on ice for 30 min. The resultant cell lysate was centrifuged at 12,000×g for 10 min, and the obtained supernatant was incubated with anti-FLAG M2 affinity gel (Sigma) at 4° C. for 16 hours. After washing five times with a wash buffer (50 mM Tris-HCl, pH 7.4, with 150 mM NaCl), the immunoprecipitated complex thus obtained was eluted with 3×FLAG peptides, so as to obtain an eluted product containing a FLAG-tagged ubiquitinated FBP2 protein.
For in vitro IRES-driven translation assay, the obtained eluted FLAG-tagged FBP2 protein (250 ng) was mixed with the following components to reach a final volume of 25 μL: 15 μL of the shFBP2-RD cell translation extract obtained above, 0.25 μg of EV71 5′UTR-FLuc reporter RNA as prepared in Example 5, a translation mixture (10 mM creatine phosphate, 50 μg/mL creatine phosphokinase, 79 mM KOAc, 0.5 mM Mg(OAc)2, 2 mM DTT, 0.02 mM hemin, 0.5 mM spermidine), 20 mM HEPES-KOH (pH 7.6), 20 μM amino acid mixture (Promega), 0.4 mM ATP (Promega) and an RNase inhibitor. The mixture thus formed was incubated at 30° C. for 90 min and then measured for firefly luciferase activity using the luciferase assay system (Promega).
The aforesaid experiments were conducted in triplicate, and the results were subjected to statistical analysis using Student's two-tailed unpaired t-test using Prism 6 software (GraphPad Software, San Diego, Calif.) where p<0.05 was considered to be statistically significant.
Results:
It was noted in Example 8 that both wild-type FBP2 and the reduced ubiquitination variant (i.e. FBP2-K109,121,122R) can associate with the EV71 5′UTR RNA. To further confirm whether the competitive capability of FBP2 against other positive ITAFs (such as FBP1) is affected by ubiquitination, the following experiments were conducted.
RD cells were transfected with the recombinant plasmid pFLAG-CMV2-FBP2 or pFLAG-CMV2-FBP2-K109,121,122R using the X-tremeGENE transfection reagent (Roche). At 48 hours post-transfection, the transfected cells were washed with PBS and lysed with a lysis buffer (containing 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF and 5 mM β-mercaptoethanol) for 30 minutes on ice. Afterwards, the resultant cell lysate was centrifuged at 10,000×g for 10 min at 4° C., and the supernatant thus formed was collected (serving as an RD cell extract) and stored at −80° C. for further analysis.
The obtained RD cell extract (200 μg) was mixed with the following components to reach a final volume of 100 μL: 12.5 pM of the biotinylated EV71 5′UTR RNA probe as obtained in Example 4, different concentrations (0, 0.5, 1 and 2 μM) of recombinant His-tagged wild-type FBP1, and an RNA mobility shift buffer (5 mM HEPES (pH 7.1), 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM DTT and 0.25 mg/mL heparin). After incubation at 30° C. for 15 min, the thus formed mixture containing protein-biotinylated RNA complexes was added to 400 μL of Streptavidin MagneSphere Paramagnetic Particles (Promega) for capturing at room temperature for 10 min. The captured product thus obtained was washed five times with a heparin-free RNA mobility shift buffer, after which 30 μL of a 2× sample buffer was added, followed by incubation for 10 min at room temperature to dissociate the protein-biotinylated RNA complexes. The thus formed sample containing the eluted protein was incubated at 95° C. for 5 min and then subjected to immunoblotting according to the procedures as described in the preceding section, entitled “4. Immunoblot analysis,” of the General Experimental Procedures. The primary and secondary antibodies used for detecting the respective protein in this example are shown in Table 10 below.
Results:
In order to investigate how the ubiquitination of FBP2 affects EV71 and CVB3 virus yield in infected RD cells, the following experiments were conducted.
A. Determination of Viral Protein Synthesis
shFBP2-RD cells obtained in Example 8 were transfected with a respective one of pFLAG-CMV2 vector as a vector control, pFLAG-CMV2-FBP2 and pFLAG-CMV2-FBP2-K109,121,122R.
At 24 hours post-transfection, 2.5×105 transfected cells were seeded into each well of a 12-well plate and incubated at 37° C. for additional 24 hours. The obtained cell culture was infected with EV71 at a m.o.i. of 10 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures. The medium was replaced with methionine-free DMEM, and incubation was continued at 37° C. for 1 hour. Afterwards, the medium was replaced with DMEM containing [35S]-methionine (50 μCi/mL) to label the newly synthesized viral proteins. After 1 hour of labeling, the cells were washed with PBS and lysed with a lysis buffer (150 mM NaCl, 50 mM Tris-Base, 1% IGEPAL CA630 (octylphenoxy poly(ethyleneoxy)ethanol), pH 8.0). The cell lysate was centrifuged at 10000×g for 10 min at 4° C. The resultant supernatant was subjected to SDS-PAGE, followed by transfer to a PVDF membrane and detection by autoradiography and immunoblotting.
Viral proteins seen from autoradiography were identified according to the protein size. The levels of [35S]-methionine-labeled VP1 and 3Cpro proteins were quantified and normalized against the actin level, based on two repeated experiments.
B. Plaque Assay
shFBP2-RD cells as obtained in Example 8 were transfected with a respective one of the following plasmids: pFLAG-CMV2 as a vector control, pFLAG-CMV2-FBP2, pFLAG-CMV2-FBP2-K109,121,122R, pFLAG-CMV2-FBP2-K109R, pFLAG-CMV2-FBP2-K121R and pFLAG-CMV2-FBP2-K122R. After incubation at 37° C. for 2 days, the transfected cells were subjected to virus infection with EV71 or CVB3 virus at a m.o.i. of 40 according to the procedures as described in the preceding section, entitled “1. Virus infection,” of the General Experimental Procedures. The EV71 and CVB3 viruses thus produced were harvested at 9 hours post-infection, and the virus titers were titrated according to the procedures as described in the preceding section, entitled “5. Viral plaque assay,” of the General Experimental Procedures. The virus titer in the FLAG vector control was set as 100%.
Statistical significance of the experimental data was analyzed by performing one-way ANOVA using Prism 6 software (GraphPad Software, San Diego, Calif.), where p<0.05 was considered to be statistically significant.
Results:
Similarly, as revealed in
All patents and references cited in this specification are incorporated herein in their entirety as reference. Where there is conflict, the descriptions in this case, including the definitions, shall prevail.
While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 106113165 A | Apr 2017 | TW | national |
| Entry |
|---|
| Kung et al. Control of the negative IRES trans-acting factor KHSRP by ubiquitination. Nucleic Acids Res. Jan. 9, 2017;45(1):271-287. Epub Nov. 28, 2016. (Year: 2017). |
| Huang et al. Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth. Nucleic Acids Res. Dec. 2011; 39(22): 9633-9648. (Year: 2011). |
| GenBank: ARV77948.1. FUBP1. (Year: 2017). |
| Zheng et al. Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site. Int J Biochem Cell Biol. Nov. 2011;43(11):1641-8. (Year: 2011). |
| Number | Date | Country | |
|---|---|---|---|
| 20180305422 A1 | Oct 2018 | US |
