Application of PTBP1 as a diagnostic biomarker and therapeutic target for Zika virus infection

Abstract

This invention belongs to the field of antiviral drug technology, and particularly relates to the application of PTBP1 as a diagnostic biomarker and therapeutic target for Zika virus infection. The study found a correlation between PTBP1 expression levels and Zika virus infection (ZIKV). With prolonged ZikaKV infection, the expression levels of PTBP1 gene and protein in host cells significantly increased. Furthermore, upregulating the expression levels of PTBP1 gene or protein in host cells effectively inhibited ZikaKV replication at both transcriptional and translational levels, blocking ZikaKV's ability to infect host cells, while knocking down PTBP1 promoted ZikaKV replication. This indicates that PTBP1 can serve as a novel diagnostic biomarker and therapeutic target for Zika virus infection, providing new ideas and strategies for the diagnostic assessment of Zika virus-related diseases and for the clinical prevention and / or treatment of various diseases caused by ZikaKV infection.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, and in particular to the application of PTBP1 as a diagnostic biomarker and therapeutic target for Zika virus infection. Background Technology

[0002] Zika virus (ZIKV) is a member of the genus Flavivirus in the family Flaviviridae. It is an insect-borne, single-stranded, positive-sense RNA virus and the most common cause of microcephaly and Guillain-Barré syndrome in newborns. In recent years, Zika virus has ravaged many countries and regions around the world, posing a serious challenge to public health security. Patients infected with Zika virus may experience mild symptoms such as fever, headache, muscle pain, conjunctivitis, and rash, while severe cases may face serious neurological complications, such as microcephaly in fetuses and Guillain-Barré syndrome in adults. Unfortunately, there are currently no specific vaccines or antiviral drugs available for the prevention and treatment of Zika virus infection.

[0003] Although multiple ZIKV vaccine candidates are under development, with some already in clinical trials, none have yet been approved for use. In terms of drug development, the design of anti-ZIKV drugs primarily focuses on antiviral targets within the virus itself or host cells; however, most remain in the clinical research stage, and no anti-ZIKV drug has yet received production approval. Therefore, drug development against ZIKV is particularly urgent and of great significance for ensuring global public health security. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a PTBP1 gene or protein whose expression level is correlated with ZIKV infection. With prolonged ZIKV infection time, the expression levels of PTBP1 gene and protein in host cells significantly increase. Furthermore, upregulating the expression level of PTBP1 gene or protein in host cells can effectively inhibit ZIKV replication at both the transcriptional and translational levels, blocking ZIKV's ability to infect host cells, while knocking down PTBP1 promotes ZIKV replication. This indicates that PTBP1 can serve as a novel diagnostic biomarker and therapeutic target for Zika virus infection, providing new ideas and strategies for the application of PTBP1 in the diagnosis and assessment of Zika virus-related diseases, as well as the clinical prevention and / or treatment of various diseases caused by ZIKV infection. This invention is specifically achieved through the following technical solutions:

[0005] The first aspect of this invention provides the application of PTBP1 as a diagnostic biomarker in the preparation of auxiliary diagnostic reagents or kits for Zika virus infection.

[0006] This invention elucidated the expression characteristics of PTBP1 in a ZIKV-infected cell model, revealing that the levels of PTBP1 mRNA and protein were significantly increased in infected cells, and that PTBP1 expression levels increased with prolonged infection time, indicating a correlation between changes in PTBP1 expression levels and ZIKV infection. Therefore, PTBP1 expression levels can serve as a diagnostic biomarker for ZIKV infection, providing a new approach for the diagnosis of ZIKV-related diseases.

[0007] Furthermore, the diagnostic reagent or kit detects the expression level of PTBP1 in the subject's in vitro sample, and an elevated PTBP1 expression level indicates that the subject is at risk of Zika virus infection.

[0008] Furthermore, the diagnostic reagent or kit includes a primer pair for specifically amplifying the PTBP1 gene, the nucleotide sequences of the upstream and downstream primers of the primer pair being as shown in SEQ ID NO. 6-7, or includes an antibody that specifically binds to the PTBP1 protein.

[0009] Based on the same inventive concept as described above, a second aspect of the present invention provides the application of a reagent for detecting PTBP1 expression levels in the preparation of auxiliary diagnostic reagents or kits for Zika virus infection.

[0010] Furthermore, the reagent for detecting the PTBP1 expression level is selected from at least one of primer pairs that specifically amplify the PTBP1 gene or antibodies that specifically bind to the PTBP1 protein.

[0011] The third aspect of this invention provides the use of PTBP1 as a therapeutic target in the preparation of drugs for the prevention and / or treatment of Zika virus infection.

[0012] This invention elucidates the effect of PTBP1 on Zika virus infection. By investigating the differences in ZIKV infectivity between cell lines overexpressing and knocking down PTBP1, it was found that upregulating PTBP1 expression levels helps reduce viral replication and infectivity, exhibiting a good antiviral effect at the cellular level; conversely, knocking down PTBP1 promotes ZIKV replication. These findings clearly demonstrate that PTBP1 plays a crucial role in inhibiting ZIKV replication. Using PTBP1 as a therapeutic target for Zika virus infection demonstrates good antiviral effects, providing a new and effective treatment for the prevention and / or treatment of ZIKV-related diseases, and has significant practical implications and application value for the clinical treatment and prevention of ZIKV infection.

[0013] Furthermore, the drug includes a reagent that upregulates the expression level of PTBP1, which is used as the active ingredient of the drug. By enhancing the expression of the PTBP1 gene or protein, it achieves the effect of resisting ZIKV infection and reducing ZIKV replication, thus providing an effective prevention and treatment approach for inhibiting ZIKV.

[0014] Based on the same inventive concept as described above, the fourth aspect of this invention provides the use of a reagent that upregulates PTBP1 expression levels in the preparation of drugs for the prevention and / or treatment of Zika virus infection.

[0015] Furthermore, the reagents for upregulating PTBP1 expression levels described above are selected from at least one of the following: reagents for increasing the expression level of PTBP1 mRNA, reagents for increasing the expression level of PTBP1 protein, or reagents for enhancing the activity of PTBP1 protein.

[0016] Furthermore, the reagent used to upregulate PTBP1 expression level is selected from PTBP1 overexpression plasmids or HIF-1α activators.

[0017] Furthermore, the PTBP1 overexpression plasmid is pLV3-CMV-PTBP1-3×FLAG-Puro, which is obtained by inserting the PTBP1 gene into the multiple cloning site of the original plasmid pLV3-CMV-MCS-3×FLAG-Puro. Exemplarily, the multiple cloning site can be between the EcoRI and BamHI restriction sites.

[0018] Furthermore, the HIF-1α activator is IOX2 (CAS No.: 931398-72-0), with the following structural formula:

[0019]

[0020] In this invention, the nucleotide sequence of the PTBP1 gene is shown in SEQ ID NO.1. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the change in PTBP1 mRNA expression level after ZIKV infection of A549 cells in an embodiment of the present invention.

[0023] Figure 2The graph shows the changes in intracellular PTBP1 protein expression levels in A549 cells after ZIKV infection in an embodiment of the present invention. From left to right, the graph represents the protein expression levels at different ZIKV infection doses and at different ZIKV infection times.

[0024] Figure 3 This is a graph showing the changes in the expression levels of ZIKV NS5, HIF-1α, and PTBP1 proteins over time after ZIKV infection of A549 cells in an embodiment of the present invention.

[0025] Figure 4 This is a graph showing the changes in HIF-1α and PTBP1 expression levels in A549 cells after pretreatment with different concentrations of IOX2 according to an embodiment of the present invention.

[0026] Figure 5 This is a graph showing the changes in the expression levels of ZIKV NS5, HIF-1α, and PTBP1 over time after ZIKV infection of A549 cells pretreated with IOX2 in an embodiment of the present invention.

[0027] Figure 6 This is a graph showing the changes in the expression levels of ZIKVNS5, HIF-1α, and PTBP1 over time after ZIKV infection of YC-1 pretreated A549 cells in an embodiment of the present invention.

[0028] Figure 7 This figure shows the verification results of PTBP1 gene overexpression in the A549 cell line stably expressing the PTBP1 gene in an embodiment of the present invention.

[0029] Figure 8 This is a graph showing the changes in the expression levels of PTBP1 and ZIKV NS5 mRNA over time in an A549 cell line overexpressing the PTBP1 gene infected with ZIKV, as an embodiment of the present invention.

[0030] Figure 9 This is a graph showing the changes in the expression levels of PTBP1 and ZIKV NS5 proteins over time in an A549 cell line overexpressing the PTBP1 gene infected with ZIKV, as an embodiment of the present invention.

[0031] Figure 10 This is a statistical graph showing the change in viral titer over time in an A549 cell line overexpressing the PTBP1 gene infected with ZIKV, as an embodiment of the present invention.

[0032] Figure 11 This figure shows the verification results of PTBP1 gene knockout in the A549 cell line with stable PTBP1 gene knockdown constructed in this embodiment of the invention.

[0033] Figure 12This is a graph showing the changes in the expression levels of PTBP1 and ZIKV NS5 mRNA over time in the A549 cell line with ZIKV-infected PTBP1 gene knockdown, as an embodiment of the present invention.

[0034] Figure 13 This is a graph showing the change in the expression levels of PTBP1 and ZIKV NS5 proteins over time in the A549 cell line with ZIKV-infected PTBP1 gene knockdown, as an embodiment of the present invention.

[0035] Figure 14 This is a statistical graph showing the change in viral titer over time in the A549 cell line with ZIKV infection and PTBP1 gene knockdown, as described in this embodiment of the invention.

[0036] Figure 15 Figure 1 shows the effect of activating HIF-1α expression on ZIKV infection in the A549 cell line with knocked-down PTBP1 gene, according to an embodiment of the present invention. Figure 2 shows the change in ZIKV NS5 mRNA expression level with infection time in cells pretreated with different concentrations of IOX2 by ZIKV infection. Figure 3 shows the change in the expression levels of PTBP1, HIF-1α and ZIKV NS5 proteins with infection time in cells pretreated with different concentrations of IOX2 by ZIKV infection. Figure 4 shows the change in viral titer with infection time in cells pretreated with different concentrations of IOX2 by ZIKV infection. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0038] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0039] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0040] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0041] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0042] The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is considered to include (i) A, (ii) B, and (iii) A and B.

[0043] The term "gene" refers to the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. Therefore, gene expression includes transcription and the stable accumulation of gene-coding RNA (mRNA) or functional RNA, and can also refer to the translation of mRNA into polypeptides or proteins.

[0044] The term "vector" refers to a self-replicating DNA molecule, often in the form of a circular double-stranded DNA molecule, used to transfer a target gene into a host organism (e.g., the A549 cells of this invention). Vectors containing the target gene are called recombinant vectors. Typical vectors include plasmids, viruses, bacteriophages, granules, and minichromosomes. Plasmids are the most common form of vector; therefore, in the context of this invention, plasmids and vectors are used interchangeably. An "expression vector" allows the inserted target gene to be expressed after introduction into a host organism. It contains regulatory elements, such as promoters and enhancers, for expression in the specified host organism.

[0045] The terms "import" or "transfer" refer to the transfer of a target gene nucleic acid molecule into a host organism, resulting in the stable inheritance of the gene. The imported nucleic acid molecule can be in plasmid form retained in the host organism or can be integrated into the host organism's genome. Nucleic acid molecules and / or vectors can be transferred into a host organism via methods such as "transfection," "transformation," or "transduction." A host organism containing the imported nucleic acid molecule is referred to as a "transgenic," "recombinant," "transformed," or "engineered" organism. Vector introduction into a host organism can be performed using conventional techniques well known to those skilled in the art.

[0046] Terms such as “overexpression,” “hyperexpression,” or “overexpression” refer to gene expression levels that exceed normal expression levels. In preferred embodiments, gene expression levels are at least 10%, 20%, 30%, 40%, 50%, 100% (2-fold), 200% (3-fold), 300% (4-fold), or even more times higher than normal expression levels.

[0047] The terms "knockdown" or "knockout" refer to techniques that reduce the expression level or inactivate the function of a specific gene through certain means. In the context of this invention, "knockdown," "knockout," "deletion," or "silencing" have the same meaning. Gene knockout methods include, but are not limited to, gene knockout technology based on homologous recombination, CRISPR-Cas9 gene knockout technology, and gene knockout using random mutations.

[0048] Compare C T The value method is a commonly used method for studying the relative expression levels of genes. The quantitative results are obtained by combining the target gene and the internal reference gene C. T The difference between values ​​(△C) T This is reflected in the term "C". T "C-value" refers to the number of cycles required for the fluorescence signal in each reaction tube to reach a set threshold. The C-value for each sample template... T The value of C is linearly related to the logarithm of the initial copy number of the template; the higher the initial copy number, the better the C value. T The smaller the value.

[0049] It is important to emphasize that, in the context of this invention, the "expression level" of a gene includes the mRNA level at the transcriptional level or the protein level at the translational level. Accordingly, unless otherwise specified, PTBP1 can refer to either a gene or a protein. For example, when PTBP1 is used as a diagnostic biomarker or a therapeutic target, it can refer to either the PTBP1 gene or the PTBP1 protein.

[0050] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will now be described in detail.

[0051] Polypyrimidine tract-binding protein 1 (PTBP1), also known as PTB or heterogeneous ribonucleoprotein I (hnRNP I), belongs to the hnRNP family. Its gene (nucleotide sequence shown in SEQ ID NO.1) is located on human chromosome 19p13.3. The coding region contains 16 exons, encoding a protein with 531 amino acids (AA) and a relative molecular mass of 57 kDa. It is a shuttle protein primarily located in the cell nucleus. The PTBP1 protein structure consists of four non-standard RNA recognition motif domains (RRM), one nucleocytoplasmic shuttle domain (NLD), and a nuclear export signal (NES) at the N-terminus of the protein. It participates in regulating mRNA splicing, translation, localization, and stability.

[0052] This invention analyzes the changes in PTBP1 expression in cells under different times and doses of Zika virus (ZIKV) infection using Western blot and qRT-PCR experiments. The results show that the levels of PTBP1 gene and protein significantly increase with prolonged ZIKV infection time, suggesting that upregulation of PTBP1 plays a crucial role in viral infection and replication. Subsequently, based on cell lines with stable PTBP1 gene expression or knockout, Western blot, qRT-PCR, and tissue culture half-maximal infectious dose (TCID50) were used to further analyze PTBP1 expression. 50 Experiments such as [list of experiments] investigated the effects of overexpression or knockdown of the PTBP1 gene on ZIKV infectivity. They found that enhancing PTBP1 expression significantly inhibited ZIKV infection, reduced the expression of ZIKV-specific mRNA and protein, suppressed the production of ZIKV progeny viruses, and reduced the viral titer of infected cells. Conversely, reducing the expression of this gene promoted viral replication, revealing that the PTBP1 gene and its protein have important functions in inhibiting ZIKV replication and possess antiviral activity.

[0053] The above findings clearly demonstrate the correlation between the PTBP1 gene or protein and Zika virus infection, making it a novel potential diagnostic biomarker and therapeutic target. Using PTBP1 as a diagnostic biomarker for Zika virus infection, and examining changes in its expression level to aid in the diagnosis of Zika virus infection, can significantly improve the sensitivity and specificity of diagnosing Zika virus-related diseases. Furthermore, using PTBP1 as a therapeutic target for Zika virus infection, upregulating the expression level of the PTBP1 gene or protein can effectively inhibit Zika virus replication at both the transcriptional and translational levels, blocking Zika virus's ability to infect host cells, exhibiting strong antiviral activity. This provides new ideas and strategies for the clinical prevention and / or treatment of various diseases caused by Zika virus infection.

[0054] Furthermore, this invention discovered that ZIKV infection may activate hypoxia-inducible factor 1α (HIF-1α) by inducing oxidative stress, thereby upregulating PTBP1 expression. Specifically, cells were pretreated with the HIF-1α activator IOX2 and the inhibitor YC-1 before being infected with ZIKV. Western blot and qRT-PCR experiments were used to detect PTBP1 expression levels in infected cells. The results showed that after activating HIF-1α expression and then infecting with ZIKV, both the mRNA and protein expression levels of PTBP1 in the cells significantly increased. Conversely, inhibiting HIF-1α expression with YC-1 significantly suppressed ZIKV-induced PTBP1 expression, revealing the specific molecular mechanism by which ZIKV infection upregulates PTBP1. Subsequently, different concentrations of the HIF-1α activator IOX2 were used to stimulate HIF-1α to upregulate PTBP1 expression or enhance its functional activity, showing that viral infection was significantly inhibited, verifying the inhibitory effect of IOX2 on ZIKV. Furthermore, IOX2 exhibited good antiviral effects at the cellular level. These results further suggest that upregulating PTBP1 expression may become an effective therapeutic approach for inhibiting ZIKV, and provide a theoretical basis for the application of HIF-1α activators in the development of anti-ZIKV drugs, potentially contributing new strength and research ideas to the fight against viral diseases.

[0055] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or conditions recommended by the manufacturer. Furthermore, unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0056] Main experimental reagents: Plasmid vectors were obtained from Addgene, independently constructed, or obtained through academic exchange. Additionally, DMEM culture medium and fetal bovine serum (FBS) were purchased from GIBCO (catalog numbers C11885500BT and 10099-141, respectively); ZIKV NS5 antibody was purchased from Genetex (catalog number GTX133312); PTBP1 protein antibody was purchased from Proteintech (catalog number 12582-1-AP); GAPDH was purchased from Gibco (catalog number G9295); HIF-1α antibody was purchased from Proteintech (catalog number 20960-1-AP); FLAG antibody was purchased from Sigma (catalog number F3165); HA antibody was purchased from ABclonal (catalog number AE025); and mouse and rabbit secondary antibodies were purchased from Proteintech. HIF1α activator IOX2 and HIF1α inhibitor YC-1 were purchased from MCE, catalog numbers FG-4592 and HY-14927, respectively; HRP luminescent substrate reaction solution was purchased from Bio-Rad, catalog number 1705061.

[0057] Major experimental instruments: Cell culture incubator (Thermofisher); Real-time quantitative PCR instrument (Roche); Vertical electrophoresis apparatus (Bio-rad); Bioluminescence analyzer (Fujifilm).

[0058] Cells and Viruses: The cells and viruses used were obtained from cell and microbial resource banks of ATCC or other relevant institutions. Specifically, the three cell types used—human embryonic kidney 293T cells (HEK293T cells, ATCC#CRL-11268), human alveolar basal epithelial cells for lung cancer (A549 cells, ATCC#CCL-185), and Aedes albopictus cells (C6 / 36, ATCC#CRL-1660)—were all purchased from the American Type Culture Collection (ATCC). Human embryonic rhabdomyosarcoma (RD) cells (resource number: 3142C0001000000321) were obtained from the China Center for Type Culture Collection (CCTCC). The ZIKV used was the Asian strain isolate z16006 (GenBank accession number: KU955589.1), obtained from the Institute of Pathogenic Microbiology, Guangdong Provincial Center for Disease Control and Prevention. It was expanded in C6 / 36 cells and aliquoted and stored at -80°C.

[0059] Genes and primers: The PTBP1 gene sequence and primers used for real-time quantitative PCR (qRT-PCR) are shown in Table 1.

[0060] Table 1 Sequence information of genes and primers used in the embodiments of the present invention.

[0061]

[0062]

[0063] 1. ZIKV infection upregulates PTBP1 expression levels

[0064] This example identifies the effect of ZIKV infection on PTBP1 expression levels in the A549 cell line, as follows:

[0065] A549 cells were seeded into 6-well plates and, when the cells reached 80-90% confluence, the medium was replaced with serum-free DMEM. The A549 cells were then infected with ZIKV at MOIs of 0, 0.25, 0.5, and 1.0 for 24 h, or with ZIKV at MOI = 1 for 12 h, 24 h, and 48 h. The virus-infected cells were cultured at 37°C in a 5% CO2 incubator for 2 h, followed by replacement with 2% FBSDMEM medium. Cells were harvested at the corresponding time points, and PTBP1 mRNA levels were detected by Western blot, while PTBP1 protein levels were detected by qRT-PCR.

[0066] Western blot analysis of PTBP1 protein content included the following steps: A549 cells were infected with ZIKV at different MOIs (MOI = 0, 0.25, 0.5, 1.0) for 24 h, or infected with A549 cells at MOI = 1 for different time periods (0 h, 12 h, 24 h, 48 h). Cells were collected, and total cellular protein was extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined using the BCA method. An SDS-PAGE gel was prepared, and protein samples and protein markers were added for vertical electrophoresis. After electrophoresis, the protein was transferred to a 0.22 μm PVDF membrane and incubated under constant current for 2 h. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then primary antibodies (PTBP1, ZIKV NS5, or GAPDH protein antibodies) diluted 1:1000 were added. The membrane was incubated overnight at 4°C, and washed three times with TBST buffer the next morning for 5 min each time. Add the corresponding species secondary antibody diluted at a ratio of 1:5000, incubate at room temperature for 1 hour, wash three times with TBST buffer for 5 minutes each time. Add the chromogenic solution and incubate for 3-5 minutes, then expose on a chemiluminescence imaging system.

[0067] The qRT-PCR experiment for detecting mRNA protein content included the following steps: A549 cells were infected with ZIKV at different MOIs (MOI = 0, 0.25, 0.5, 1.0) for 24 h, or infected with A549 cells at MOI = 1 for different time periods (0 h, 12 h, 24 h, 48 h). Cells were then collected, and total RNA was extracted using TRIzol reagent. The concentration and purity of total RNA were detected using a Nanodrop micro-spectrophotometer. RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Beijing TransGen Biotech Co., Ltd.). The reaction program was: incubation at 50℃ for 5 min, heating at 85℃ for 2 min, and infinite cycling at 4℃. qRT-PCR experiments were performed using a Roche real-time quantitative PCR instrument. Primers used for qRT-PCR are listed below.

[0068] Table 1 shows SEQ ID NO. 6-11, and the reaction procedure is shown in Table 2. Gene expression data were analyzed using the 2-ΔΔCt method, with three replicates for each sample as parallel experiments.

[0069] Table 2 qRT-PCR reaction procedure

[0070]

[0071] Figure 1 The changes in intracellular PTBP1 mRNA expression levels after ZIKV infection of A549 cells are shown. The top figure, from left to right, shows the detection results of PTBP1 mRNA and ZIKV mRNA expression levels in A549 cells at different ZIKV infection doses (MOI). The bottom figure, from left to right, shows the detection results of PTBP1 mRNA and ZIKV mRNA expression levels in A549 cells at different infection times (h pi). GAPDH was used as an internal reference gene.

[0072] Figure 2 The changes in intracellular PTBP1 protein expression levels after ZIKV infection of A549 cells are shown. From left to right, the graphs show the detection results of intracellular PTBP1 protein expression levels in A549 cells under different ZIKV infection doses (MOI) and different ZIKV infection times (h pi). In the graphs, ZIKV-NS5 represents the NS5 protein of ZIKV, which is used to indicate the ZIKV virus level, PTBP1 is the PTBP1 protein, and GAPDH is the internal reference protein.

[0073] The results showed that with the increase of ZIKV infection dose or infection time, the transcription level of PTBP1 gene in cells showed a significant upward trend, and the protein expression level was consistent with the trend of mRNA level. This indicates that ZIKV infection can upregulate the expression of PTBP1 gene and protein in cells, suggesting that PTBP1 may play an important role in the regulation of ZIKV infection.

[0074] 2. ZIKV upregulates PTBP1 expression levels by activating HIF-1α.

[0075] Studies have shown that ZIKV infection can trigger hypoxia and cellular stress responses, a process that appears to be closely related to the mediation of hypoxia-inducible factor 1-alpha (HIF-1α). Under hypoxic conditions, HIF-1α expression remains stable and binds to HIF-1β, forming HIFs with activity regulating downstream signaling pathways and gene expression, thereby modulating cellular adaptive and inflammatory responses. Studies have also found that under hypoxic conditions, HIF-1α can directly bind to the promoter region of mouse PTBP1, thereby upregulating PTBP1 expression. Based on the aforementioned research, this invention hypothesizes that ZIKV infection may activate HIF-1α by inducing oxidative stress, thereby upregulating PTBP1 expression.

[0076] In this embodiment, A549 cells were pretreated with a HIF-1α activator and inhibitor by ZIKV infection, and then the intracellular PTBP1 expression level was measured to explore the specific molecular mechanism by which ZIKV infection leads to PTBP1 upregulation. The following procedures were performed:

[0077] (1) A549 cells were infected with ZIKV at MOI=1. Cell samples were collected at 12h, 24h, and 48h post-infection. The protein levels of ZIKV NS5, HIF-1α, and PTBP1 were detected by Western blot to investigate the effect of viral infection on HIF-1α expression. The results are shown in […]. Figure 3 .

[0078] (2) After stimulating A549 cells with different concentrations of IOX2 (0, 20, 40 μM) for 12 h, qRT-PCR and Western blot were used to detect the PTBP1 mRNA level and HIF-1α and PTBP1 protein levels, respectively, to investigate the effect of HIF-1α activation on PTBP1 expression. The results are shown in […]. Figure 4 . Figure 4 In the middle, the left image shows the qRT-PCR detection of PTBP1 mRNA levels, and the right image shows the Western blot detection of HIF-1α and PTBP1 protein levels.

[0079] (3) A549 cells were pretreated with HIF-1α activator IOX2 (40 μM) for 12 h, followed by infection with ZIKV at MOI=1 for 24 h. Cells were then collected, and PTBP1 mRNA levels and ZIKV NS5, HIF-1α, and PTBP1 protein levels were detected by qRT-PCR and Western blot, respectively. Results are shown in [Figure number missing]. Figure 5 . Figure 5 In the middle, the left image shows the qRT-PCR detection of PTBP1 mRNA levels, and the right image shows the Western blot detection of ZIKV NS5, HIF-1α, and PTBP1 protein levels. DMSO represents the untreated group, which is compared with the IOX2 group as a control.

[0080] (4) A549 cells were pretreated with the HIF-1α inhibitor YC-1 (20 μM) for 12 h, followed by infection with ZIKV at MOI=1 for 24 h. Cells were then collected, and PTBP1 mRNA levels and ZIKV NS5, HIF-1α, and PTBP1 protein levels were detected by qRT-PCR and Western blot, respectively. Results are shown in [Figure number missing]. Figure 6 . Figure 6 In the middle, the left image shows the qRT-PCR detection of PTBP1 mRNA levels, and the right image shows the Western blot detection of ZIKV NS5, HIF-1α, and PTBP1 protein levels. DMSO represents the untreated group, which is compared with the IOX2 group as a control.

[0081] The results showed that the protein expression levels of both HIF-1α and PTBP1 were significantly upregulated at 24 and 48 hours after ZIKV infection. Furthermore, in the uninfected state, activation of intracellular HIF-1α expression with an HIF-1α activator resulted in a significant increase in both PTBP1 mRNA and protein expression levels with increasing IOX2 concentration. These results demonstrate that activating HIF-1α expression effectively upregulates PTBP1 expression. Subsequent cell pretreatment with HIF-1α activators and inhibitors to regulate HIF-1α levels revealed that after HIF-1α activation followed by ZIKV infection, both intracellular PTBP1 mRNA and protein expression levels significantly increased further, while ZIKV protein expression was inhibited. Furthermore, inhibition of HIF-1α expression with YC-1 significantly suppressed the ZIKV-induced upregulation of PTBP1 expression. This further confirms that ZIKV indeed upregulates PTBP1 expression by activating HIF-1α and can influence the ZIKV infection process. This also suggests that using drugs to stimulate HIF-1α to upregulate the expression level of PTBP1 or enhance its functional activity may become an effective treatment for ZIKV.

[0082] 3. Overexpression of the PTBP1 gene inhibits the replication of ZIKV in cells.

[0083] To further explore the potential association between PTBP1 and ZIKV replication, this embodiment constructed an A549 cell line stably expressing PTBP1 and used this cell line to conduct ZIKV infection experiments to examine the specific effect of PTBP1 overexpression on ZIKV expression. The procedure is as follows:

[0084] 1) Construction of overexpression vector: PTBP1 amplification primers (see SEQ ID NO. 2-3 in Table 1) were designed using Snapgene software and synthesized at Guangzhou Qingke Biotechnology Co., Ltd. Using A549 cell cDNA as a template, the PTBP1 gene was amplified by PCR. The PCR reaction system consisted of: 1 μL cDNA, 2 μL each of forward and reverse primers (10 μmol / L), 10 μL 2×PhantaMax MasterMix, and ddH2O to a final volume of 20 μL. The PCR program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min. Subsequently, the vector pLV3-CMV-MCS-3×FLAG-Puro was digested with restriction endonucleases EcoRI and BamHI. Subsequently, the amplified PTBP1 gene fragment was homologously recombinated with the enzyme-digested pLV3-CMV-MCS-3×FLAG-Puro vector to obtain the overexpression vector pLV3-CMV-PTBP1-3×FLAG-Puro. The ligation product was then transformed into DH5α competent cells. After plating, the cells were incubated overnight. The next day, single colonies were picked for PCR verification, and positive colonies were sent for sequencing.

[0085] 2) Construction of an A549 cell line stably expressing the PTBP1 gene: The overexpression vector pLV3-CMV-PTBP1-3×FLAG-Puro, verified by sequencing, was extracted and transfected into HEK293T cells using Lipo2000 transfection reagent at a ratio of 3.75:1.25:5. The psPAX2 packaging plasmid, pMD2.G envelope plasmid, and pLV3-CMV-PTBP1-3×FLAG-Puro recombinant plasmid (or corresponding empty vector) were then transfected. Forty-eight hours after transfection, the protein expressed by the plasmid assembled into lentivirus within the cells. The supernatant was collected and centrifuged to obtain a clear viral solution. Subsequently, 1 mL of lentivirus solution, 1 mL of 10% FBSDMEM medium, and an appropriate amount of polybrene (1:1000 ratio) were mixed and used to infect A549 cells for 48 hours. Cell selection was performed by culturing in puro-containing medium for one week until no cell death was observed, confirming successful selection. On the third day of screening, cell samples were collected, and their expression efficiency was detected using Western blot. The results are shown below. Figure 7 In this context, LV-Vector represents A549 cells transfected with the empty vector, and LV-PTBP1 represents A549 cells transfected with the PTBP1 gene. It is evident that the PTBP1 protein FLAG tag was clearly detected in the PTBP1-transfected cells, while it was not detected in the Vector control cells, demonstrating the successful construction of a stable A549 cell line overexpressing PTBP1.

[0086] 3) ZIKV infection of LV-PTBP1 cell lines: PTBP1 stable cells and Vector control cells were infected with ZIKV at MOI=1. Cell samples were collected at 24h and 48h post-infection. qRT-PCR and Western blot were used to detect changes in the expression of ZIKV and PTBP1 (FLAG-tagged) at the mRNA and protein levels. Additionally, viral supernatants were collected from PTBP1 stable cells and Vector control cells 24h and 48h after ZIKV infection and used to infect RD cells. The number of cell wells showing lesions was observed and counted, and the viral titer (PFU) was calculated.

[0087] TCID 50 The experimental determination of viral titer includes the following steps: (i) Seeding RD cells in 96-well cell culture plates and culturing overnight until the cells reach 80%-90% cell growth. (ii) Diluting the viral solution 10-fold to 8 concentration gradients using serum-free DMEM medium, i.e., diluting ZIKV 10-fold. 1 Up to 10 8The diluted ZIKV was added to 96-well plates according to a concentration gradient and placed in a 37°C, 5% CO2 incubator. The cells were infected with the virus for 2 hours. After 2 hours, the virus inoculation solution was discarded, and 100 μL of DMEM medium containing 2% FBS was added to each well. The plates were then incubated for 3-5 days. Eight replicates were set up for each dilution, and a DMEM group was set up as a negative control. (iii) Every 24 hours after virus inoculation, cell markers were observed in wells showing lesions, and records were kept until no new lesion wells were observed for 3 consecutive days. Observation was then stopped, and the number of lesion wells was counted. The PFU of ZIKV was calculated using the Reed-Muench method.

[0088] Figure 8-9 The graphs show the changes in mRNA and protein expression levels of PTBP1 (left) and ZIKV NS5 (right) in PTBP1-stable cells over time. Figure 9 In the diagram, + indicates that the corresponding intervention (LV-Vector or LV-PTBP1) was used, and - indicates that the corresponding intervention was not used. Figure 10 The statistical results of PFU at different time points are shown for ZIKV-infected PTBP1 stable cells and Vector control cells.

[0089] The results showed that in PTBP1-transfected cells, both the mRNA level and ZIKV NS5 protein expression were significantly lower than in the control group, indicating that PTBP1 overexpression significantly inhibited ZIKV infection within cells. Furthermore, ZIKV titers decreased significantly with PTBP1 gene overexpression. These results confirm that PTBP1 overexpression inhibits ZIKV infection and the replication of ZIKV progeny viruses.

[0090] 4. Knockdown of the PTBP1 gene promotes ZIKV replication in cells.

[0091] To further verify the effect of PTBP1 on ZIKV replication, this embodiment constructed an A549 cell line with knocked-down PTBP1 gene expression and used this cell line to conduct ZIKV infection experiments to detect the specific effect of PTBP1 knockdown on ZIKV expression. The specific procedures are as follows:

[0092] 1) Gene Knockout Vector Construction: sh-RNA (see SEQ ID NO. 4-5 in Table 1) was designed on the Sigma website and synthesized at Guangzhou Qingke Biotechnology Co., Ltd. The primer annealing system consisted of 5 μL each of the forward and reverse shRNA primers (10 μmol / L), 5 μL of Buffer II (NEB), and ddH2O to a final volume of 50 μL. The annealing program was as follows: 83 cycles from 99℃ to 16℃, 30 s / cycle, with a 1℃ decrease per cycle. After annealing, the PLKO.1 vector was digested with restriction endonucleases Age I and EcoRI. Subsequently, the shRNA hairpin structure was ligated to the digested PLKO.1 vector using T4 ligase, and the ligation product was transformed into DH5α competent cells. The cells were plated and cultured overnight. The next day, single colonies were picked for PCR verification to ensure successful insertion of the shRNA into the vector. Finally, positive bacterial cultures were sent for sequencing.

[0093] 2) Construction of A549 cell lines with knocked-down PTBP1 gene expression: Subsequent steps were the same as for overexpression-stabilized cell lines, including lentivirus packaging, cell infection with lentivirus, selection via puro, and detection of knockdown efficiency by Western blot. Compared to shNC control cells (which expressed the PTBP1 gene normally), the PTBP1 protein expression level in the PTBP1 knockdown cells in the experimental group was significantly reduced (see...). Figure 11 This confirmed that the expression of the PTBP1 gene was successfully knocked down in this cell line.

[0094] 3) ZIKV infection of PTBP1 knockdown cells: PTBP1 knockdown cells and shNC control cells were infected with ZIKV at MOI=1, and cell samples were collected at 24h and 48h post-infection. qRT-PCR and Western blot were used to detect changes in the expression of ZIKV and PTBP1 at the mRNA and protein levels. Additionally, viral supernatants were collected from PTBP1 knockdown cells and shNC control cells 24h and 48h after infection and used to infect RD cells. The number of cell wells showing lesions was observed and counted, and PFU were calculated.

[0095] Figure 12-13 The graphs show the changes in mRNA and protein expression levels of PTBP1 (left) and ZIKV NS5 (right) in PTBP1 knockdown cells over time. Figure 12 In the diagram, + indicates that the corresponding intervention (shNC or shPTBP1) was used, and - indicates that the corresponding intervention was not used. Figure 14 The statistical results of PFU at different time points are shown for ZIKV-infected PTBP1 knockdown cells and shNC control cells.

[0096] The results showed that knocking down the PTBP1 gene significantly increased the mRNA level and ZIKV NS5 protein expression level of ZIKV compared to the control group, indicating that PTBP1 knockdown promotes ZIKV infection in cells. Furthermore, knocking down PTBP1 significantly increased the intracellular ZIKV titer, suggesting that PTBP1 knockdown promotes the replication process of ZIKV progeny.

[0097] 5. Knocking down the PTBP1 gene weakens the inhibitory effect of stimulating HIF-1α expression on ZIKV.

[0098] The above examples revealed that ZIKV infection simultaneously upregulated the expression of HIF-1α and PTBP1, and that ZIKV upregulated PTBP1 expression by activating HIF-1α expression. Therefore, to further explore the relationship between PTBP1, HIF-1α, and ZIKV, and whether the upregulation of PTBP1 by the HIF-1α activator IOX2 through the HIF-1α pathway has an inhibitory effect on ZIKV infection, this example used different concentrations of IOX2 (0, 30, 40, 50 μM) to stimulate PTBP1 knockdown cells and shNC control cells. After pretreating cells with IOX2 for 12 h, these cells were infected with ZIKV at MOI=1 and cultured for another 24 h. Subsequently, cell samples were collected, and the mRNA and protein expression levels of PTBP1 and ZIKV NS5 were detected and analyzed by qRT-PCR and Western blot. The viral supernatant from the PTBP1 knockdown cells and shNC control cells infected in the above experiments was collected, diluted and infected in 96-well plates containing RD cells, and the PFU was calculated by observing and counting the number of wells with lesions.

[0099] Figure 15 The effect of PTBP1 gene knockdown on HIF-1α expression activation on ZIKV infection is shown. Figures A, C, and D respectively show the expression levels of ZIKV NS5 mRNA and protein in PTBP1 knockdown cells pretreated with different concentrations of IOX2 and the changes in viral titer over time.

[0100] The results showed that in shNC control cells, PTBP1 expression was further enhanced after IOX2 stimulation followed by ZIKV infection, while ZIKV mRNA levels were significantly reduced, indicating that viral infection was suppressed. However, in PTBP1 knockdown cells, ZIKV mRNA levels were significantly higher than in the control group, a finding further confirmed by Western blot results. Furthermore, TCOID... 50The experimental results showed that after IOX2 stimulation and ZIKV infection, the ZIKV titer in shNC control cells was significantly lower than that in PTBP1 knockdown cells. This indicates that IOX2 can inhibit ZIKV progeny replication, further validating the inhibitory effect of IOX2 on ZIKV. These results suggest that IOX2 upregulates PTBP1 expression by stimulating HIF-1α expression, thereby inhibiting ZIKV, the infection process, and its replication within cells.

[0101] In summary, this invention investigated the differences in ZIKV infectivity between cell lines overexpressing and knocking down PTBP1. The findings revealed that increased PTBP1 expression effectively inhibited ZIKV replication and reduced viral titers in host cells, while knocking down PTBP1 significantly promoted ZIKV replication. This discovery fully demonstrates that PTBP1 plays a crucial role in inhibiting ZIKV replication. Further investigation revealed that activating the HIF-1α pathway to upregulate PTBP1 expression exhibited good antiviral effects at the cellular level, providing a theoretical basis for the development of HIF-1α activators as anti-ZIKV drugs and potentially contributing new strength and research ideas to the fight against viral diseases.

[0102] It should be noted that in all the figures of this invention, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001; and so on; all statistical data are obtained by calculating the mean and error value in three or more parallel calculations.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a reagent that upregulates PTBP1 expression levels in the preparation of drugs for treating Zika virus infection, wherein the reagent that upregulates PTBP1 expression levels is 10X2.

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