Use of host itch as a drug target in preparation of anti-flaviviridae virus infection drugs

Abstract

This invention discloses the application of host ITCH as a drug target in the preparation of drugs against normal flavivirus infection, belonging to the field of biomedical technology. This invention is the first to propose and verify the host E3 ubiquitin ligase ITCH as a key target for anti-normal flavivirus infection, and provides an antiviral strategy based on interfering with the interaction between host ITCH and normal flavivirus capsid proteins or inhibiting the expression level / ubiquitin ligase activity of host ITCH. Unlike existing mainstream viral target strategies, this invention is a novel antiviral approach based on host factors, providing a new target and theoretical basis for developing broad-spectrum, low-drug-resistance anti-normal flavivirus drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the host E3 ubiquitin ligase ITCH as a drug target in the preparation of drugs against normal flavivirus infection. Background Technology

[0002] Yellow virus ( Orthoflavivirus As a member of the Flaviviridae family ( Flaviviridae A significant class of pathogens, primarily transmitted by arthropods such as mosquitoes and ticks, these vector-borne viruses have a wide infectivity, encompassing birds, ruminants, ungulates, and small mammals. Humans often serve as incidental or definitive hosts. Against the backdrop of global climate change, population growth, and accelerated urbanization, the continued expansion of arthropod habitats has led to the spillover of zoonotic vector-borne viruses from forest ecosystems into human settlements, posing an increasingly serious public health threat.

[0003] Based on the different transmission vectors, orthoflaviruses can be mainly divided into mosquito-borne orthoflaviruses and tick-borne orthoflaviruses. Among mosquito-borne orthoflaviruses, Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) are typical examples. In recent years, ZIKV has spread rapidly in the Western Hemisphere, and the congenital Zika syndrome (CZS) it causes poses a serious threat to the health of newborns and has received widespread global attention. DENV infects as many as 400 million people annually, and more than a quarter of the world's population lives in dengue-endemic areas; infection can cause diseases of the internal organs and central nervous system in humans. WNV, as a neurotropic virus, is mainly widespread in the Western Hemisphere, and human infection can develop into serious neurological diseases such as encephalitis, meningitis, or acute flaccid paralysis. Although vaccines against YFV and JEV are currently approved, both continue to spread. YFV, primarily prevalent in Africa and South America, causes severe yellow fever (YF). According to the World Health Organization (WHO), there are approximately 200,000 clinical cases of YF annually, with a mortality rate as high as 30%.

[0004] Currently, there are no specific antiviral drugs for most oroflavone infections, and treatment mainly focuses on symptomatic and supportive care. Antiviral strategies for oroflavone infection currently revolve around the following three main directions:

[0005] (1) Direct antiviral strategy. This strategy aims to develop small molecules or biological agents that directly target key stages of the viral life cycle. For example, targeting viral NS2B-NS3 proteases, RNA-dependent RNA polymerase (RdRp), or viral envelope proteins (E proteins) to inhibit viral entry, replication, or assembly.

[0006] (2) Host-based antiviral strategies. This strategy indirectly inhibits viruses by intervening in host cytokines or pathways on which viral replication depends, theoretically possessing advantages in broad spectrum and low drug resistance. For example, regulating lipid metabolism, the life cycle of flaviviruses is highly dependent on the lipid metabolism of host cells. Studies have found that certain polyphenolic compounds (such as N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its regiomeric derivatives) can effectively inhibit the replication of West Nile virus, Usutu virus, dengue virus, and Zika virus by inhibiting ceramide desaturase (Des1), leading to the accumulation of dihydrosphingomyelin (dhSM) in cells. Exogenous addition of dhSM or use of the Des1 inhibitor GT-11 can also produce similar antiviral effects, confirming the therapeutic potential of this pathway.

[0007] (3) Vaccine strategy. This is currently the most important means of preventing flavivirus infection, and there are various technical platforms. For Zika virus, several candidate vaccines, including DNA vaccines, mRNA vaccines, and live attenuated vaccines, have entered clinical trials. Dengvaxia (a chimeric live attenuated vaccine) has been approved, but its applicable population is limited to serologically positive individuals, highlighting the serious challenge of antibody-dependent enhancement (ADE). West Nile virus vaccines for humans are still in Phase II clinical trials. Japanese encephalitis vaccines have evolved from traditional inactivated / live attenuated vaccines to candidate vaccines based on novel platforms such as virus-like particles (VLPs) and mRNA. The yellow fever vaccine YF17D is a successful example of a live attenuated vaccine.

[0008] For orthoflavir infection, existing direct antiviral strategies often face the risk of drug resistance due to the virus's high mutation rate, while vaccine strategies are constrained by core issues such as antibody-dependent enhancement (ADE), uneven immune responses, and concerns about safety in specific populations. Furthermore, the well-defined tissue tropism of orthoflavir (e.g., ZIKV's invasion of neural and placental tissues) suggests that its pathogenic mechanism is closely related to host factors, but the specific regulatory network between the two remains unclear.

[0009] Viral infection is essentially a complex game between the virus and the host at the molecular level. Therefore, developing host-targeted therapies by analyzing virus-host interaction networks is considered a novel antiviral strategy with broad potential and low risk of drug resistance. However, a systematic analysis of existing technologies reveals that this strategy faces a series of fundamental challenges and limitations in practical development, specifically: 1) Challenges in Target Selection and Validation. The core of host-targeted therapy lies in identifying host factors that are crucial for viral replication but have a relatively minor impact on normal host cell function. However, the specific mechanisms of action of many identified host factors (such as aryl hydrocarbon receptors, heat shock factor 1, and kinesin family member 20A) in the viral life cycle are not yet fully elucidated, and their functional localization within complex cellular signaling networks remains unclear, directly affecting the precise design of targeted drugs. Furthermore, cellular pathways often exhibit redundancy; inhibiting a single host factor may lead to viral replication through alternative pathways, thereby weakening the therapeutic effect.

[0010] 2) Core limitations of safety and cytotoxicity. The most significant limitation of host-targeted therapies lies in their potential cytotoxicity. Because they target the host's own proteins or pathways, there is a high risk of interfering with their normal functions, potentially affecting cellular homeostasis. For example, strategies targeting autophagy (e.g., using HM-013 inhibitors) or heat shock proteins (e.g., targeting Hsp70i), while effective in inhibiting viruses, highlight the fundamental role these pathways play in maintaining cellular homeostasis, responding to stress, and clearing abnormal proteins. Long-term or potent inhibition may adversely affect normal cells, especially rapidly dividing or highly metabolic cells (such as immune cells and neurons). Many candidate compounds often have a narrow therapeutic window in vivo, with effective antiviral concentrations being close to toxic concentrations, limiting clinical translation.

[0011] 3) Challenges in drug development and delivery. Developing highly selective, potent, and low-toxicity small-molecule modulators targeting host proteins is more complex than targeting viral enzymes. Furthermore, accurately delivering drugs to target tissues or organelles (such as neurons or specific cell compartments) and maintaining effective concentrations remains a significant technological bottleneck.

[0012] 4) Potential risks of efficacy and drug resistance. The broad-spectrum antiviral efficacy of host-targeting strategies may vary depending on the virus species or strain. More importantly, viruses may still reduce their dependence on specific host factors through adaptive mutations, thereby creating a de facto "treatment escape," leading to decreased or non-durable efficacy.

[0013] 5) Practical obstacles to clinical translation and application. The lack of ideal animal models that can fully simulate human diseases (especially congenital infections or severe dengue) introduces uncertainty into the extrapolation of preclinical data. Furthermore, designing combination therapies combining host-targeted drugs and direct-acting antiviral agents will further increase the complexity and cost of research and development.

[0014] In summary, the core challenge of existing host-targeted antiviral strategies lies in how to design efficient, safe, and non-viral-escape-prone intervention programs based on the accurate identification of key host pathways on which viral replication depends, while overcoming the practical obstacles of drug delivery and clinical translation.

[0015] Therefore, developing new and effective interventions against orthoflavin, especially those that can address multiple orthoflavins and overcome the shortcomings of existing strategies, is of great scientific significance and clinical need.

[0016] ITCH, also known as AIP4, is an E3 ubiquitin ligase with a HECT domain, belonging to the NEDD4 family. It plays a central regulatory role in various cellular processes, and its function is highly dependent on its unique domain composition and dynamic interactions with other proteins. The ITCH / AIP4 protein structure includes C2, WW, and HECT domains, which collectively determine its subcellular localization, substrate recognition, and catalytic activity. ITCH interacts with different proteins through its domains, participating in the regulation of multiple key biological processes, from cell growth and differentiation to the viral life cycle. Previous studies have shown that ITCH plays multiple roles in the infection cycles of various viruses: it can directly regulate viral proteins, promote the maturation and assembly of EBV particles, and the budding process of Ebola virus. However, the role of ITCH in flaviviruses has not yet been reported. Summary of the Invention

[0017] In view of the technical problems existing in the background art, and in order to overcome the systemic bottleneck in the fight against orthoflavir infection, the purpose of this invention is to discover and validate a novel host target that plays a key role in the early stages of infection, and based on this, to provide a novel antiviral strategy that can circumvent the shortcomings of existing technologies. This invention aims to address the key deficiencies of existing host-targeted therapies in terms of target mechanism clarity, host safety, broad-spectrum efficacy and durability, and clinical translation feasibility, providing a new foundation for the development of next-generation antiflavir drugs.

[0018] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention discloses the application of host ITCH as a drug target in the preparation of drugs against normal flavivirus infection, wherein normal flavivirus includes, but is not limited to, viruses such as ZIKV, DENV, WNV, YFV and JEV.

[0019] This invention is the first to demonstrate that the host E3 ubiquitin ligase ITCH plays a key inhibitory role in the early stages of normal flavivirus infection. This role can effectively intervene in the process of the virus successfully establishing infection in the host, providing a clear host target for early intervention.

[0020] Furthermore, in the aforementioned applications, drugs developed using host ITCH as a drug target achieve anti-flavin infection by blocking the ubiquitination modification of the normal flavivirus capsid protein (i.e., C protein) by host ITCH. Viral uncoating is a key step in the release of the viral genome, involving the degradation of the viral C protein. This invention demonstrates that ITCH can directly act on viral particles, specifically promoting its degradation through ubiquitination modification by interacting with the viral C protein. This mechanism directly promotes the release of the viral genome (vRNA) into the cytoplasm, revealing the core principle of ITCH promoting normal flavivirus infection at the molecular level. This invention further elucidates that host ITCH specifically regulates the ubiquitination modification of the 5th lysine residue of the ZIKV C protein. This site is highly conserved among different ZIKV strains; therefore, intervention strategies targeting this site can significantly reduce the risk of drug resistance due to viral gene mutations, ensuring the long-term effectiveness of the target strategy. Concurrent studies also confirmed that host ITCH has a similar effect on the 6th lysine residue of the DENV C protein.

[0021] Furthermore, in the above applications, anti-flavin infection drugs are developed by targeting the E3 ubiquitin ligase active site of the host ITCH. For human ITCH (amino acid sequence shown in SEQ ID NO.1), its ubiquitin ligase activity depends on its 830th cysteine ​​residue. Simultaneously, experiments in this invention have shown that this site is the active center regulating the degradation of the Flavin C protein. Therefore, highly selective, potent, and low-toxicity small molecule inhibitors can be precisely designed targeting this key active site to achieve antiviral objectives.

[0022] Furthermore, in the above applications, drugs developed using host ITCH as a drug target achieve anti-flavin infection by reducing the expression level or activity of host ITCH. In vitro cell and in vivo animal experiments of this invention both demonstrate that reducing host ITCH expression can inhibit flavivirus infection; moreover, animal experiments show that ITCH gene knockout did not have an observable impact on the normal growth and development of mice, indicating that intervention targeting this ITCH expression level may have high host safety and good prospects for translational applications.

[0023] Furthermore, in some embodiments of the present invention, the substance that reduces the host's ITCH expression level is an RNA molecule.

[0024] Secondly, the present invention provides a drug for treating flavivirus infection, which contains at least one of the following: 1) Substances that block the ubiquitination modification of the normal flavivirus capsid protein by the host ITCH; 2) Substances that reduce the expression level of ITCH in the host.

[0025] Furthermore, the aforementioned drugs also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to pharmaceutical excipients widely used in the pharmaceutical manufacturing industry, specifically one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0026] This invention validated the value of developing antiflavin infection drugs targeting the host ITCH in various rigorous in vivo infection models, including the Zika virus immune healthy pregnant mouse model and the Japanese encephalitis virus intraperitoneal challenge model. The experimental results consistently showed that inhibiting the ITCH-capsid protein pathway has clear therapeutic potential for resisting various flavivirus infections.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel host factor-based anti-flavin strategy, distinct from existing mainstream viral target strategies, offering a new target and theoretical basis for developing broad-spectrum, low-resistance anti-flavin drugs. Specifically, this antiviral strategy has the following core advantages: (1) Novel target and clear mechanism: The novel mechanism of host ITCH regulating uncoating by ubiquitination and degradation of viral capsid proteins is discovered and elucidated for the first time, providing a clear action link (early infection) and molecular basis for antiviral drug design.

[0028] (2) High host safety: Animal experiments have shown that, ITCH Gene knockout mice developed normally, and no obvious physiological defects were observed, suggesting that intervention targeting this pathway may have a good window of host safety and tolerability.

[0029] (3) Broad-spectrum antiviral potential: The host ITCH interacts with a variety of orthoflavirus capsid proteins and promotes their degradation, indicating that this target has the potential for broad-spectrum anti-orthoflavirus application.

[0030] (4) Low risk of drug resistance: The core of the strategy is to target highly conserved and functionally critical amino acid sites on the viral capsid protein. The virus is unlikely to lose its infectivity by mutating these sites, thus greatly reducing the risk of treatment escape. Attached Figure Description

[0031] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0032] Figure 1 The host cell in Example 1 ITCH The effect of gene knockout on ZIKV in vitro replication, where A is... ITCH Validation of KOJEG-3 cell knockout effect, B represents detection using qRT-PCR. ITCH Results of the effect of gene knockout on ZIKV viral RNA levels, C represents the effect detected by plaque assay. ITCH Results of the effect of gene knockout on ZIKV viral titer; p <0.05 indicates that the result is statistically significant. p <0.01 indicates a significant result. p <0.001 indicates that the result is highly significant, and ns indicates that the result is not statistically significant.

[0033] Figure 2 The host cell in Example 2 ITCH The effects of gene knockout on ZIKV binding, internalization, and early infection events, where A represents... ITCH The effect of gene knockout on viral adsorption, B is ITCH The effect of gene knockout on viral internalization, C is ITCH The impact of gene knockout on early viral infection; p <0.05 indicates that the result is statistically significant. p <0.01 indicates a significant result, and ns indicates a result without statistical significance.

[0034] Figure 3 The host cell in Example 3 ITCH Analysis of gene knockout on uncoating of flavivirus, where A is... ITCH The results of gene knockout inhibiting ZIKV C protein degradation are shown in Figures B, C, DE, and F. B represents the localization analysis of early ZIKV infection vRNA, C represents the localization analysis of early DENV infection vRNA, DE represents the immunoblotting results of ITCH deficiency inhibiting ZIKV C protein degradation, and F represents the immunoblotting results of ITCH deficiency inhibiting DENV, YFV, WNV, and JEV capsid protein degradation, respectively.

[0035] Figure 4The analysis of the direct interaction between ITCH and ZIKV C protein in Example 4 is shown in Figure A, which shows the results of co-immunoprecipitation (Co-IP) of the direct interaction between ITCH and ZIKV C protein; Figure B shows the results of confocal microscopy observation of the co-localization of ITCH and ZIKV C protein; and Figure C shows the results of Co-IP of the direct interaction between ITCH and DENV, YFV, WNV, and JEV C proteins, respectively.

[0036] Figure 5 This is an evaluation of the ubiquitination modification of ZIKV C protein by the ITCH enzyme active site in Example 5. A represents the effect of ITCH overexpression and ITCH enzyme active site mutation on ubiquitination modification of ZIKV C protein, B represents the effect of ITCH enzyme active site mutation on ZIKV C protein degradation, and CF represent the effect of ITCH overexpression on ubiquitination modification of DENV, YFV, WNV, and JEV C proteins, respectively.

[0037] Figure 6 This is a screening and functional validation of key sites of the ZIKV and DENV C proteins targeted by ITCH in Example 6. A is a schematic diagram of the ZIKV C mutant, and B shows... ITCH The effects of knockout on the expression levels of ZIKV C protein and its mutants were investigated. C showed the role of the K5 site in the ubiquitination modification of the ZIKV capsid protein. D showed the effect of the C-K5R point mutation on the pathogenicity of ZIKV. EF showed the role of the K6 site in the ubiquitination modification of the DENV C protein. G showed the effect of the C-K6R point mutation on the infectivity of DENV. p <0.05 indicates that the result is statistically significant. p <0.01 indicates a significant result, and ns indicates a result without statistical significance.

[0038] Figure 7 For the systemic mouse infection in Example 7 Itch The effects of gene knockout on ZIKV and JEV infection and pathogenicity, where A is a schematic diagram of the construction and detection process of the pregnant mouse infection model, and B is... Itch - / - Comparison of mouse embryos with wild-type mice, CD values ​​were respectively Itch - / - Comparison of embryo weight and abnormality rate between pregnant and wild-type mice before and after ZIKV infection, with EF values ​​of [missing data]. Itch - / - Comparison of viral load in the spleen and placenta of pregnant and wild-type mice after ZIKV infection, with GH levels of [missing data]. Itch - / -Comparison of body weight and mortality rates in mice and wild-type mice infected with JEV, IJ respectively Itch - / - Comparison of viral load and pathological damage in the brain of mice and wild-type mice after infection with JEV; p <0.05 indicates that the result is statistically significant. p <0.01 indicates a significant result. p <0.001 indicates that the result is highly significant, and ns indicates that the result is not statistically significant. Detailed Implementation

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having” and any variations thereof are intended to cover non-exclusive inclusion.

[0040] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0041] Example 1 This example tests... ITCH The functions of the gene in ZIKV infection are as follows: Using CRISPR / Cas9 gene editing technology, a new type of gene was constructed in the human chorionic villus carcinoma cell line JEG-3. ITCH Stable cell lines with gene knockout (KO) Figure 1 A), denoted as ITCH KO JEG-3 cells. The specific construction method was as follows: JEG-3 cells were seeded in 6-well plates and transduced with lentiviruses containing non-targeting sgRNA (i.e., not targeting any gene) and two sgRNAs targeting ITCH (sequences shown in SEQ ID NO. 2-3, respectively), along with 10 µg / ml polybrene. After 24 h of transduction, cells were digested with trypsin and screened with puromycin. Single-clonal cell lines were selected using the limiting dilution method and verified by Western blot to obtain NC and [missing information - likely cell lines]. ITCHKO JEG-3 cell line.

[0042] Make NC and ITCH KO JEG-3 cells were infected with ZIKV (MOI=0.1), and cell samples were collected at different time points after infection. Viral RNA levels and viral titers were detected by qRT-PCR and plaque assays. The results are as follows: Figure 1 B and appendix Figure 1 As shown in C, the result indicates that: ITCH The absence of [specific component] significantly inhibited ZIKV infection in JEG-3 cells, confirming [the following]. ITCH Genes are crucial for ZIKV infection.

[0043] Example 2 This example explores ITCH The specific experimental steps regarding the role of genes in viral infection are as follows: First, the test was performed. ITCH The effect of gene knockout on viral adsorption and internalization processes. NC and... ITCH KOJEG-3 cell lines were inoculated with ZIKV (MOI=1) at 4°C for 1 h to detect adsorption, followed by incubation at 37°C for 1 h. Then, the cells were treated with proteinase K to remove adsorbed viral particles from the surface, and internalization was detected. qRT-PCR results showed that... ITCH Gene knockout promotes viral adsorption ( Figure 2 A) and internalization ( Figure 2 B) None of them had a significant effect.

[0044] Furthermore, cells were pretreated for 2 h with DMEM containing 50 μg / mL cyclohexylimide (CHX, a protein biosynthesis inhibitor that blocks protein translation) or an equal volume of DMSO. After washing with DMEM, cells were inoculated with ZIKV (MOI=1) and incubated at 37°C for 1 h. Unbound virus was then washed away with DMEM, and the cells were cultured in fresh medium (containing 2% FBS) containing CHX or DMSO. Detection was performed 6 h post-infection. qRT-PCR results showed that under conditions where viral protein synthesis was blocked, ITCH Gene knockout still limits ZIKV infection on JEG-3 cells. Figure 2 C).

[0045] The above results indicate that ITCH Genes play an important role in the early infection process of ZIKV.

[0046] Example 3 This example explores ITCH The influence of genes on the viral uncoating process is illustrated in the following experiments: First, its effect on the stability of C protein in the early stage of ZIKV infection was examined. NC and ITCH KO JEG-3 cell lines were transfected with the GFP-Rab7 eukaryotic expression plasmid, and infected with ZIKV (MOI=50) 24 h after transfection. Cells were fixed at 30 min, 1 h, and 2 h post-infection, stained with anti-ZIKV C protein antibody, and the dynamic changes of C protein in the early stages of ZIKV infection were observed using confocal fluorescence microscopy. Immunofluorescence results showed that ZIKV co-localized with late endosomes at 30 min post-infection, and the C protein signal gradually weakened as infection progressed, suggesting the occurrence of uncoating. ITCH KO inhibited the degradation process of ZIKV C protein ( Figure 3 A). Therefore, the above experiment suggests... ITCH Gene knockout inhibits the viral uncoating process by blocking the degradation of the C protein in the early stages of Zika virus infection.

[0047] The final marker of viral uncoating is the release of the viral genome from the endosome into the cytoplasm. To further elucidate... ITCH To investigate the role of genes in regulating the uncoating process of ZIKV and other normal flaviviruses (such as DENV), this study employed high-resolution, single-molecule level π-FISH (prime flow RNA fluorescence in situ hybridization) technology, combined with Rab7 immunofluorescence staining, to observe the localization of intracellular vRNA in the early stages of infection. Following ZIKV infection, 3 hours after infection in NC cells, vRNA was observed to be primarily distributed in the cytoplasm. Figure 3 B), confirming the release of the viral genome into the cytoplasm. Conversely, ITCH vRNA signaling in KO JEG-3 cells remained in Rab7-positive endosomes, indicating that genome release was blocked. Figure 3 B). Simultaneously, knockdown in Huh7 cells. ITCH DENV was infected, and similar π-FISH and vRNA localization analyses were performed. The results also showed that ITCH deficiency inhibited the release of the DENV genome. Figure 3 C). This further confirms that ITCH regulates the uncoating process of normal flavivirus from the endosome by promoting capsid protein degradation, which is a conserved regulatory mechanism.

[0048] To further investigate whether ITCH has a regulatory effect on the expression of exogenous normal flavivirus capsid proteins, this example constructs... ITCH HEK-293T cell lines with gene knockout. Constructed using the same method as JEG-3 cells in Example 1, resulting in sgNC and sg... ITCHHEK-293T cell line. sgNC and sgITCH HEK-293T cell lines were transfected with exogenous FLAG-ZIKVC plasmids. Immunoblotting experiments demonstrated that, regardless of the presence or absence of CHX (CHX treatment as described in Example 2), ITCH deficiency inhibited the degradation of exogenous C protein. Figure 3 DE). Simultaneously, following the above method, by exogenously transfecting the C protein of various other orthoflaviruses (DENV, YFV, WNV, and JEV), it was confirmed that... ITCH Gene knockout can effectively inhibit the degradation of C proteins in various orthoflavir viruses. Figure 3 FI).

[0049] Example 4 To investigate the mechanism by which ITCH regulates the uncoating of normal flavivirus, this study tested whether ITCH directly interacts with the normal flavivirus C protein. The specific experiment is as follows: HEK-293T cells were co-transfected with FLAG-ITCH and HA-ZIKV C plasmids. Twenty-four hours after transfection, cells were treated with 15 μM proteasome inhibitor MG132 for six hours. Cells were then collected for Co-IP experiments. The results showed a direct interaction between ITCH and ZIKV C protein. Figure 4 A). Further co-transfection of exogenous ITCH and ZIKV C into HeLa cells, observed using confocal microscopy, confirmed the co-localization of the two genes, both located on late endosomes. Figure 4 B). Furthermore, Co-IP experiments confirmed that ITCH interacts with the C proteins of DENV, YFV, WNV, and JEV. Figure 4 C).

[0050] Example 5 Given that ITCH is an E3 ubiquitin ligase, this study investigated whether ITCH mediates the ubiquitination and degradation of the C protein in dependence of its E3 enzyme activity.

[0051] HEK-293T cells were transfected with the corresponding eukaryotic expression plasmid. Twenty-four hours after transfection, cells were treated with 15 μM MG132 for 6 hours. Cells were then collected and cell lysates were prepared. Quantitatively quantified cell lysates were incubated with anti-FLAG M2 magnetic beads for 2 hours, washed, and analyzed by Western blot using the specified antibody. The results showed that ZIKV C protein can be ubiquitinated, and overexpression of ITCH significantly enhanced this modification. Figure 5 A).

[0052] Existing techniques indicate that the ubiquitin ligase activity of ITCH depends on its cysteine ​​residue at position 830 (C830). In this case, by constructing an alanine mutant (C830A) at this site, it was found that this mutant completely loses the ability to enhance C protein ubiquitination. Figure 5 A) and promoting its degradation ( Figure 5 B) demonstrates that the E3 ubiquitin ligase activity of ITCH is essential.

[0053] Similarly, ITCH overexpression can also promote ubiquitination of other normal flavivirus C proteins. Figure 5 CF).

[0054] The above results indicate that ITCH, through its E3 ubiquitin ligase activity, specifically promotes the ubiquitination modification of the normal flavivirus C protein, thereby driving its subsequent degradation process.

[0055] Example 6 To identify key sites for ITCH-mediated ubiquitination modification of the ZIKV C protein, this study used BDM-PUB software to predict potential ubiquitination sites in the C protein and selected nine potential ubiquitination sites for validation based on the scoring results. For subsequent functional validation, these nine lysine residues (K2, K5, K6, K7, K31, K75, K86, K101, and K102) were replaced with arginine (R) to construct corresponding FLAG-tagged ZIKV C mutants. Figure 6 A).

[0056] To screen for the specific lysine sites on the C protein that ITCH acts upon, its effect on the expression of these mutants was first evaluated. (sgNC and sg) ITCH HEK-293T cell lines were transfected with plasmids expressing FLAG-ZIKV C and its nine mutants. Western blot was used to detect the effect of ITCH on the expression of different C protein mutants. Results are as follows: Figure 6 As shown in B, ITCH Gene knockout led to upregulation of expression levels of ZIKV wild-type C protein and other mutants; however, the K5R mutation completely eliminated the regulatory effect of ITCH on its expression.

[0057] To verify the crucial role of the 5th lysine (K5) site in C protein ubiquitination, expression plasmids of FLAG-tagged ZIKV C, ZIKV C-K5R, and HA-Ub were transfected into HEK-293T cells, and ubiquitination assays were performed. Figure 6 C). Co-IP assays showed that the ubiquitination level of the K5R mutant was significantly reduced compared to the wild-type C protein. Figure 6(C), indicating that the 5th lysine residue of the ZIKV C protein is the key site for ITCH regulation of its ubiquitination modification.

[0058] To further explore the physiological significance of the ITCH-capsid protein regulatory axis in ZIKV infection, this study rescued a ZIKV C-K5R point mutant strain using reverse genetics. The strain was then tested in A129 mice. Ifnar1 - / - The model was inoculated via intraperitoneal injection of 1×10 3 PFU was administered via ZIKV-WT, ZIKV C-K5R mutant virus, or an equal volume of DMEM as a control, followed by daily monitoring of mouse body weight, clinical symptoms, and survival. Results are as follows: Figure 6 As shown in D, compared to the 45% mortality rate in mice caused by ZIKV-WT infection, the ZIKV C-K5R mutant strain did not cause mouse death. Figure 6 (D) This confirms that ITCH-mediated ubiquitination modification at the K5 site of the ZIKV capsid protein plays a decisive role in the infectivity and pathogenicity of ZIKV.

[0059] Similarly, the above conclusions were confirmed in another flavivirus, DENV, where experiments demonstrated that ITCH also mediates the ubiquitination and degradation of lysine (K6) at position 6 of the DENV capsid protein. Figure 6 EF), and the infectivity of the constructed DENV C-K6R mutant virus was significantly weakened ( Figure 6 G). This indicates that ITCH, through ubiquitination modification targeting conserved lysine sites on the flavivirus capsid protein, is a broadly applicable antiviral mechanism.

[0060] Example 7 To evaluate ITCH targeting as an effective strategy for treating orthoflavin infection, this study constructed a systemic cytogenetic model in the C57BL / 6J background. Itch Gene knockout Itch - / - The mouse, specifically constructed as follows: in Itch Three sgRNAs (sequences shown in SEQ ID NO.4-6) were designed for editing the fourth exon region of the gene. ItchThese sgRNAs were transcribed in vitro using the MEGAshortscript™ T7 kit and purified using the MEGAclear™ kit, then stored at -80°C until microinjection. Single-cell embryos were isolated from C57BL / 6J mice and microinjected at the E0.5 stage, with each embryo receiving 50 ng of sgRNA and 100 ng of Cas9 mRNA. Embryos that developed to the 2-cell stage were then transplanted into the oviducts of pseudopregnant recipient mice. The target genome of F0 mice was amplified by PCR and sequenced. The resulting chimeras (F0 mice) were then mated with wild-type C57BL / 6J mice to produce F1 generation mice. The F1 generation heterozygous mice were then mated to produce homozygous knockout mice.

[0061] right Itch - / - Morphological and weight analysis of mouse embryos showed no significant difference compared to wild-type mouse embryos. Figure 7 B) indicates that the absence of ITCH during development is well tolerated by the body and does not cause obvious developmental defects.

[0062] By establishing a ZIKV infection model in pregnant mice, the study confirmed that Zika virus infection can cause fetal developmental abnormalities (such as microcephaly and miscarriage). Figure 7 CD). But compared to wild-type pregnant mice. Itch - / - The rate of developmental abnormalities in embryos born from pregnant mice was significantly lower (21% vs 41%), and the degree of abnormalities was also milder. Figure 7 CD). Virus load detection shows, Itch - / - The viral load in the spleen and placenta of pregnant mice was significantly lower than that in wild-type pregnant mice. Figure 7 EF).

[0063] Similarly, the above conclusions were confirmed in the Japanese encephalitis virus (JEV) infection model. Itch - / - The mortality rate of mice was lower than that of wild-type mice. Figure 7 GH), and alleviated the more severe viral load and pathological damage in the brain caused by JEV (Gastrointestinal Encephalopathy). Figure 7 IJ).

[0064] The above in vivo experiments show that ITCH specifically regulates the infection and pathogenesis of Flavivirosis viruses.

[0065] In summary, this invention clarifies the crucial role of host ITCH in the early stages of normal flavivirus infection, effectively blocking viral infection. It also elucidates the molecular mechanism by which host ITCH directly targets the viral C protein. Furthermore, through the identification of ubiquitination modification sites, key active sites of ITCH, and multi-model analysis, it provides structural biology evidence for drug design and confirms that the resulting drug possesses broad-spectrum antiviral potential, low risk of drug resistance, and high safety, thus offering a novel approach for developing drugs against normal flavivirus infection.

[0066] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of host ITCH as a drug target in the preparation of drugs against normal flavivirus infection.

2. The application according to claim 1, characterized in that, The yellow fever virus includes at least one of Zika virus, dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus.

3. The application according to claim 1, characterized in that, The drug is designed to target the E3 ubiquitin ligase active site of the host ITCH.

4. The application according to claim 3, characterized in that, The drug blocks the host ITCH from ubiquitinizing the capsid protein of the normal flavivirus.

5. The application according to claim 1, characterized in that, The drug reduces the expression level or activity of ITCH in the host.

6. The application according to claim 5, characterized in that, The drug is an RNA molecule that reduces the expression level of ITCH in the host.

7. The application according to claim 1, characterized in that, The drug contains a pharmaceutically acceptable carrier.

8. A drug for treating flavivirus infection, characterized in that, It contains at least one of the following: A substance that blocks the ubiquitination modification of the capsid protein of the normal flavivirus by the host ITCH; Substances that reduce the expression level or activity of ITCH in the host.

9. The medicament according to claim 8, characterized in that, The yellow fever virus is selected from Zika virus, dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus.

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