Anti-influenza A virus micropeptide uYIPF2 coded by upstream open reading frame of YIPF2 gene and application thereof

Abstract

The invention discloses an anti-influenza A virus micropeptide uYIPF2 coded by an upstream open reading frame of a YIPF2 gene and application thereof, and relates to the technical field of biological medicines. The micropeptide uYIPF2 is coded by an upstream open reading frame (uORF) of a YIPF2 gene, contains 87 amino acids, and has a specific sequence as shown in SEQ ID No.1. A hemagglutination test and a plaque test prove that the micropeptide uYIPF2 can significantly reduce the HA titer and the virus load of the influenza A virus, and can significantly inhibit the replication of the influenza A virus. The invention reveals that the uYIPF2 micro-peptide has an anti-influenza A virus biological function for the first time, and provides application of the uYIPF2 micro-peptide in preparation of drugs for preventing and treating influenza A virus infection. The invention provides a brand-new drug target and a treatment strategy for the field of flu prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a micropeptide uYIPF2 encoded by the open reading frame upstream of the YIPF2 gene for resisting influenza A virus and its applications. Background Technology

[0002] Upstream open reading frames (uORFs), as important regulatory elements of the mRNA 5' untranslated region (5'UTR), play a crucial role in gene expression through translational regulation mechanisms. Cross-species comparative studies have shown that uORFs exhibit high sequence conservation in mammals, plants, and yeast, suggesting they play a core biological role in evolution. These elements can dynamically respond to environmental changes such as nutrient deficiency and oxidative stress, achieving rapid reprogramming of gene expression by regulating the translation efficiency of the main open reading frame (mORF). Notably, mutations or aberrant regulation of uORFs are closely associated with various diseases, such as tumorigenesis caused by oncogene translational dysregulation.

[0003] In recent years, breakthroughs in high-throughput technologies have greatly advanced the exploration of the encoding potential of uORFs. Ribosome profiling (Ribo-seq), by capturing ribosome protective fragments (RPFs), has confirmed that over 90% of uORFs possess translational activity during Drosophila development, while high-sensitivity mass spectrometry (MS) has identified numerous functional micropeptides. Currently known uORF-encoded micropeptides exhibit significant functional diversity, including the MP31 micropeptide regulating mitochondrial quality control in glioblastoma, the RNF10-derived immunomodulatory peptide serving as a T-cell antigen, and the endometrial microprotein SLC35A4 involved in metabolic regulation. These micropeptides can influence key physiological processes through interventions in signal transduction (such as virulence factors encoded by PRRSV viral uORFs) and cellular metabolic reprogramming.

[0004] The YIPF2 (Yip1 domain family member 2) gene encodes a Golgi-associated protein and belongs to the Yip1 domain family. Located on human chromosome 19q13.33, it encodes a 224-amino acid (25 kDa) protein containing a typical Yip1 domain. At the cellular functional level, YIPF2 not only mediates protein transport between the endoplasmic reticulum and the Golgi apparatus but also participates in DNA damage repair by maintaining genome stability. Disease-related studies show that YIPF2 has a dual regulatory role in tumors: on the one hand, it enhances the chemosensitivity of non-small cell lung cancer by promoting TNFRSF10B membrane localization; on the other hand, it abnormally activates the CIE glycoprotein transport pathway in liver cancer.

[0005] Influenza A virus (IAV), a representative member of the Orthomyxoviridae family, has a genome consisting of eight segmented negative-sense RNAs encoding at least 11 viral proteins. This virus has continuously evolved through antigenic drift and antigenic shift mechanisms involving surface hemagglutinin and neuraminidase, enabling it to spread across species in humans and animals (such as poultry and pigs), causing seasonal epidemics and large-scale outbreaks, posing a serious threat to livestock farming and human health. IAV control still faces significant challenges. The virus's high mutation rate leads to frequent vaccine escape and drug resistance, while the molecular mechanisms by which host factors regulate viral replication remain incompletely understood. Of particular note is the current lack of research on the role of uORF-encoded micropeptides in viral infection.

[0006] In summary, existing Ribo-seq data suggest the existence of a conserved uORF in the 5'UTR of the YIPF2 gene, but its coding potential has not yet been verified; the known membrane transport function of YIPF2 is potentially associated with influenza virus vesicle transport; however, there are currently no reports of uORF micropeptides regulating influenza virus replication. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a novel micropeptide encoded by an open reading frame upstream of the YIPF2 gene. This micropeptide can inhibit the replication of influenza A virus and can serve as a target for the design of anti-influenza virus drugs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] One objective of this invention is to provide a micropeptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] Furthermore, the nucleic acid sequence encoding the amino acids of the micropeptide is shown in SEQ ID No. 2.

[0011] Furthermore, the peptide is uYIPF2, which is encoded by the open reading frame upstream of the YIPF2 gene.

[0012] A second objective of this invention is to provide the application of any of the above-described micropeptides in the preparation of products that inhibit influenza virus replication.

[0013] A third objective of this invention is to provide the application of any of the above-described micropeptides in the preparation of products for the prevention and treatment of influenza.

[0014] Furthermore, the influenza virus includes influenza A virus.

[0015] Furthermore, the influenza A virus includes the H1N1 subtype influenza virus.

[0016] Furthermore, the H1N1 subtype influenza virus includes strain A / PR / 8 / 34 (ATCC VR-1469).

[0017] The fourth objective of this invention is to provide a product that inhibits the replication of influenza virus, wherein the product contains any of the micropeptides described above, and the product may be in the form of a drug, vaccine, reagent, or kit.

[0018] The fifth objective of this invention is to provide a product for combating and / or preventing influenza, wherein the product contains any of the micropeptides described above, and the product may be in the form of a drug, vaccine, reagent, or kit.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Hemagglutination and plaque assays revealed that, compared to the control group, overexpression of the micropeptide uYIPF2 from this invention significantly reduced the HA titer and viral load of influenza virus. This invention is the first to identify a micropeptide encoded by YIPF2 that inhibits influenza virus replication. This micropeptide can serve as a novel target for anti-influenza virus drugs, providing a completely new option for the preparation of anti-influenza virus drugs. Attached Figure Description

[0021] Figure 1 This is the detection result of Example 1 of the present invention, which determined that the YIPF2 gene contains a uORF with coding ability.

[0022] Figure 2 The results of detecting the encoding ability of uYIPF2 in Example 1 of this invention are shown. A shows that uYIPF2 can drive the re-expression of mutated and non-expressed GFP, emitting green fluorescence; B shows the fusion expression of uYIPF2 and GFP.

[0023] Figure 3This is the detection result of the endogenous micropeptide uYIPF2 in A549 cells by mass spectrometry in Example 1 of the present invention.

[0024] Figure 4 The results of the detection of the inhibitory ability of uYIPF2 on influenza A virus replication in Example 2 of this invention are shown. A shows the hemagglutination titer of the virus in the cell culture supernatant of the control group and the uYIPF2 overexpression group after infection with influenza A virus; B shows the viral load in the cell culture supernatant of the control group and the uYIPF2 overexpression group after infection with influenza A virus. * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01). Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0026] The cell sources used in this invention are as follows:

[0027] 293T cells (human embryonic kidney cell line): American Type Culture Collection (ATCC), catalog number CRL-3216.

[0028] A549 cells (human lung cancer cell line): American Type Culture Collection (ATCC), catalog number CCL-185.

[0029] MDCK cells (canine kidney cell line): American Type Culture Collection (ATCC), catalog number CCL-34.

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0031] Example 1: Identification of the micropeptide encoded by the open reading frame upstream of the YIPF2 gene

[0032] By combining Ribo-seq and RNA-seq data (already entered into the GEO database, accession numbers: GSE252920 and GSE252713, respectively), a uORF located upstream of the YIPF2 gene with the potential to encode a micropeptide was discovered. To verify its coding ability, it was studied and analyzed from both exogenous and endogenous perspectives.

[0033] First, the uORF was constructed into the pLVX3 vector (containing a 3×Flag tag) (Chi X, Huang G, Wang L. A small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy[J]. PLoS Pathogens, 2024, 20(8):e1012461), and the plasmid was named pLVX3-uYIPF2. It was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After verifying the sequencing results, subsequent cell experiments were conducted. The plasmid was transiently transfected into 293T cells, and the ability of the uORF to encode micropeptides was detected through exogenous expression. The specific steps are as follows:

[0034] 293T cells were used at a rate of 1×10 5 Cells were seeded at a density of 1 / well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum (Nanjing Novizan, catalog number F103-03), 100 U / mL penicillin, and streptomycin at 37°C and 5% CO2 until cell confluence reached 80-90%. Following the Lipofectamine 8000 transfection reagent (Beyotime, catalog number C0533) instructions, 2 μg of the empty pLVX3 vector or pLVX3-uYIPF2 plasmid was transiently transfected into the cells. 24 hours after transfection, cells were washed with PBS, collected by cell scraping, and centrifuged at 4500 rpm for 5 minutes at 4°C. After discarding the supernatant, 100 μL of lysis buffer containing 2 mM PMSF was added, and lysis was performed on ice for 20 minutes (vortexing for 10 seconds every 5 minutes). After centrifugation at 12000 rpm for 10 minutes at 4°C, the supernatant was mixed with loading buffer at a 4:1 volume ratio, and the mixture was incubated at 100°C for 5 minutes to denature the proteins. Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to an NC membrane. The membrane was blocked with 5% skim milk for 1 hour, and then sequentially incubated with Flag primary antibody (TransGold, catalog number HT201-01, 1:1000 dilution) and HRP-labeled secondary antibody (Sanying Biotech, catalog number SA00001-1, 1:5000 dilution). Detection was then performed using chemiluminescence immunoassay. Figure 1As shown, the uORF-encoded product with the 3×Flag tag exhibits a specific band at approximately 13 kDa (β-actin as an internal control), consistent with the theoretically predicted molecular weight. Three independent experiments confirmed that this uORF encodes a micropeptide. This uORF contains 264 bases and encodes a micropeptide composed of 87 amino acids. We named this micropeptide uYIPF2, and its amino acid sequence and the nucleic acid sequence encoding the amino acid are shown below:

[0035] The amino acid sequence of the micropeptide uYIPF2 (SEQ ID No. 1):

[0036] MRWRRRVTRPRSCRSSSSSSSRDSGPSATIRASLTWTPHRSWTGSKAHCCPGLATTLCGTICGIGRICMAPSGSVPRWPLSWPSLAT

[0037] Nucleic acid sequence encoding uYIPF2 (SEQ ID No. 2):

[0038] ATGAGGTGGAGGAGGAGAGTGACAAGGCCGCGCTCCTGCAGGAGCAGCAGCAGCAGCAGCAGCCGGGATTCTGGACCTTCAGCTACTATCAGAGCTTCTTTGACGTGGACACCTCACAGGTCCTGGACCGGA TCAAAGGCTCACTGCTGCCCCGGCCTGGCCACAACTTTGTGCGGCACCATCTGCGGAATCGGCCGGATCTGTATGGCCCCTTCTGGATCTGTGCCACGTTGGCCTTTGTCCTGGCCGTCACTGGCAACCTGA

[0039] Note: SEQ ID No. 1 is a single-letter abbreviation for amino acids. Its three-letter form is: MetArgTrpArgArgArgVal ThrArg Pro Arg Ser Cys Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser ArgAsp Ser Gly Pro Ser Ala Thr Ile Arg Ala Ser Leu Thr Trp Thr Pro His ArgSer Trp Thr Gly Ser Lys Ala His Cys Cys Pro Gly Leu Ala Thr Thr Leu Cys GlyThr Ile Cys Gly Ile GlyArg Ile Cys MetAla Pro Ser Gly Ser Val Pro Arg Trp ProLeu Ser Trp Pro Ser LeuAla Thr

[0040] Furthermore, the YIPF2 uORF was constructed into the pEGFP-N1 vector (named pEGFP-N1-mut) with a GFP start codon mutation. After transfection into cells, the expression of GFP green fluorescence and GFP protein was detected to determine whether the uORF could initiate the translation of mutant GFP, thereby determining its translational ability. The technical solution of this method is as follows:

[0041] YIPF2 uORF was constructed into the pEGFP-N1-mut vector, inserted at the front of the GFP sequence (Chi X, Huang G, Wang LA small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy[J]. PLoSPathogens, 2024, 20(8):e1012461), and the plasmid was named pEGFP-N1-mut-uYIPF2 and sequenced for verification. When the confluence of 293T cells in 6-well plates reached 80-90%, 2 μg of pEGFP-N1, 2 μg of pEGFP-N1-mut, and 2 μg of pEGFP-N1-mut-uYIPF2 were transfected into 293T cells according to the Lipofectamine 8000 transfection reagent instructions. After 24 h of culture, the presence or absence of cell fluorescence was observed by fluorescence microscopy. Figure 2As shown in Figure A, green fluorescence was observed in both cells transfected with the pEGFP-N1 plasmid and cells transfected with the pEGFP-N1-mut-uYIPF2 plasmid, but no fluorescence was observed in cells transfected with the pEGFP-N1-mut plasmid, indicating that uYIPF2 can initiate the translation of downstream mutant GFP.

[0042] Cells were then harvested, protein samples were prepared, and GFP expression was detected using Western blot analysis. The results are as follows: Figure 2 As shown in Figure B, GFP protein expression was detected in the pEGFP-N1 group, while no corresponding band was observed in the pEGFP-N1-mut group. In contrast, GFP expression was detected in the pEGFP-N1-mut-uYIPF2 group, and its molecular weight increased by approximately 10 kDa compared to wild-type GFP, indicating that uYIPF2 and GFP formed a fusion protein.

[0043] Finally, the endogenous expression of the micropeptide uYIPF2 in cells was detected by mass spectrometry. A549 cells cultured in 10cm culture dishes were harvested, lysed, and total protein lysate was obtained. SDS-PAGE was performed, and gel fragments below 15kDa were cut, followed by enzymatic digestion and LC-MS / MS mass spectrometry analysis. Results are as follows: Figure 3 As shown, mass spectrometry identified a peptide segment that matches the micropeptide uYIPF2: DSGPSATIRASLTWTPHRSWTGSK (SEQ ID No. 3), confirming the actual presence of uYIPF2 in the cellular endogenous system.

[0044] The above method can accurately determine that YIPF2 uORF has the ability to encode micropeptides.

[0045] Example 2: The peptide uYIPF2 encoded by the open reading frame upstream of the YIPF2 gene can inhibit the replication of influenza A virus.

[0046] To investigate the function of uYIPF2 in influenza A virus replication, this embodiment used a lentiviral expression vector system to construct an A549 cell model stably overexpressing uYIPF2 (Chi X, Huang G, Wang LA small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy[J]. PLoS Pathogens, 2024, 20(8):e1012461). The experimental steps are as follows:

[0047] When the cell culture confluence reaches 90%, wash the cells 2-3 times with PBS, add 1 mL of virus maintenance medium (serum-free DMEM containing 2 μg / mL trypsin) and H1N1 influenza virus A / PR / 8 / 34 with an MOI of 1, and incubate in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration for 1 hour, shaking the 6-well plate every 15 minutes. After 1 hour, discard the virus maintenance medium, wash the cells again with PBS 2-3 times, add 2 mL of fresh virus maintenance medium to each well, and continue culturing.

[0048] Different samples are collected depending on the different subsequent testing requirements:

[0049] Hemagglutination assay was used to detect influenza virus replication in cells: 200 μL of cell supernatant was collected at 16 h, 18 h, 20 h, 22 h, and 24 h after viral infection for hemagglutination assay. Results are as follows: Figure 4 As shown in Figure A, the hemagglutination titer of the experimental group was significantly lower than that of the control group, confirming that uYIPF2 can inhibit the replication of influenza virus in cells.

[0050] Plaque assay for detecting influenza virus viral load in cells: Cell supernatant was collected 16 hours after influenza virus infection treatment, and a plaque assay was performed. The specific steps are as follows:

[0051] MDCK cells were seeded in 6-well plates for later use (cell density 5 × 10⁶ cells / well). 5 (For best results, use a 6-well plate). Serially dilute the collected cell supernatant 10-fold with virus maintenance medium. After washing MDCK cells three times with PBS, add 0.1 mL of the diluted virus solution to each well of a 6-well plate, followed by 0.9 mL of virus maintenance medium to each well. Set up three parallel wells for each dilution. Incubate the cells in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 for 1 hour to allow virus adsorption (shaking the 6-well plate every 15 minutes). During virus adsorption, microwave-thaw 3% low-melting-point agarose (Promega, product number V2111-25g) and mix it with phenol red-free DMEM (HYCLONE, product number SH30284.01) at a 1:5 (volume ratio). Add TPCK-trypsin (Sigma, product number T8802-50MG) to a final concentration of 1 μg / mL for later use. One hour after virus adsorption, discard the supernatant, wash cells three times with PBS, and after aspirating as much PBS as possible, quickly add 2 mL of the prepared mixture to each well. Place the 6-well plate in a 4°C freezer for 1 hour to allow the gel to solidify, then invert it and incubate in a 37°C cell culture incubator for 2-3 days. When white spots appear, count the empty spots (or stain with crystal violet before counting). Results are as follows. Figure 4As shown in Figure B, the viral load of influenza in the experimental group was significantly lower than that in the control group, further confirming that the micropeptide uYIPF2 can significantly inhibit the replication of influenza virus.

[0052] Through the above experiments, this embodiment confirms the function of uYIPF2 in inhibiting influenza virus replication.

[0053] In summary, this invention provides a novel micropeptide, and experiments have demonstrated that this novel micropeptide can significantly reduce the HA titer and viral load of influenza A virus, and can significantly inhibit the replication of influenza A virus. This novel micropeptide provides a novel drug target and treatment strategy for the field of influenza prevention and control, and is of great significance in the field of influenza prevention and control.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A micropeptide, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The micropeptide according to claim 1, characterized in that, The nucleic acid sequence encoding its amino acids is shown in SEQ ID No.

2.

3. The micropeptide according to claim 2, characterized in that, The peptide is uYIPF2, which is encoded by the open reading frame upstream of the YIPF2 gene.

4. The use of the micropeptide according to any one of claims 1 to 3 in the preparation of a product that inhibits influenza virus replication.

5. The use of the micropeptide according to any one of claims 1 to 3 in the preparation of products for the prevention and treatment of influenza.

6. The application according to claim 5, characterized in that, The influenza viruses mentioned include influenza A viruses.

7. The application according to claim 6, characterized in that, The influenza A virus includes the H1N1 subtype of influenza virus.

8. The application according to claim 7, characterized in that, The H1N1 subtype influenza virus includes strain A / PR / 8 / 34.

9. A product for inhibiting influenza virus replication, characterized in that, The product contains the micropeptide according to any one of claims 1 to 3, and the product form includes pharmaceuticals, vaccines, reagents, or kits.

10. A product for treating and / or preventing influenza, characterized in that, The product contains the micropeptide according to any one of claims 1 to 3, and the product form includes pharmaceuticals, vaccines, reagents, or kits.

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