Application of deubiquitinase in prediction target of immune reconstitution insufficiency of AIDS patient

Abstract

The application of the deubiquitinase to the immune reconstitution incomplete prediction target of the AIDS patient comprises the following steps: identifying that the ubiquitinase is YOD1 and has obvious interaction with HIV virus protein Nef; identifying that the YOD1 stabilizes the HIV auxiliary protein Nef through the protein level instead of the RNA level; identifying that the YOD1 can prolong the half-life period of the HIV auxiliary protein Nef; the YOD1 is identified to enhance the expression level of Nef in virus particles of an HIV NL4-3 clone strain by reducing the ubiquitination of the Nef protein of HIV-1 (Human Immunodeficiency Virus 1) and influence the ubiquitination of the K6 site of the Nef protein; identifying that YOD1 inhibits ubiquitination of Nef protein and stabilizes the Nef protein; yOD1 is identified to be in positive correlation with Nef under the HIV infection condition, and the positive correlation is increased along with the increase of the infection dose or the infection time; it is identified that YOD1 promotes virus replication by stabilizing the Nef protein level and aggravates loss of CD4 + T cells, and the expression level of YOD1 is in positive correlation with immune imperfection of an HIV-1 infected person; it is identified that the vitamin C can effectively lower the expression level of YOD1 and Nef protein, and then the virus virulence is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to the application of deubiquitinases in the prediction, diagnosis and treatment of incomplete immune reconstitution (INRs) in AIDS patients. It specifically covers prediction methods, detection kits and inhibitor screening systems targeting specific deubiquitinases. Specifically, it relates to the application of deubiquitinases as predictive targets for incomplete immune reconstitution in AIDS patients. Background Technology

[0002] (I) Current Status of HIV / AIDS Treatment and the Dilemma of Immune Reconstitution: Human immunodeficiency virus (HIV) infection is one of the major public health problems facing the world. HIV primarily attacks CD4 cells. + T cells. A typical characteristic of people living with HIV (PLWH) is the presence of CD4+ cells in their bodies. + The number, types, and functions of T cells suffer irreversible damage. Therefore, for PLWH, achieving ideal virological suppression—that is, plasma viral RNA levels below the detection limit—and obtaining a good quality of life, depends on highly active antiretroviral therapy (HAART). This therapy can continuously increase the patient's CD4 count. + T cell levels, and systemic restoration of immune function. Regarding CD4... + In infected individuals whose T-cell levels recovered to 500 cells / μl, their immune function was well reconstituted, meaning a significantly reduced risk of opportunistic infections. However, the immune reconstitution process is not always successful; CD4+... + The recovery of T cell counts varies significantly among individuals. Studies have reported that even with HAART initiation and effective suppression of viral replication over several years, approximately 10%-40% of infected individuals still fail to achieve satisfactory CD4 counts. + Despite maintaining low T-cell levels, severe impairment of the immune response still occurs, leading to metabolic disorders, kidney damage, opportunistic infections, malignant tumors, and other HIV-related and non-HIV-related events, ultimately resulting in death. This group is known as immunocompromised individuals (or immune non-responders), and this phenomenon is defined as incomplete immune reconstitution.

[0003] Currently, there is considerable controversy globally regarding the definition of "poorly immune response." One standard is to consider CD4 counts after 4-7 years of continuous antiviral therapy. + Individuals with T cell levels remaining below 200 cells / μl are defined as having a "poor immunological response." The World Health Organization defines these patients as those with CD4+ levels two years after effective treatment, compared to baseline.+ T cell increase was less than 100 cells / μl. In contrast, CD4... + A T cell count >500 cells / μl is considered a successful immune reconstitution result. Overall, CD4 counts after treatment... + Failure to restore T cell count to normal is considered incomplete immune reconstitution. Rita Rb-Silva et al., through a systematic review of numerous original studies, proposed that the criteria for "immune non-responder" is "CD4 count after 24 months of antiretroviral therapy." + An absolute T-cell count below 350 cells / mL helps identify patients with immune non-response (INR) whose CD4+ T-cell count should have reached maximum potential but is still below expected. Currently, incomplete immune reconstitution can be attributed to the complex interaction of multiple mechanisms. These mechanisms may involve impaired lymphoid organ and thymus function, persistent replication of the HIV reservoir, high levels of CCR5-induced activation, co-infection, gut microbiota translocation, and CD4+ T-cell counts. + Decreased T cells, etc. Among the above factors, post-HIV infection CD4... + The destruction of T cells and the excessive activation of the immune system caused by the viral reservoir have a significant impact on incomplete immune reconstitution.

[0004] (II) The role of HIV-1 accessory protein Nef in viral reservoir establishment and incomplete immune reconstitution: To evade host antiviral factors and replicate using host cells, the HIV-1 genome encodes three structural proteins (Gag, Pol, Env) and two regulatory proteins (Tat, Rev), as well as several accessory proteins (Nef, Vif, Vpr, and Vpu). Each protein expressed by the viral gene performs a corresponding function according to its role. In the in vivo state, accessory proteins can create an intracellular environment conducive to viral amplification, promote viral transmission, and enhance pathogenicity. Among them, the accessory protein Nef (Negative Regulation Factor) is a highly conserved early viral replication gene product present in the genomes of HIV-1, HIV-2, and SIV (simian immunodeficiency virus), and is also the most immunogenic of all HIV-1 accessory proteins. It plays an important role in the pathogenesis, disease progression, and immune reconstitution of HIV-1. Nef has long been thought to reduce resting CD4 counts. +Nef plays a role in T cell activation threshold, promoting cell activation, and HIV-1 infection, and is involved in the establishment and maintenance of the viral reservoir after latent HIV infection. Nef protein can downregulate HLA-A / B, SERINC5 (Serine Incorporator 5) protein, and the CD3-TCR (T Cell Receptor) complex, thereby promoting viral infection, replication, and immune evasion. It has been reported that Nef can stimulate the immune system via lipid rafts, thereby helping the virus in the reservoir to trigger immune activation, leading to CD4+ activation. + T cells die via apoptosis, while Nef stimulates the immune system via lipid rafts. Recent research has found that Nef inhibitors, when used in combination with Latency-Reversing Agents (LRAs) and therapeutic vaccines, can reduce or eliminate the HIV reservoir.

[0005] (III) The regulatory role of ubiquitination modification in the HIV-1 infection cycle: The ubiquitin / proteasome pathway widely mediates cellular protein degradation, interactions, and signal transduction. During the viral infection cycle, ubiquitination is closely related to HIV-1 transcription and latency, as well as reverse transcription and antiviral restriction. Databases show that approximately 17% of the 103 identified deubiquitinases are associated with HIV-1. For E3 ubiquitin ligases, approximately 95 (about 25%) are associated with HIV-1. The viral infectivity factor Vif (VIF) is involved in inducing polyubiquitination and degradation of APOBEC3G (apolipoprotein-B-mRNA-editing enzyme catalytic polypeptide-like 3G). The E3 ubiquitin ligase MDM2 is involved in both Vif degradation and enhancing Tat transcriptional activity. A 2017 study reported that the E3 ubiquitin ligase Praja-2 (PJA2) can ubiquitinate Tat in a non-degradative manner. Besides transcription, ubiquitination is also involved in the synthesis and release of viral particles. The E3 ubiquitin ligase NEDD4-like protein (NEDD4L) interacts with Gag to promote HIV viral particle release. The E3 ubiquitin ligase MARCH8 can limit viral particle formation by downregulating various host transmembrane proteins. For DUBs in HIV viral synthesis, broad-spectrum inhibitors DUBi and small-molecule inhibitors of single DUBs have been explored. Specific inhibitors of USP7 and USP47, P22077 and PR-619, can interfere with the processing of the structural protein GAG and affect the infectivity of released HIV viral particles. However, the identification and regulatory mechanisms of HIV-1 accessory protein-related deubiquitinating enzymes remain largely unexplored.

[0006] Existing prediction methods have significant limitations: CD4 + A T-cell count below 50 cells / μl is merely a generalization risk factor and cannot effectively distinguish whether immune reconstitution is successful; the degree of viral load decline is also not significantly associated with the occurrence of incomplete immune reconstitution (INRs).

[0007] Therefore, it is necessary to provide an application of deubiquitinating enzymes as a predictive target for incomplete immune reconstitution in AIDS patients to solve the above problems. Summary of the Invention

[0008] The purpose of this invention is to provide an application of deubiquitinating enzymes as a predictive target for incomplete immune reconstitution in AIDS patients. By analyzing the ubiquitination modification network, the core mechanism of INRs is revealed, and a novel predictive model is constructed accordingly. This provides a novel molecular target with high specificity and sensitivity, enabling early and accurate identification and intervention of INRs, thereby providing a key decision-making basis for personalized treatment and having significant clinical application value.

[0009] The technical solution is as follows: Application of a deubiquitinating enzyme as a predictive target for incomplete immune reconstitution in AIDS patients, including: 1) The ubiquitinase was identified as YOD1, which exhibits significant interaction with the HIV viral protein Nef. 2) The YOD1 stable HIV accessory protein Nef is identified at the protein level rather than the RNA level: 3) Identification showed that YOD1 significantly prolongs the half-life of the HIV accessory protein Nef and affects the ubiquitination at the K6 position of the Nef protein: 4) It was determined that YOD1 significantly enhanced the expression level of Nef in HIV NL4-3 clone viral particles by reducing the ubiquitination of HIV-1 Nef protein; 5) Identify that YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. 6) It was found that under HIV infection conditions, YOD1 was positively correlated with Nef, and increased with increasing infection dose or infection time; 7) It was found that YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and that the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. 8) It was found that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence.

[0010] Furthermore, 1) specifically includes: HEK293T cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were harvested, and Flag-Nef was immunoprecipitated. The Myc-His-YOD1 protein was detected by Western blotting. HeLa cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were washed with 1×PBS, fixed on ice with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. Subsequently, the cells were incubated with anti-Myc and anti-FLAG antibodies in 1% BSA. After three washes with 1×PBS, the cells were stained with 488 goat anti-mouse IgG Alexa Fluor, A11001 or 594 goat anti-rabbit IgG Alexa Fluor, A11012; the cell nuclei were stained with DAPI. Finally, fluorescence images were captured using a Nikon A1 laser confocal microscope to observe the colocalization of Myc-His-YOD1 and Flag-Nef.

[0011] Furthermore, 2) specifically includes: HEK293T cells were transfected with Myc-His-YOD1 and co-transfected with Flag-Nef plasmid. After 48 hours of transfection, the cells were lysed and protein samples were collected. The results of HIV-1 expression of Nef protein were detected by Western blotting. HEK293T cells were transfected with Flag-Nef and simultaneously co-transfected with YOD1 knockdown plasmid. After 72 hours of transfection, the Flag-Nef protein was detected by Western Blot. We simultaneously transfected HEK293T cells with HIV NL4-3 and YOD1 knockdown plasmids. After 72 hours of transfection, we used RT-qPCR to detect the changes in the expression level of Nef gene mRNA transcribed by HIV NL4-3 in the cells. Furthermore, 3) specifically includes: HEK293T cells were transfected with blank plasmid and Myc-YOD1 plasmid, and simultaneously co-transfected with Flag-Nef. 24 hours after transfection, the cells were treated with DMSO or CHX 100 mg / mL for 0, 12 and 24 hours. After harvesting the cells, the stability of Flag-Nef protein was detected by Western Blot. HEK293T cells were transfected with plasmids shYOD1, Flag-Nef and HA-Ub. Flag-Nef protein was co-immunoprecipitated with M2 antibody, and the ubiquitination results of Flag-Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids Myc-His-YOD1, Flag-Nef, and HA-K6Ub, HA-K11Ub, HA-K27Ub, HA-K29Ub, HA-K33Ub, HA-K48Ub, and HA-K63Ub. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the polyubiquitination type of Nef protein was detected by Western blotting. HEK293T cells were transfected with plasmids Flag-Nef and Myc-YOD1. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the K6 ubiquitination of Flag-Nef protein was detected by Western blotting.

[0012] Furthermore, 4) specifically includes: HEK293T cells were transfected with Myc-YOD1 and Flag-Nef plasmids. Proteins were harvested 48 hours later, and changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD1 and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD11#;2#;3#;4# and Flag-Nef. After 72 hours, the protein was harvested, and the changes in endogenous Nef protein were detected by Western blotting.

[0013] Furthermore, 5) specifically includes: HEK293T cells were transfected with Myc-YOD1 wild-type WT and mutant CS, and Flag-Nef plasmid. After 48 hours, the protein was harvested and the changes in Flag-Nef protein were detected by Western Blot. HEK293T cells were transfected with plasmids Myc-YOD1 WT and CS and HIV NL4-3. After cell harvesting, the changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with Myc-YOD1 WT, CS, and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting.

[0014] Furthermore, 6) specifically includes: In PMA-treated THP1 cells, different doses of HIV-1 NL4-3 virus were used to infect the cells for 24 hours. The changes in Nef and YOD1 proteins were detected by Western Blot. In PMA-treated THP1 cells, HIV-I NL4-3 was used to infect cells at different time points, and the changes in Nef and YOD1 proteins were detected by Western blotting. The results are sorted from low to high based on the detected viral titer: 0 for undetectable virus, 102 for viral load, and 105 for viral load. Peripheral blood samples were collected from AIDS patients using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Changes in YOD1 and Nef proteins were detected using Western blotting. PMA-activated T cells were infected with patient serum with a viral load of 105 copies / mL, and the levels of YOD1, Nef, and Tubulin proteins were detected by Western blotting.

[0015] Furthermore, 7) specifically includes: HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. After 72 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. Cell culture supernatants were collected at 24, 48, and 72 hours after transfection. After centrifugation, the p24 antigen level in the cell supernatant at different time points was detected by ELISA. HEK293T cells were transfected with PX462, VSVG, and FH-YOD1 plasmids. After 48 hours, the supernatant was discarded, and the cells were cultured for another 48 hours. The supernatant was then collected to infect THP-1 cells. HIV virus generated by pHIV-1 (NL4-3) was then added to infect PMA-activated THP-1 cells. After 48 hours, the cell culture supernatant was collected. HIV-1 p24 antigen was detected using an ELISA kit. HEK293T cells were simultaneously transfected with HIV NL4-3, FH-YOD1 WT, and CS plasmids. After 48 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Patients were divided into two groups based on the number of CD4+ T cells: Group A had <200 CD4+ T cells / μL, and Group B had >200 CD4+ T cells / μL. The expression levels of YOD1 in the serum of patients in the incomplete and good immune reconstitution groups were investigated using ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. RNA was extracted from cells by Trizol lysis, and YOD1 mRNA results were investigated in patients with incomplete and good immune reconstitution using RT-qPCR.

[0016] Furthermore, 8) specifically includes: PMA-activated THP1 cells were induced with vitamin C (VitC) at 25µM and 50µM for 24 hours, and the changes in YOD1 protein levels were detected by Western Blot. HEK29T cells and PMA-activated THP1 cells were treated with 50µM Vitamin C for 12 and 24 hours, and the changes in YOD1 protein levels were detected by Western blotting. In PMA-activated THP1 cells, after induction with pHIV-1 (NL4-3) for 12, 24, and 48 hours, the cells were treated with 50 µM of Vitamin C for 24 hours. The changes in Nef protein levels were detected by Western blotting.

[0017] Compared with existing technologies, this invention reveals a novel mechanism and therapeutic significance of the deubiquitinating enzyme YOD1 in the HIV-1 infection process. YOD1 can specifically recognize and bind to the HIV-1 Nef protein, inhibiting its degradation via the proteasome pathway by removing ubiquitination at its K6 site, thereby stabilizing Nef protein levels, promoting viral replication, and exacerbating CD4+ T cell depletion. Functional experiments confirmed that when YOD1 undergoes a loss-of-function point mutation, the stability of the Nef protein and its mediated viral virulence are significantly suppressed. The expression level of YOD1 is positively correlated with INRs of incomplete immune reconstitution in HIV-1 infected individuals, indicating that it can serve as a potential biomarker for predicting the recovery of immune function. The association between the essential nutrient vitamin C (CVitC) and this pathway was established, confirming that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence and providing a novel adjuvant therapy strategy for the immune recovery of AIDS patients. Attached Figure Description

[0018] Figure 1 This is one of the schematic diagrams used to identify the ubiquitinase as YOD1 and its significant interaction with the HIV viral protein Nef. Figure 2 This is the second schematic diagram illustrating the identification of the ubiquitinase as YOD1 and its significant interaction with the HIV viral protein Nef. Figure 3 This is the third schematic diagram illustrating the identification of the ubiquitinase as YOD1 and its significant interaction with the HIV viral protein Nef. Figure 4 This is one of the schematic diagrams illustrating the identification of the stable HIV accessory protein Nef (YOD1) at the protein level rather than the RNA level; Figure 5 This is the second diagram illustrating the identification of the stable HIV accessory protein Nef (YOD1) at the protein level rather than the RNA level. Figure 6 This is the third diagram illustrating the identification of the stable HIV accessory protein Nef (YOD1) at the protein level rather than the RNA level. Figure 7 This is one of the schematic diagrams used to identify that YOD1 can significantly prolong the half-life of the HIV accessory protein Nef and affect the ubiquitination of the Nef protein at the K6 position. Figure 8 This is the second schematic diagram illustrating that YOD1 can significantly prolong the half-life of the HIV helper protein Nef and affect the ubiquitination of the Nef protein at the K6 position. Figure 9 This is the third schematic diagram illustrating that YOD1 can significantly prolong the half-life of the HIV helper protein Nef and affect the ubiquitination of the Nef protein at the K6 position. Figure 10 This is the fourth schematic diagram illustrating that YOD1 can significantly prolong the half-life of the HIV helper protein Nef and affect the ubiquitination of the Nef protein at the K6 position. Figure 11 This is one of the schematic diagrams used to identify how YOD1 significantly enhances the expression level of Nef in HIV NL4-3 clone viral particles by reducing ubiquitination of HIV-1 Nef protein; Figure 12 This is the second schematic diagram illustrating how YOD1 significantly enhances the expression level of Nef in HIV NL4-3 clone viral particles by reducing ubiquitination of HIV-1 Nef protein. Figure 13 This is the third schematic diagram illustrating how YOD1 significantly enhances the expression level of Nef in HIV NL4-3 clone viral particles by reducing ubiquitination of HIV-1 Nef protein. Figure 14 This is one of the schematic diagrams used to identify how YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. Figure 15 This is the second schematic diagram illustrating how YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. Figure 16 This is the third schematic diagram illustrating how YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. Figure 17 This is one of the schematic diagrams used to identify a positive correlation between YOD1 and Nef under HIV infection conditions, which increases with increasing infection dose or infection time; Figure 18 This is the second schematic diagram illustrating the positive correlation between YOD1 and Nef under HIV infection conditions, which increases with increasing infection dose or infection time. Figure 19 This is the third schematic diagram illustrating the positive correlation between YOD1 and Nef under HIV infection conditions, which increases with increasing infection dose or infection time; Figure 20 This is the fourth diagram illustrating the positive correlation between YOD1 and Nef under HIV infection conditions, which increases with increasing infection dose or infection time. Figure 21 This is one of the schematic diagrams used to identify that YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and that the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 22This is the second schematic diagram that identifies YOD1 as promoting viral replication by stabilizing Nef protein levels and exacerbating the loss of CD4+ T cells, and that YOD1 expression levels are positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 23 This is the third schematic diagram that identifies YOD1 as promoting viral replication by stabilizing Nef protein levels and exacerbating the loss of CD4+ T cells, and that YOD1 expression levels are positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 24 This is the fourth illustration showing that YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and that the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 25 This is the fifth diagram illustrating how YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and how the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 26 This is the sixth diagram illustrating how YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and how the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. Figure 27 This is one of the schematic diagrams used to demonstrate that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence. Figure 28 This is the second schematic diagram illustrating how vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence. Figure 29 This is the third diagram illustrating how vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence. Detailed Implementation Example

[0019] This embodiment demonstrates the application of a deubiquitinating enzyme as a predictive target for incomplete immune reconstitution in AIDS patients, including: 1) The ubiquitinase was identified as YOD1, which exhibits significant interaction with the HIV viral protein Nef. 2) The YOD1 stable HIV accessory protein Nef is identified at the protein level rather than the RNA level: 3) Identification showed that YOD1 significantly prolongs the half-life of the HIV accessory protein Nef and affects the ubiquitination at the K6 position of the Nef protein: 4) It was determined that YOD1 significantly enhanced the expression level of Nef in HIV NL4-3 clone viral particles by reducing the ubiquitination of HIV-1 Nef protein; 5) Identify that YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. 6) It was found that under HIV infection conditions, YOD1 was positively correlated with Nef, and increased with increasing infection dose or infection time; 7) It was found that YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and that the expression level of YOD1 is positively correlated with incomplete immune reconstitution (INRs) in HIV-1 infected individuals. 8) It was found that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence.

[0020] in: See Figures 1-3 1) Specifically includes: HEK293T cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were harvested, and Flag-Nef was immunoprecipitated. The Myc-His-YOD1 protein was detected by Western blotting. HeLa cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were washed with 1×PBS, fixed on ice with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. Subsequently, the cells were incubated with anti-Myc and anti-FLAG antibodies in 1% BSA. After three washes with 1×PBS, the cells were stained with 488 goat anti-mouse IgG Alexa Fluor, A11001 or 594 goat anti-rabbit IgG Alexa Fluor, A11012; the cell nuclei were stained with DAPI. Finally, fluorescence images were captured using a Nikon A1 laser confocal microscope to observe the colocalization of Myc-His-YOD1 and Flag-Nef.

[0021] See Figures 2-6 2) Specifically includes: HEK293T cells were transfected with Myc-His-YOD1 and co-transfected with Flag-Nef plasmid. After 48 hours of transfection, the cells were lysed and protein samples were collected. The results of HIV-1 expression of Nef protein were detected by Western blotting. HEK293T cells were transfected with Flag-Nef and simultaneously co-transfected with YOD1 knockdown plasmid. After 72 hours of transfection, the Flag-Nef protein was detected by Western Blot. We simultaneously transfected HEK293T cells with HIV NL4-3 and YOD1 knockdown plasmids. After 72 hours of transfection, we used RT-qPCR to detect the changes in the expression level of Nef gene mRNA transcribed by HIV NL4-3 in the cells. See Figures 7-10 3) Specifically includes: HEK293T cells were transfected with blank plasmid and Myc-YOD1 plasmid, and simultaneously co-transfected with Flag-Nef. 24 hours after transfection, the cells were treated with DMSO or CHX 100 mg / mL for 0, 12 and 24 hours. After harvesting the cells, the stability of Flag-Nef protein was detected by Western Blot. HEK293T cells were transfected with plasmids shYOD1, Flag-Nef and HA-Ub. Flag-Nef protein was co-immunoprecipitated with M2 antibody, and the ubiquitination results of Flag-Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids Myc-His-YOD1, Flag-Nef, and HA-K6Ub, HA-K11Ub, HA-K27Ub, HA-K29Ub, HA-K33Ub, HA-K48Ub, and HA-K63Ub. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the polyubiquitination type of Nef protein was detected by Western blotting. HEK293T cells were transfected with plasmids Flag-Nef and Myc-YOD1. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the K6 ubiquitination of Flag-Nef protein was detected by Western blotting.

[0022] See Figures 11-13 4) Specifically includes: HEK293T cells were transfected with Myc-YOD1 and Flag-Nef plasmids. Proteins were harvested 48 hours later, and changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD1 and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD11#;2#;3#;4# and Flag-Nef. After 72 hours, the protein was harvested, and the changes in endogenous Nef protein were detected by Western blotting.

[0023] See Figures 14-16 5) Specifically includes: HEK293T cells were transfected with Myc-YOD1 wild-type WT and mutant CS, and Flag-Nef plasmid. After 48 hours, the protein was harvested and the changes in Flag-Nef protein were detected by Western Blot. HEK293T cells were transfected with plasmids Myc-YOD1 WT and CS and HIV NL4-3. After cell harvesting, the changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with Myc-YOD1 WT, CS, and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting.

[0024] See Figures 17-20 6) Specifically includes: In PMA-treated THP1 cells, different doses of HIV-1 NL4-3 virus were used to infect the cells for 24 hours. The changes in Nef and YOD1 proteins were detected by Western Blot. In PMA-treated THP1 cells, HIV-I NL4-3 was used to infect cells at different time points, and the changes in Nef and YOD1 proteins were detected by Western blotting. The results are sorted from low to high based on the detected viral titer: 0 for undetectable virus, 102 for viral load, and 105 for viral load. Peripheral blood samples were collected from AIDS patients using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Changes in YOD1 and Nef proteins were detected using Western blotting. PMA-activated T cells were infected with patient serum with a viral load of 105 copies / mL, and the levels of YOD1, Nef, and Tubulin proteins were detected by Western blotting.

[0025] See Figures 21-26 7) Specifically includes: HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. After 72 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. Cell culture supernatants were collected at 24, 48, and 72 hours after transfection. After centrifugation, the p24 antigen level in the cell supernatant at different time points was detected by ELISA. HEK293T cells were transfected with PX462, VSVG, and FH-YOD1 plasmids. After 48 hours, the supernatant was discarded, and the cells were cultured for another 48 hours. The supernatant was then collected to infect THP-1 cells. HIV virus generated by pHIV-1 (NL4-3) was then added to infect PMA-activated THP-1 cells. After 48 hours, the cell culture supernatant was collected. HIV-1 p24 antigen was detected using an ELISA kit. HEK293T cells were simultaneously transfected with HIV NL4-3, FH-YOD1 WT, and CS plasmids. After 48 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Patients were divided into two groups based on the number of CD4+ T cells: Group A had <200 CD4+ T cells / μL, and Group B had >200 CD4+ T cells / μL. The expression levels of YOD1 in the serum of patients in the incomplete and good immune reconstitution groups were investigated using ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. RNA was extracted from cells by Trizol lysis, and YOD1 mRNA results were investigated in patients with incomplete and good immune reconstitution using RT-qPCR.

[0026] See Figures 27-29 8) Specifically includes: PMA-activated THP1 cells were induced with vitamin C (VitC) at 25µM and 50µM for 24 hours, and the changes in YOD1 protein levels were detected by Western Blot. HEK29T cells and PMA-activated THP1 cells were treated with 50µM Vitamin C for 12 and 24 hours, and the changes in YOD1 protein levels were detected by Western blotting. In PMA-activated THP1 cells, after induction with pHIV-1 (NL4-3) for 12, 24, and 48 hours, the cells were treated with 50 µM of Vitamin C for 24 hours. The changes in Nef protein levels were detected by Western blotting.

[0027] Specific implementation: I. Materials and Methods: (1) Cell culture and reagents: HEK293T, HeLa, and THP1 cells were all obtained from the American Collection of Standard Microbial Cultures. HEK293T and HeLa cells were cultured at 37°C and 5% CO2 using Durbeck Modified Eagle Medium (DMEM; HyClone); THP1 cells were cultured at 37°C and 5% CO2 using RPMI supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0028] (2) Plasmids and reagents: Plasmids expressing HIV-1 viral proteins, including the Flag-tagged Nef (Flag-Nef) and HIV-1 (pNL4-3), were provided by Dr. Binlian Sun (Jianghan University, China). The Myc-His-YOD1 plasmid was provided by Ubisoft Bio. HA-Ub, HA-R6K, HA-R11K, HA-R27K, HA-R29K, HA-R33K, HA-R48K, and HA-R63K (all lysine residues in the ubiquitin genes were mutated to arginine, except for the corresponding lysine residues) were provided by Professor Hui Zheng (University of Electronic Science and Technology of China, China). All gRNAs were purchased from Vigen Biosciences.

[0029] (3) RNA isolation and RT-qPCR: Total RNA was extracted from cells using TRIzol reagent (Invitrogen). All cDNAs were synthesized from 600 ng of total RNA using a 5× All-In-One RT Master Mix (Abcam, product number G490). mRNA levels were subsequently analyzed by RT-qPCR using a 2× SYBR Green qRCR Master Mix (Selleck, product number B21202). Primer sequences are as follows: The forward primer for the HIV-1 Nef gene is 5′-GCCTGGCTAGAAGCACAAGAA-3′, and the reverse primer is 5′-GTGATGAAATGCTAGGCGGC-3′.

[0030] YOD1: Forward primer, 5′-CAATGGGGATACCATTCTGG-3′; Reverse primer, 5′-ACCACGGTTCTGGTAAGC-3′.

[0031] β-actin: The forward primer is 5′-ACCAACTGGGACGACATGGAGAAA-3′, and the reverse primer is 5′-ATAGCACAGCCTGGATAGCAACG-3′.

[0032] The relative expression levels of the target gene were standardized using β-actin mRNA as a reference, and the results were analyzed based on the results of three independent experiments. The results are presented as mean ± standard deviation.

[0033] (4) Immunoblotting and immunoprecipitation: Cells were collected using 1% cell lysis buffer. The lysis buffer contained 150 mM sodium chloride, 0.5 mM EDTA, 20 mM Tris-HCl (pH 7.4), and 1% Nonidet P-40 (NP-40). Before cell collection, a mixture of PMSF (50 μg / ml) and protease inhibitors (Sigma) was added to the lysis buffer. Cell lysates were centrifuged at 12,000 g for 15 min, and protein concentration was determined using a microplate reader. Based on the calculated concentration, an equal volume of protein lysate was used for Western blotting and immunoprecipitation. Specific antibodies were added to the immunoprecipitated protein samples, and the mixture was incubated overnight at 4°C on a rotor. Subsequently, protein G agarose beads (Millipore, #16-266) were added to the overnight protein samples, and the mixture was incubated for 4 h at 4°C on a rotor. The immunoprecipitated protein samples were washed five times with lysis buffer. When protein ubiquitination was confirmed, cells were collected in 1% lysis buffer containing N-ethylmaleimide (10 mM), and the immunoprecipitate was washed once with high-salt (500 mM sodium chloride) wash buffer and four times with normal (150 mM sodium chloride) wash buffer. The co-precipitated protein samples were then eluted with loading buffer containing β-mercaptoethanol for 10 min. SDS-PAGE gel electrophoresis was then performed, transferring the protein bands onto a PVDF membrane. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 h. Primary antibody was incubated overnight, followed by incubation with anti-mouse or anti-rabbit (Bioworld) secondary antibody. Immunoreaction bands were visualized using the SuperSignalWest Dura Extended kit (Thermo Scientific).

[0034] (5) Mass spectrometry (MS): HEK293T cells were transfected with either an empty vector or a vector expressing Flag-Nef. Forty-eight hours after transfection, cells were harvested in NP-40 lysis buffer. Flag-Nef and its interacting proteins were extracted from whole-cell lysates using a Flag M2 affinity gel (A2220; Sigma-Aldrich). SDS-PAGE gels were stained with Coomassie Brilliant Blue. Gel bands from the control and experimental groups were carefully excised and digested with trypsin. The resulting trypsin peptides were purified using a C18 Zip Tip and then analyzed using an Orbitrap Elite hybridization mass spectrometer coupled to a Dionex LC (Thermo Fisher Scientific). Spectral data were compared with the UniProt protein database using Proteome Discoverer 1.4.

[0035] (6) Immunofluorescence microscopy: HEK293T cells were transfected with two vectors to express Myc-His-YOD1 and Flag-Nef. After washing with 1×PBS, cells were fixed on ice with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. Subsequently, cells were incubated in 1% BSA with anti-Myc and anti-FLAG antibodies. After three washes with 1×PBS, cells were stained with either 488 goat anti-mouse IgG (Alexa Fluor, A11001) or 594 goat anti-rabbit IgG (Alexa Fluor, A11012). Cell nuclei were stained with DAPI. Finally, fluorescence images were captured using a Nikon A1 laser confocal microscope.

[0036] (7) HIV-1 Virus Stock Preparation and HIV-1 Infection: Regarding HIV-1 replication capacity, HEK293T cells cultured in 10 cm culture dishes were transfected with 30 μg of plasmid pHIV-1 (NL4-3) to produce HIV-1 viral particles. Cell culture supernatant was collected 48 hours after transfection, clarified by centrifugation at 3,500 rpm for 10 min, filtered through a 0.45 μm filter, and then concentrated by ultracentrifugation at 30,000 rpm for 1.5 h at 4 °C. Aliquots were prepared and HIV-1 viral particles were stored at -80 °C. PMA (80 ng / ml) activated THP-1 cells or peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 (NL4-3) virus at a fold increase of 0.5 or 1.0 for 6 h at 37 °C in a culture medium containing Polybrene (8 μg / mL) and intermittently mixed. Cells were then washed twice with 1X PBS. PBMCs were cultured in complete medium containing 20 U / mL of recombinant human IL-2 (R&D) until harvest. The culture supernatant of infected cells was collected and virus production was detected by p24 antigen capture ELISA.

[0037] (8) Patient samples, data, and ethics: This study included samples from 90 HIV-infected patients who received antiretroviral therapy at the Affiliated Infectious Disease Hospital of Soochow University between 2022 and 2023. Patients were categorized as having good or incomplete immune reconstitution based on their antiretroviral therapy status after two years. Immune reconstitution was defined as the CD4+ T cell count (CD4 < 200 indicated incomplete reconstitution). Peripheral blood samples were used prior to the first treatment. Informed consent was obtained from each participant, and the study was approved by the Ethics Committee of the Fifth People's Hospital of Suzhou (K-2024-005-01).

[0038] (9) Cycloheximide Pulse Chase Assay: The stability of FLAG-Nef protein was determined using the CHX pulse tracking assay. Briefly, HEK293T cells were co-transfected with two vectors expressing FLAG-Nef and Myc-YOD1. Twenty-four hours post-transfection, cells were treated with either DMSO or CHX (100 mg / mL), then harvested and subjected to Western blot analysis. Data analysis was performed using ImageJ from three independent experiments and is presented as mean ± standard deviation.

[0039] Statistical analysis: Two-tailed t-tests were used to analyze statistical significance for intergroup comparisons. ∗P<0.05, ∗P<0.01, and ∗∗P<0.001 were considered statistically significant. NS indicates no significance. Data were analyzed using Graph Pad Prism 8.3.0 software. II. Experimental Results

[0040] (1) Interaction between YOD1 and Nef: We transfected HEK293T cells with CON and Flag-Nef, and co-precipitated Flag-Nef protein using M2 antibody. LC-MS was then used to identify proteins interacting with Nef. The results showed that several deubiquitinating enzymes, including YOD1, USP25, STAMBP, and CYLD, could interact with Nef protein (Fig. 1A). Furthermore, we performed a dual exogenous assay in cells to confirm their interaction. We transfected HEK293T cells with Myc-YOD1 and Flag-Nef plasmids, and co-precipitated Myc-YOD1 using Myc antibody. Western blotting analysis revealed a significant interaction between Myc-YOD1 and Flag-Nef (Fig. 1B). Immunofluorescence microscopy further demonstrated the co-localization of Flag-Nef and Myc-YOD1 (Fig. 1C). These data confirm the physical interaction between Nef and YOD1.

[0041] (2) YOD1 stabilizes Nef protein expression: HEK293T cells were graded-transfected with the FH-YOD1 plasmid and co-transfected with the Flag-Nef plasmid to examine the effect of the deubiquitinating enzyme YOD1 on Nef protein levels. The results are shown in Figure 2A. YOD1 significantly stabilized Nef protein levels, and this effect increased with increasing dosage. Furthermore, the knockdown plasmid shYOD1 of the deubiquitinating enzyme YOD1 was constructed. HEK293T cells were transfected with both Flag-Nef and shYOD1 (1... # ,2 # 3 # 4 #The results showed that knocking down endogenous YOD1 expression in host cells significantly suppressed Nef protein expression (Fig. 2B). Based on these findings, we investigated whether YOD1 stabilizes Nef by affecting its transcriptional level. HEK293T cells were transfected with HIV NL4-3 and shYOD1 plasmids (1#, 2#, 3#). After 72 hours, changes in Nef gene mRNA levels were detected. We found that specific knockdown of YOD1 did not affect Nef mRNA levels, but YOD1 transcription was significantly suppressed (Fig. 2C). These findings indicate that cellular YOD1 is a positive regulator of HIV-1 Nef protein expression, and this effector pathway is not a change in transcriptional levels.

[0042] (3) YOD1 antagonizes Nef ubiquitination, half-life, and degradation by targeting the K6-linked ubiquitin chain: The results showed that YOD1-mediated upregulation of Nef expression did not enhance Nef transcription, suggesting that YOD1 may be involved in Nef's post-translational modification. We then used cyclohexanone (CHX), an inhibitor of protein biosynthesis, in a CHX pulse-chase assay to determine the stability of HIV-1 Nef protein in cells. In HEK293T cells transfected with CON and YOD1, we observed that YOD1 overexpression significantly inhibited Nef protein stability, thereby upregulating HIV-1 Nef protein levels and prolonging its half-life (Fig. 3A).

[0043] As is well known, during protein deubiquitination, deubiquitinating enzymes regulate the protein levels of substrate proteins by removing ubiquitin molecules from the substrate protein. As a member of the DUB ovarian tumor (OUT) family, we hypothesized that YOD1 might affect the ubiquitination modification of Nef. Therefore, we analyzed the ubiquitination level of Nef protein after knocking out YOD1. We transfected HEK293T cells with either shCON or shYOD1 (1... # ,2 # 3 # 4 #Simultaneously, Flag-Nef and HA-Ub were co-transfected, and after 72 hours, Flag-Nef protein was immunoprecipitated using M2 antibody. The results showed that knocking down YOD1 significantly enhanced the ubiquitination level of Flag-Nef protein (Fig. 3B). These results indicate that YOD1 stabilizes Nef protein levels by inhibiting its ubiquitination. So, through what ubiquitination form does YOD1 regulate the deubiquitination of Nef protein? Next, we further analyzed the deubiquitination type of the HIV-1 expression gene Nef protein induced by YOD1. In HEK293T cells, Myc-His-YOD1 and the Flag-Nef expression vector were overexpressed, and HA-Ub (K6, K11, K27, K29, K33, K48, and K63) were co-transfected. Using immunoprecipitation and Western blotting, we found that overexpression of YOD1 specifically weakened the formation of the K6 polyubiquitin chain of Nef, thereby enhancing its stability (Fig. 3C). Next, we transfected Flag-Nef or Myc-YOD1 plasmids to further confirm that the level of YOD1 stabilizing Nef protein depends on the formation of K6 polyubiquitin chains (Fig 3D). These data show that cellular YOD1, as a deubiquitinating enzyme, cleaves the K6-linked ubiquitin chain of the HIV-1 Nef protein, preventing it from subsequently causing Nef protein degradation via the proteasome pathway.

[0044] Figure 4 shows that YOD1 plays an active role in regulating the expression level of viral Nef protein in the HIV-1 NL4-3 infection model.

[0045] We further utilized the HIV-1 clone HIV-NL4-3 to generate viral particles in cells to simulate the transmission virulence of HIV-1. Results analysis showed that overexpression of YOD1 significantly enhanced the expression level of Nef protein produced by HIV-1 (NL4-3) (Fig. 4A). Conversely, in YOD1-deficient cells, HIV-1 (NL4-3 strain) produced lower levels of Nef protein (Fig. 4B). Specifically, we discovered that specific knockdown of YOD1 (shYOD1 1)... # ,2 # 3 # 4 # Following this, YOD1 enhanced the intracellular ubiquitination level of Nef protein produced by HIV-1 (NL4-3) (Fig 4C), confirming that cellular YOD1 can reduce the ubiquitination modification of HIV-1 viral Nef protein, thereby leading to the upregulation of Nef protein expression. This indicates that intracellular YOD1 reduces the ubiquitination of HIV-1-produced Nef protein, thereby leading to the upregulation of Nef protein.

[0046] Figure 5. The enzyme activity of YOD1 is crucial for the stability of Nef: We conducted exogenous experiments by constructing the YOD1 enzyme-active mutant plasmid Myc-YOD1 C110S. The results showed that, consistent with previous studies, YOD1 can stabilize the Nef protein. When Myc-YOD1 loses its enzyme activity, it loses its stabilizing effect on the Nef protein (Fig. 5A). Simultaneously, the Nef protein produced by HIV-1 virus also became unstable in the presence of the mutant. In contrast, wild-type YOD1 can stabilize the Nef level produced by HIV-1 (NL4-3) (Fig. 5B). Previous results showed that YOD1 can reduce Nef ubiquitination, thereby mediating an increase in protein levels. Therefore, we investigated whether the YOD1 active mutant affects Nef ubiquitination levels. Overexpression of HIV NL4-3 and Myc-YOD1 (WT / CS) in HEK293T cells showed that, compared to the wild-type Myc-YOD1 strain, the intracellular Myc-YOD1 C110S mutant no longer reduced the endogenous ubiquitination level of the Nef protein. Therefore, the previous conclusions were further validated: when YOD1 mutates, its deubiquitinase function is lost, thus failing to promote the deubiquitination of the endogenous Nef protein in HIV-1 (Fig. 5C). These experimental results collectively demonstrate that YOD1 inhibits the protein degradation process of Nef by mediating its deubiquitination, and this function depends on its deubiquitinase active site. In conclusion, inhibiting YOD1 expression during HIV-1 infection can promote the degradation of the Nef protein, thus potentially becoming a novel strategy for inhibiting HIV-1 replication.

[0047] Figure 6. YOD1 stabilizes the Nef protein, enhancing viral replication. To further investigate the correlation between YOD1 and Nef in HIV-1 infection, we infected PMA-treated THP1 cells with different doses of HIV-1 NL4-3 virus. We found that YOD1 and Nef gradually increased with the increase of viral particle count (Fig. 6A). We also found that under HIV-1 NL4-3 infection conditions, the expression of YOD1 and Nef was time-dependent (Fig. 6B).

[0048] To verify the above results, we sorted the HIV viral load in the blood samples of AIDS patients according to the level (from undetectable as 0, to 10² copies / mL, up to 10...). 5(Copies / mL). Peripheral blood mononuclear cells (PBMCs) were isolated. Western blot results showed a positive correlation between viral load and YOD1 protein expression level. Simultaneously, YOD1 and Nef protein expression levels in the patient's peripheral blood samples also showed a consistent positive correlation (Fig. 6C). Furthermore, with prolonged infection time, the protein expression levels of both YOD1 and Nef gradually increased (Fig. 6D). These results indicate that during the HIV infection process, with prolonged infection time and increased viral load, YOD1 may affect the patient's infection status and subsequent immune status by regulating the stability of the Nef protein.

[0049] Figure 7. YOD1 influences the efficacy of antiretroviral therapy and incomplete immune reconstitution in HIV patients: Previous results have confirmed that YOD1 stabilizes Nef levels. Given that Nef protein deficiency weakens HIV-1 virulence in infecting macrophages or T cells, we will next assess the effect of YOD1 on HIV-1 infectivity based on changes in p24 protein levels (a commonly used marker for assessing HIV-1 viral particle levels) in the supernatant of HEK293T cell culture. We transfected HEK293T cells with shCON or shYOD1 (1... # 2 # 3 # 4 # Simultaneously, the cells were co-transfected with the HIV-1 NL4-3 plasmid, and the expression of p24 antigen in the cell culture supernatant was detected. The results showed that knocking out YOD1 significantly decreased HIV-1 p24 antigen expression (Fig. 7A), consistent with changes in p24 antigen in peripheral blood PBMCs from PLWH patients. Furthermore, the rate of p24 antigen decrease after YOD1 knockout was significantly higher than that in the blank control group with prolonged infection time (Fig. 7B). In addition, we observed that overexpression of YOD1 increased the virulence of HIV-1 viral particles in a dose-dependent manner (Fig. 7C), suggesting that YOD1 promotes HIV-1 viral replication. However, the Myc-YOD1 C110S mutant lost the ability to promote viral particle virulence (Fig. 7D). Therefore, during HIV-1 infection, YOD1 promotes viral pathogenicity, which may hinder the recovery of immune function in AIDS patients.

[0050] We analyzed the differences in YOD1 levels in patients with different immune reconstitution outcomes. We further examined serum YOD1 protein expression levels in blood samples from both groups of infected individuals. Results showed that YOD1 protein expression was present in CD4+. +In the group with poor immune reconstitution (T cells <200 cells / μL), the YOD1 protein content was significantly higher than that in the group with good immune reconstitution (p<0.05), suggesting that YOD1 may be involved in reducing CD4 molecule expression (Fig. 7E). Transcriptional analysis revealed that CD4... + In infected individuals with T cell counts <200 cells / μL, the expression level of YOD1 mRNA was significantly higher than that of CD4. + Infected individuals with T cell counts >200 cells / μL (Fi6F). Based on this, we hypothesize that YOD1 may affect the efficacy of antiretroviral therapy and incomplete immune reconstitution in HIV patients.

[0051] Figure 8. Vitamin C enhances its anti-HIV-1 efficacy by degrading YOD1: We have found that YOD1 plays a crucial role in mediating the enhanced infectivity of HIV-1 viral particles by Nef, and developing pathways to reduce YOD1 levels may aid in the body's antiviral response. Studies have shown that the essential nutrient vitamin C (VitC, L-ascorbic acid, ascorbate) can downregulate ACE2 protein levels and enhance defense against SARS-CoV-2 and its variants. The mechanism is mainly that VitC significantly inhibits viral infection by blocking the interaction between USP50 and ACE2, leading to ACE2 degradation. Therefore, based on the hypothesis that VitC can affect YOD1 expression levels, we conducted a preliminary investigation. Under normal physiological conditions, the concentration of VitC in human plasma is approximately 50-70 μM. Our study found that a concentration of 50 μM VitC significantly reduced YOD1 levels (Fig. 8A). Interestingly, at this concentration, in HEK293T cells and THP-1 cells, YOD1 protein levels gradually decreased over time after VitC treatment (Fig. 8B). We also found that, compared with the control group, the expression of Nef protein in the HIV-1 (NL4-3) strain was significantly reduced after treatment with Vitamin C (Fig. 8C). In conclusion, Vitamin C, an essential nutrient, can downregulate the levels of YOD1 and Nef proteins, thereby enhancing the efficacy of anti-HIV-1 therapy.

[0052] Compared with existing technologies, this invention reveals a novel mechanism and therapeutic significance of the deubiquitinating enzyme YOD1 in the HIV-1 infection process. YOD1 can specifically recognize and bind to the HIV-1 Nef protein, inhibiting its degradation via the proteasome pathway by removing ubiquitination at its K6 site, thereby stabilizing Nef protein levels, promoting viral replication, and exacerbating CD4+ T cell depletion. Functional experiments confirmed that when YOD1 undergoes a loss-of-function point mutation, the stability of the Nef protein and its mediated viral virulence are significantly suppressed. The expression level of YOD1 is positively correlated with INRs of incomplete immune reconstitution in HIV-1 infected individuals, indicating that it can serve as a potential biomarker for predicting the recovery of immune function. The association between the essential nutrient vitamin C (CVitC) and this pathway was established, confirming that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence and providing a novel adjuvant therapy strategy for the immune recovery of AIDS patients.

[0053] For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of this invention, and these all fall within the protection scope of this invention.

Claims

1. The application of a deubiquitinating enzyme as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: include: 1) The ubiquitinase was identified as YOD1, which exhibits significant interaction with the HIV viral protein Nef. 2) The YOD1 stable HIV accessory protein Nef is identified at the protein level rather than the RNA level: 3) Identification showed that YOD1 significantly prolongs the half-life of the HIV accessory protein Nef and affects the ubiquitination at the K6 position of the Nef protein: 4) It was determined that YOD1 significantly enhanced the expression level of Nef in HIV NL4-3 clone viral particles by reducing the ubiquitination of HIV-1 Nef protein; 5) Identify that YOD1 acts as a deubiquitinating enzyme to inhibit the ubiquitination of Nef protein, thereby stabilizing Nef protein. 6) It was found that under HIV infection conditions, YOD1 was positively correlated with Nef, and increased with increasing infection dose or infection time; 7) It was found that YOD1 promotes viral replication by stabilizing Nef protein levels and exacerbates the loss of CD4+ T cells, and that the expression level of YOD1 is positively correlated with incomplete immune reconstitution in HIV-1 infected individuals. 8) It was found that vitamin C can effectively downregulate the expression levels of YOD1 and Nef proteins, thereby reducing viral virulence.

2. The application of the deubiquitinating enzyme according to claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 1) Specifically includes: HEK293T cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were harvested, and Flag-Nef was immunoprecipitated. The Myc-His-YOD1 protein was detected by Western blotting. HeLa cells were co-transfected with Myc-His-YOD1 and Flag-Nef. After 48 hours, the cells were washed with 1×PBS, fixed on ice with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. Subsequently, the cells were incubated with anti-Myc and anti-FLAG antibodies in 1% BSA. After three washes with 1×PBS, the cells were stained with 488 goat anti-mouse IgG Alexa Fluor, A11001 or 594 goat anti-rabbit IgG Alexa Fluor, A11012; the cell nuclei were stained with DAPI. Finally, fluorescence images were captured using a Nikon A1 laser confocal microscope to observe the colocalization of Myc-His-YOD1 and Flag-Nef.

3. The application of a deubiquitinating enzyme as described in claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 2) Specifically includes: HEK293T cells were transfected with Myc-His-YOD1 and co-transfected with Flag-Nef plasmid. After 48 hours of transfection, the cells were lysed and protein samples were collected. The results of HIV-1 expression of Nef protein were detected by Western blotting. HEK293T cells were transfected with Flag-Nef and simultaneously co-transfected with YOD1 knockdown plasmid. After 72 hours of transfection, the Flag-Nef protein was detected by Western Blot. In HEK293T cells, HIV NL4-3 and YOD1 knockdown plasmids were simultaneously transfected. After 72 hours of transfection, RT-qPCR was used to detect changes in the expression level of Nef gene mRNA transcribed by HIV NL4-3 in the cells.

4. The application of the deubiquitinating enzyme according to claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 3) Specifically includes: HEK293T cells were transfected with blank plasmid and Myc-YOD1 plasmid, and simultaneously co-transfected with Flag-Nef. 24 hours after transfection, the cells were treated with DMSO or CHX 100 mg / mL for 0, 12 and 24 hours. After harvesting the cells, the stability of Flag-Nef protein was detected by Western Blot. HEK293T cells were transfected with plasmids shYOD1, Flag-Nef and HA-Ub. Flag-Nef protein was co-immunoprecipitated with M2 antibody, and the ubiquitination results of Flag-Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids Myc-His-YOD1, Flag-Nef, and HA-K6Ub, HA-K11Ub, HA-K27Ub, HA-K29Ub, HA-K33Ub, HA-K48Ub, and HA-K63Ub. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the polyubiquitination type of Nef protein was detected by Western blotting. HEK293T cells were transfected with plasmids Flag-Nef and Myc-YOD1. Flag-Nef protein was co-immunoprecipitated using M2 antibody, and the K6 ubiquitination of Flag-Nef protein was detected by Western blotting.

5. The application of a deubiquitinating enzyme as described in claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 4) Specifically includes: HEK293T cells were transfected with Myc-YOD1 and Flag-Nef plasmids. Proteins were harvested 48 hours later, and changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD1 and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with plasmids shYOD11#;2#;3#;4# and Flag-Nef. After 72 hours, the protein was harvested, and the changes in endogenous Nef protein were detected by Western blotting.

6. The application of the deubiquitinating enzyme according to claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 5) Specifically includes: HEK293T cells were transfected with Myc-YOD1 wild-type WT and mutant CS, and Flag-Nef plasmid. After 48 hours, the protein was harvested and the changes in Flag-Nef protein were detected by Western Blot. HEK293T cells were transfected with plasmids Myc-YOD1 WT and CS and HIV NL4-3. After cell harvesting, the changes in endogenous Nef protein were detected by Western blotting. HEK293T cells were transfected with Myc-YOD1 WT, CS, and HIV NL4-3. Nef protein was co-precipitated using Nef antibody, and the ubiquitination results of endogenous Nef protein were detected by Western blotting.

7. The application of the deubiquitinating enzyme according to claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 6) Specifically includes: In PMA-treated THP1 cells, different doses of HIV-1 NL4-3 virus were used to infect the cells for 24 hours. The changes in Nef and YOD1 proteins were detected by Western Blot. In PMA-treated THP1 cells, HIV-I NL4-3 was used to infect cells at different time points, and the changes in Nef and YOD1 proteins were detected by Western blotting. The virus titers are sorted from low to high based on the detected viral load, with 0 being undetectable, 102 being viral load, and 105 being viral load. Peripheral blood samples were collected from AIDS patients using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Changes in YOD1 and Nef proteins were detected using Western blotting. PMA-activated T cells were infected with patient serum with a viral load of 105 copies / mL, and the levels of YOD1, Nef, and Tubulin proteins were detected by Western blotting.

8. The application of a deubiquitinating enzyme as described in claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 7) Specifically includes: HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. After 72 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. HEK293T cells were simultaneously transfected with two knockdown plasmids, shYOD1 1#, 2#, 3#, and 4#, of HIV NL4-3 and YOD1. Cell culture supernatants were collected at 24, 48, and 72 hours after transfection. After centrifugation, the p24 antigen level in the cell supernatant at different time points was detected by ELISA. HEK293T cells were transfected with PX462, VSVG, and FH-YOD1 plasmids. After 48 hours, the supernatant was discarded, and the cells were cultured for another 48 hours. The supernatant was then collected to infect THP-1 cells. HIV virus generated by pHIV-1 (NL4-3) was then added to infect PMA-activated THP-1 cells. After 48 hours, the cell culture supernatant was collected. HIV-1 p24 antigen was detected using an ELISA kit. HEK293T cells were simultaneously transfected with HIV NL4-3, FH-YOD1 WT, and CS plasmids. After 48 hours of transfection, the cell culture supernatant was collected, centrifuged, and the p24 antigen level in the cell supernatant was detected by ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. Patients were divided into two groups based on the number of CD4+ T cells: Group A had <200 CD4+ T cells / μL, and Group B had >200 CD4+ T cells / μL. The expression levels of YOD1 in the serum of patients in the incomplete and good immune reconstitution groups were investigated using ELISA. Peripheral blood samples from patients with PLWH were collected using EDTA-K2 anticoagulant tubes, and PBMCs were isolated using lymphocyte separation fluid. RNA was extracted from cells by Trizol lysis, and YOD1 mRNA results were investigated in patients with incomplete and good immune reconstitution using RT-qPCR.

9. The application of a deubiquitinating enzyme as described in claim 1 as a predictive target for incomplete immune reconstitution in AIDS patients, characterized in that: 8) Specifically includes: PMA-activated THP1 cells were induced with vitamin C (VitC) at 25µM and 50µM for 24 hours, and the changes in YOD1 protein levels were detected by Western Blot. HEK29T cells and PMA-activated THP1 cells were treated with 50 µM of Vitamin C for 12 and 24 hours, and the changes in YOD1 protein levels were detected by Western Blot. In PMA-activated THP1 cells, after induction with pHIV-1 (NL4-3) for 12, 24, and 48 hours, the cells were treated with 50 µM of Vitamin C for 24 hours. The changes in Nef protein levels were detected by Western blotting.

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