MG132-mediated inhibition of rabies virus replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway

MG132-mediated inhibition of rabies virus replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MG132-mediated inhibition of rabies virus replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway Ying lin Chi, Nuo Yang, Yuan Xie, Xiaoyan Tao, Pengcheng Yu, Qian Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8283741/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2026 Read the published version in Virology Journal → Version 1 posted 9 You are reading this latest preprint version Abstract Rabies virus (RABV), a fatal neurotropic virus, still lacks effective antiviral drugs for treatment once symptoms appear. MG132 acts as a novel therapeutic agent against viral infection, inhibits the degradation of nuclear factor erythroid-derived 2-like 2 (Nfe2l2; Nrf2), and enhances SQSTM1 expression in mouse hypothalamic organotypic cultures. Rapamycin-induced autophagy and Parkin-mediated mitophagy could also be inhibited by MG132. Autophagy signaling was triggered by RABV replication, but the antiviral effects of MG132 against RABV remained unclear. Here, we showed that MG132 exerted potent inhibitory activity against different viral strains, with an efficacy comparable to that of ribavirin and higher than that of T705 in vitro. We further demonstrated that MG132 inhibited RABV replication by disrupting the Nrf2/SQSTM1/PINK1/Parkin pathway, with SQSTM1 acting as a key mediator. MG132 also prevented SQSTM1 degradation and enhanced colocalization between SQSTM1 and PINK1/Parkin. In vivo, MG132 (10 mg/kg) attenuated body weight loss and prolonged survival of RABV-infected mice by 40%. Overall, our findings indicated that MG132 inhibited RABV replication via the Nrf2/SQSTM1/PINK1/Parkin-related autophagy pathway and highlighted MG132 as a potential therapeutic agent for rabies. MG132 RABV Nrf2 SQSTM1 PINK1 Parkin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), responsible for approximately 60,000 deaths globally each year, imposing a substantial disease burden worldwide [1, 2]. Post-exposure immunization remains the only effective treatment for rabies [3], and there is still no effective antiviral drug against RABV. Consequently, the search for effective antiviral therapies remains a central focus of rabies research. MG132 is a reversible competitive proteasome inhibitor that is widely utilized in antiviral therapy research. It primarily inhibits the chymotrypsin-like activity of the proteasome, preventing the degradation of misfolded or damaged proteins. Current treatment strategies for virus-induced neurodegenerative diseases focus on maintaining proteasomal balance and stability in the body [4]. Recent studies suggest that MG132 inhibits viral replication by blocking proteasomal degradation of viruses such as classical swine fever virus (CSFV) [5], herpes simplex virus type 1 (HSV-1) [6], and hepatitis B virus (HBV) [7]. However, the antiviral effect of MG132 on RABV remains unclear. MG132 also inhibits the degradation and promotes the accumulation and nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nfe2l2; Nrf2), which suppresses inflammation and oxidative damage [8]. In vitro studies have confirmed that RABV infection induces the accumulation of reactive oxygen species (ROS), leading to a strong oxidative stress response [9]. Nrf2 is a critical transcription factor that regulates several genes related to the oxidative stress response, such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) [10, 11]. Previous studies have indicated that MG132 promotes both the mRNA and protein levels of SQSTM1 in hypothalamic organotypic cultures and induces high SQSTM1 expression in embryonic fibroblast (MEF) cells [12, 13]. We previously found that enhancing Nrf2 expression could inhibit RABV replication and was accompanied by an elevation of SQSTM1 expression [14]. Studies have shown that Nrf2 cooperates with SQSTM1 to regulate mitochondrial autophagy and maintain the expression levels of H2AX and ataxia-telangiectasia mutated (ATM), thereby inhibiting Epstein-Barr virus (EBV)-driven B-cell oncogenesis [15]. SQSTM1 directly interacts with Kelch-like ECH-associated protein 1 (Keap1) and disrupts the association between Keap1 and Nrf2, resulting in the stabilization and nuclear accumulation of Nrf2 [16]. Meanwhile, SQSTM1 is known as a target of Nrf2, and Nrf2 activation induces SQSTM1 production[17-19]. Moreover, the synergy between SQSTM1 and Nrf2 also regulates NQO1, enhancing antioxidant production and preventing muscle atrophy caused by oxidative stress [20]. These findings underscore the crucial role of the Nrf2–SQSTM1 pathway in modulating disease progression. As a scaffold protein, SQSTM1 is responsible for recognizing specific organelles and protein aggregates and regulating autophagy, a process that plays important roles in various neurodegenerative diseases [21-23]. The structure of SQSTM1 contains several functional domains, including the LC3-interacting region (LIR), which collaborates with related proteins to maintain cellular activity and homeostasis [24]. Furthermore, SQSTM1 also plays a significant regulatory role in viral infections. Studies have found that GAL9, a member of the galectin-9 family, interacts with the antiviral factor viperin to guide the autophagic degradation of HBV-HBc. Viperin interacts with HBc, inducing its accumulation in cytoplasmic foci, enhancing the self-ubiquitination of RNF13, and promoting the recruitment of SQSTM1 and the formation of LC3-positive autophagosomes, thereby participating in the regulation of viral infection. Targeting SQSTM1 aggregation mediated by viperin and GAL9 may reveal viral infection mechanisms and provide reliable targets for antiviral therapy [25]. Previous studies have shown that RABV can influence autophagy by regulating changes in SQSTM1 and LC3, thereby promoting viral replication [26]. Inhibition of SQSTM1- and LC3-mediated autophagic pathways reduces RABV replication [27]. PGG upregulates SQSTM1 to reduce LC3-mediated autophagy, significantly decreasing the accumulation of RABV M protein in autophagic structures[28]. Multiple lines of evidence suggest that SQSTM1 plays a pivotal regulatory role in autophagy induced by RABV. Ample evidence indicates that RABV infection induces ROS generation, leading to oxidative stress, mitochondrial dysfunction, and neuronal injury [29]. SQSTM1 can be recruited to damaged mitochondria to participate in their clearance, and interactions with the mitochondrial autophagy proteins PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) play a major role in this mitophagy process [30]. During the clearance of damaged mitochondria, PINK1 accumulates on the damaged mitochondrial surface and recruits Parkin, marking the damaged mitochondria for autophagic degradation [31, 32]. Human T-cell leukemia virus type 1 (HTLV-1) can elevate its replication by promoting PINK1–Parkin-dependent mitophagy and recruiting the autophagy receptor SQSTM1 to damaged mitochondria [33]. During respiratory syncytial virus (RSV) infection, its nonstructural protein (NS1) promotes the degradation of SQSTM1 to initiate PINK1/Parkin-independent mitophagy, thereby accelerating disease progression [34]. In addition, Newcastle disease virus (NDV) can regulate host cell metabolism by activating mitophagy through the SQSTM1/PINK1/Parkin pathway [35]. Therefore, SQSTM1 plays an important role in regulating PINK1/Parkin-mediated mitophagy and antiviral infection. However, the role of the autophagic mechanism mediated by the Nrf2/SQSTM1/PINK1/Parkin axis in RABV replication remains unclear. This study investigated the effects of MG132 on RABV replication both in vitro and in vivo and explored the mechanism by which MG132 influences RABV replication through autophagy mediated by the Nrf2/SQSTM1/PINK1/Parkin pathway. 2. Materials and methods 2.1. Cell culture and reagents The mouse neuroblastoma cell line N2a (CCL-131) and Golden Syrian hamster kidney cells BSR were purchased from ATCC and maintained in ATCC-formulated Dulbecco’s modified Eagle medium (DMEM, ATCC) with 10% fetal bovine serum (FBS, Gibco) at 37℃ in a humidified 5% CO₂ atmosphere. MG132, ribavirin, T705, bardoxolone methyl (CDDO-Me), retinoic acid (ATRA), rapamycin (Rapa), 3-methyladenine (3-MA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Med-Chem-Express. Stock solutions were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Each stock solution was freshly diluted with culture medium to a final concentration of 0.05% DMSO (v/v). Control cells were treated with 0.05% DMSO. 2.2. Cell viability assay The Cell Counting Kit-8 assay (CCK-8; Dojindo, Mashikimachi, Japan) was used to analyze cell viability in the absence or presence of MG132 in culture medium according to the manufacturer’s instructions. Briefly, N2a or BSR cells were seeded and grown to approximately 70-80% confluence, and the culture medium was replaced with freshly prepared MG132 solutions that had been diluted to the appropriate concentrations (1 μM, 2 μM, 5 μM, 10 μM, 20 μM or 25 μM) in culture medium. N2a or BSR cells treated with DMSO served as controls in the experiment. The commercially available CCK-8 assay was used to evaluate the cytotoxic effect of MG132. The absorbance of the supernatants at 450 nm was measured with a FLUOstar Omega instrument (BMG LABTECH, USA). All experiments were performed in triplicate. 2.3. Virus strains Two RABV strains were used in vitro, CVS-11 and CTN. These strains were chosen for this study because their isolation backgrounds, genetic characteristics, and genomic information were known and complete. The RABV strains were maintained in our laboratory. CVS-11 was a classic fixed RABV strain adapted from the CVS strain through BHK-21 cells, with strong neurotoxicity. It was used as the standard challenge strain in the rapid fluorescent focus inhibition test and was preserved in our laboratory [36, 37]. 2.4. Viral titration N2a cells or BSR cells were plated in 96-well culture plates. The cells were incubated with different strains at a multiplicity of infection (MOI) of 1 suspended in test medium for 1 h at 37℃, washed, and incubated with culture medium. At 48 h post infection (hpi), the viral titer in N2a or BSR cells was determined using a focus assay as described previously [38] . Viral foci were detected using a FITC-conjugated anti-RABV N antibody (Fujirebio Diagnostics, Inc, Malvern, PA, USA). The foci were counted to determine the viral titer (FFU/mL). 2.5. Antiviral assays N2a or BSR cells were used for antiviral assays, as they are standard models for RABV [39]. Cells were cultured to 70-80% confluence, then infected with RABV (CVS-11 or CTN) at an MOI of 1. MG132 was added at varying concentrations (1 μM, 2 μM, 5 μM, 10 μM, 20 μM or 25 μM) in triplicate. DMSO-treated virus-infected cells served as controls. At 48 h post infection, virus concentrations in the culture supernatants were measured using a direct fluorescent antibody (DFA) assay. Cells were fixed and stained with FITC-conjugated anti-RABV N antibody [40], and fluorescence signals were detected with an Olympus IX70 microscope (Olympus, Tokyo, Japan). Ribavirin and T705 were used as reference drugs. The antiviral effects of MG132, ribavirin, and T705 were assessed in N2a cells at 48 hpi, with virus titers determined. Stored supernatants were titrated as described previously [41]. The 50% cytotoxic concentration (CC 50 ) and 50% effective concentration (EC 50 ) were calculated via dose–response regression analysis. The 50% selective index (SI 50 ) was calculated by dividing the CC 50 for each compound and virus by the corresponding EC 50 as described previously [42]. All experiments were repeated in triplicate. 2.6. Western blot N2a cells were seeded in six-well plates, infected with CVS-11 or CTN for 1 h, and then cultured with MG132 for 48 hpi. After adding proteinase and phosphatase inhibitors (Roche; Indianapolis, IN, USA), cells were lysed using the M-PER mammalian protein extraction kit (Pierce; Rockford, IL, USA). Proteins were separated by SDS‒PAGE, transferred to PVDF membranes, blocked with 5% nonfat milk in TBST, and incubated with primary antibodies (anti-Nrf2 (#12721; Cell Signaling Technology), anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#6946; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-Tomm20 (#AB186735; Abcam), anti-LC3 (#12741; Cell Signaling Technology)), a mouse monoclonal antibody (mAb) against the RABV P protein (kindly provided by Professor Shoufeng Zhang’s laboratory), and anti-β-actin (#4967; Cell Signaling Technology) as a loading control. Membranes were then incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using Super Signal West Dura and analyzed with Image Lab 3.0 (Bio-Rad). Mouse brain tissues were lysed with the T-PER Tissue Protein Extraction kit (Pierce; Rockford, IL, USA) [43]. The brain tissue was ground and prepared as a 10% homogenate and then boiled, and western blotting was performed as described above. N2a cells were seeded in six-well plates and pretreated with 2 µM Rapa or 5 mM 3-MA for 2 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 hpi [27]. Additionally, N2a cells were pretreated with 10 µM ATRA for 6 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 hpi. Another group of N2a cells was infected with CVS-11 or CTN at an MOI of 1 for 1 h, followed by treatment with 0.5 µM CDDO-Me , MG132 (5 μM) respectively, or in combination for 48 hpi [14], and then cell lysates were collected. Antibodies against Nrf2, SQSTM1 and β-actin were used. Western blotting steps were performed as described above. Each experiment was conducted with three independent replicates. siSQSTM1 (Santa Cruz, #A3024) and siNC (Santa Cruz, #SC-37007) were transfected into N2a cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA). After 24 h of culture, the cells were infected with CVS-11 or CTN strains for 1 h, followed by 24 hpi of incubation. Cell lysates were collected for subsequent western blotting, which was performed as described above. Each experiment was conducted with three independent replicates. 2.7 Immunofluorescence staining experiment N2a cells were seeded into a Millicell® EZ 8-well plate and cultured for 24 h until 60-70% confluence. Then, CVS-11 at an MOI of 1 was added for a 1-h infection. A control group of uninfected cells and a mitochondrial autophagy-positive control (CCCP for 6 h) were prepared. Another set of experiments involved one group supplemented with 5 μM MG132 1 h after CVS-11 infection. After blocking, the cells were incubated with anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#517353; Santa Cruz), and anti-Parkin (#66674-1; Proteintech) primary antibodies overnight at 4℃. After overnight incubation, the cells were then probed with Alexa Fluor 488-labeled goat anti-rabbit (#4412; Cell Signaling Technology) and Alexa Fluor 647-labeled goat-derived anti-mouse secondary antibodies (#4410; Cell Signaling Technology) for 1 h. The cells were stained with the nuclear dye DAPI for 30 min at room temperature and then treated with a fluorescent mounting medium to protect against photobleaching. Finally, the cells were observed under a Leica confocal laser microscope (Leica, TCS SP8, Germany), and the confocal images were analyzed using ImageJ. Each experiment was conducted with three independent replicates. Paraffin-embedded brain tissue sections were dewaxed and then subjected to antigen retrieval in a solution prepared by dissolving 0.4 g citric acid and 3 g sodium citrate in 1000 mL distilled water for 30 min. The sections were then permeabilized with 0.3% Triton X-100 at room temperature for 30 min. After removing the permeabilization solution, goat serum blocking solution was applied for 1 h at room temperature. Then, diluted anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#517353; Santa Cruz), and anti-Parkin (#66674-1; Proteintech) antibodies were added for overnight incubation. The subsequent steps were the same as those used for the cell experiments. 2.8. In vivo assay of MG132 anti-RABV effects Animal experiments were conducted according to the animal ethics review protocol approved by the Virus Disease Prevention and Control Institute of the China CDC (permit number: 20250331045). SC16, the viral strain used for the animal experiments, was isolated from the brain of a rabid dog in Sichuan in 2016. After passaging in mouse brains and adaptation to growth in N2a cells, it reached a high titer and was preserved in our laboratory. BALB/c mice were randomly divided into five groups (n = 5 per group). Four groups of mice were injected with 50 LD50 of SC16 in both hind leg muscles [44], followed by intraperitoneal injections 1 h later with saline, 5 mg/kg MG132, 10 mg/kg MG132, or 30 mg/kg ribavirin once daily. The same volume of saline was used for mock controls. All animals were observed daily for 28 days post infection (dpi). Disease progression was evaluated as described in our previous study [45]. 2.9. Statistical analysis Data were analyzed with SPSS 17.0 and GraphPad Prism software (version 9.0). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the signal densities of western blot bands. One-way analysis of variance (ANOVA) was used, and differences were considered statistically significant at p < 0.05. 3. Results 3.1. Cytotoxicity and antiviral activity of MG132 in vitro Cytotoxicity assays in N2a cells showed that MG132 did not exhibit significant cytotoxicity at concentrations up to 5 µM (Figure 1A). We also found that MG132 demonstrated inhibitory effects on RABV at concentrations ranging from 1 µM to 25 µM, with the most effective inhibition of viral replication observed at 5 µM (Figure 1B, C). Cytotoxicity and antiviral efficacy in BSR cells were similar to those observed in N2a cells (data not shown). Combining these findings with cytotoxicity experiments, we preliminarily determined that 5 µM was the optimal working concentration of MG132 in this study. MG132 displayed effective inhibitory effects on different viral strains at different stages in vitro. To further evaluate the antiviral activity of MG132, we used 1000 μM T705 and 25 μM ribavirin as positive controls in vitro. We treated CVS-11- or CTN-infected N2a cells with 5 μM MG132 for 48 hpi, and the results showed that 5 μM MG132 exerted stronger antiviral activity against RABV and its progeny than 1000 μM T705 and produced an inhibitory effect similar to that of 25 μM ribavirin (Figure 2). 3.2 MG132 regulates the Nrf2/SQSTM1/PINK1/Parkin pathway in N2a cells during RABV infection To determine and evaluate the effects of MG132 at different concentrations on the Nrf2/SQSTM1/PINK1/Parkin pathway in RABV-infected N2a cells, we detected the levels of pathway components by western blot. RABV-infected cells treated with MG132 showed increased expression of Nrf2, SQSTM1, and Tomm20 and significantly reduced levels of PINK1, Parkin, and LC3B proteins compared with the RABV-infected control group, for both the virulent strain CVS-11 (Figure 3A, B) and the attenuated strain CTN (Figure 3C, D). Both the activity of the Nrf2/SQSTM1/PINK1/Parkin pathway and the expression of the RABV P protein were progressively decreased by MG132 in a dose-dependent manner. 3.3 MG132 inhibits RABV replication via accumulation of SQSTM1 and regulation of the Nrf2/SQSTM1/PINK1/Parkin pathway in N2a cells We treated CVS-11- or CTN-infected N2a cells with 5 µM MG132 to evaluate the Nrf2/SQSTM1/PINK1/Parkin pathway and viral replication. MG132 treatment downregulated PINK1, Parkin, LC3B, and RABV P protein while upregulating Nrf2, SQSTM1, and Tomm20, thereby reducing CVS-11 or CTN replication (Figure 4). We further tested the role of Nrf2 by treating cells with Nrf2 activator CDDO-Me or inhibitor ATRA after MG132 treatment. CDDO-Me enhanced the inhibitory effect of MG132, while ATRA blocked it, regardless of the viral strain (Figure 5). The Nrf2/SQSTM1 pathway is essential for antioxidant defense and cell survival [8] [46]. MG132 has been reported to promote SQSTM1 expression, and in our system we confirmed its role in regulating the Nrf2/SQSTM1/PINK1/Parkin pathway. Autophagy activator rapamycin and inhibitor 3-MA were used with MG132 treatment. Combined treatment with rapamycin and MG132 increased the RABV titer and the expression of RABV P protein, whereas combined treatment with 3-MA and MG132 decreased them, indicating that Nrf2 and SQSTM1 significantly influence MG132-mediated regulation of this pathway and RABV progeny (Figure 6). 3.4 MG132 enhances the interaction between SQSTM1 and PINK1/Parkin in RABV-infected N2a cells Previous studies have shown that SQSTM1 plays a crucial role in mitochondrial damage and autophagy in neuronal cells through the PINK1/Parkin pathway [30]. We investigated whether suppressing SQSTM1 could enhance the PINK1/Parkin pathway in RABV-infected N2a cells. Knocking down SQSTM1 before infection with CVS-11 or CTN significantly reduced the expression of SQSTM1 and Tomm20, while increasing the levels of PINK1, Parkin, LC3B, and RABV P protein (Figure 7A, C). Furthermore, siSQSTM1 enhanced RABV replication (Figure 7E, F). To confirm the effect of MG132, we treated cells with MG132 and observed colocalization of SQSTM1 and PINK1/Parkin. The results showed that MG132 inhibited SQSTM1 degradation and enhanced colocalization, strengthening the interaction between SQSTM1 and PINK1/Parkin and ultimately affecting the Nrf2/SQSTM1/PINK1/Parkin pathway and RABV replication (Figure 8). 3.5 Antiviral effect of MG132 in vivo BALB/c mice were infected with the SC16 strain and injected intraperitoneally with ribavirin (30 mg/kg) or MG132 (5 or 10 mg/kg) for 28 days. Mice were monitored for weight and mortality. As shown in Figure 9, the 10 mg/kg MG132 and 30 mg/kg ribavirin groups showed reduced weight loss and gradual recovery from 11 to 16 dpi. The 5 mg/kg MG132-treated group had a 20% survival rate, the 10 mg/kg group had a 40% survival rate, and the ribavirin group had an 80% survival rate. Western blot analysis showed that MG132 treatment decreased PINK1, Parkin, LC3B, and RABV P protein expression and increased Nrf2, SQSTM1, and Tomm20 in mouse brain tissues, similar to the results observed in vitro (Figure 10). Immunofluorescence staining revealed enhanced co-localization of SQSTM1 and PINK1/Parkin in MG132-treated brain tissues (Figure 11). These findings suggest that MG132 inhibits RABV replication by enhancing the interaction between SQSTM1 and PINK1/Parkin, showing potential for rabies treatment. 4. Discussion Despite the availability of effective vaccines, rabies remains a public health challenge, and there is currently no specific antiviral drug for therapy [39]. Some novel anti-RABV drugs, such as CDDO-Me, BCX4430 and PGG, can inhibit RABV replication by enhancing Nrf2 expression or inhibiting autophagy [14, 27, 28]. Additionally, antiviral drugs like Z-limonene, β-d-N4-hydroxycytidine and λ-carrageenan P32 have demonstrated potent anti-RABV activity [47-49]. Ribavirin and T705 have also shown promising antiviral effects, but they cause mild liver and kidney toxicity [50, 51]. Therefore, current research is focused on identifying new antiviral drugs for rabies. MG132 is a widely used proteasome inhibitor that has been proven effective in antiviral research. Furthermore, MG132 exerts antiviral effects on HSV-1 and PRRSV by inhibiting proteasomal degradation and thereby suppressing viral replication [6, 52]. In this study, our data showed that the SI 50 values of MG132 in CVS-11 infection were 13.094, 7.693, and 6.030 at 24, 48, and 72 hpi, respectively. In CTN infection, the SI 50 values were 7.837, 5.224, and 3.778 at 24, 48, and 72 hpi, with no cytotoxicity observed in N2a cells at concentrations below 5 μM. These results are consistent with previous studies showing that MG132 inhibits the replication of HSV-1 [6] or HCV [53]. We also investigated the antiviral activity of MG132 against RABV in N2a cells. The results showed that MG132 exhibited antiviral activity similar to ribavirin and superior to T705 in N2a cells. These results suggest that MG132 has anti-RABV potential. MG132 leads to the accumulation of oxidized proteins and activates the Nrf2 pathway. Nrf2 is a critical antioxidant transcription factor that regulates the expression of genes associated with preventing oxidative stress to help cells cope with oxidative damage. MG132 can enhance cellular resistance to viral infection and oxidative damage by activating Nrf2 [54]. Previously, we found that enhancing Nrf2 could effectively reduce RABV replication [14]. In this study, we demonstrated that MG132 treatment increased Nrf2 expression and reduced RABV replication by regulating the Nrf2/SQSTM1/PINK1/Parkin pathway. Nrf2 is essential for antiviral defense by controlling cellular antioxidant mechanisms and facilitating the elimination of viral particles [55]. Furthermore, we utilized Nrf2 activator CDDO-Me and Nrf2 inhibitor ATRA to investigate the regulatory effects of MG132 on Nrf2 and SQSTM1 during RABV infection. Indeed, our data showed that the inhibitory effect of MG132 on RABV replication was enhanced when Nrf2 expression was induced, whereas this inhibition was abolished by the Nrf2 inhibitor for both the virulent strain CVS-11 and the attenuated strain CTN. We propose that Nrf2 plays a crucial role in the MG132-mediated Nrf2/SQSTM1/PINK1/Parkin pathway and in the regulation of RABV replication in vitro. Previous studies indicate that activating the Nrf2 pathway can enhance the protective effects of antioxidant defenses on neurons by upregulating autophagy-related genes [56]. Moreover, RABV infection induces different degrees of increase in autophagy and reduces the level of SQSTM1 in SK cells [26], with ROS accumulation leading to oxidative damage and mitochondrial dysfunction [29]. Nrf2-mediated induction of SQSTM1 activates the noncanonical Keap1-Nrf2 pathway under lipotoxic conditions [57]. Our previous research also suggested that the accumulation of Nrf2 reduced the degradation of SQSTM1 during RABV infection and effectively inhibited RABV replication [14]. MG132 increased both SQSTM1 mRNA and protein expression in mouse hypothalamic organotypic cultures in a dose dependent manner [12]. MG132 has been described as a proteasome inhibitor that induces autophagy in tumor cells in response to proteotoxic and ER stress, supporting a role for MG132 as an autophagy inducer in cancer cells [58, 59]. Other studies have shown that exposure to the proteasome inhibitor MG132 inhibits both basal and rapamycin-induced autophagy when polyubiquitinated proteins markedly accumulate in Bombyx cells, indicating that the attenuation of autophagy in these cells results from a distinct suppression of autophagosome formation after MG132 treatment [60]. These observations are consistent with our findings in MG132-treated, RABV-infected neuronal cells. In this study, we showed that autophagy was enhanced when Nrf2 and SQSTM1 were knocked down, and the RABV titer increased following combined MG132 and rapamycin treatment during RABV infection. This increase was reversed by MG132 and 3-MA treatment in RABV-infected cells. These data also imply that SQSTM1 acts as a regulator of the MG132-mediated Nrf2/SQSTM1 autophagy pathway and of RABV replication in vitro. Notably, we observed that the accumulation of SQSTM1 negatively regulated the expression of the mitophagy-related factors PINK1 and Parkin, leading to increased RABV replication. Besides, MG132 effectively inhibited the degradation of SQSTM1 and enhanced the colocalization between SQSTM1 and PINK1/Parkin. These results are in line with previous research. Parkin-mediated degradation of outer mitochondrial membrane proteins and mitofusins is prevented by the proteasome inhibitor MG132 [61-63]. Meanwhile, SQSTM1 and Parkin possibly have overlapping functions in mitochondrial ubiquitination [64], and the ubiquitination of Parkin-dependent mitophagy requires the ubiquitin-autophagy adaptor protein SQSTM1[30]. Here, we confirmed that MG132 increased the level of SQSTM1 and strengthened the interaction of SQSTM1 with PINK1 and Parkin. Therefore, our results suggest that MG132 treatment of RABV-infected cells increases Nrf2 accumulation, alleviates oxidative stress, inhibits SQSTM1 degradation, modulates SQSTM1-mediated mitophagy, enhances mitochondrial function, and ultimately reduces RABV replication in vitro. Furthermore, we investigated the effect of MG132 on RABV infection in vivo. The survival rate of mice in the MG132 (10 mg/kg)-treated group increased by 40% during RABV infection, and these mice also showed significant improvements in body weight during the treatment period. These results suggest that MG132 holds therapeutic potential for rabies treatmentin vivo. Similarly, we found that MG132 enhanced the expression of Nrf2 and SQSTM1, reduced PINK1 and Parkin levels, and significantly increased the colocalization of SQSTM1 with PINK1/Parkin in vivo. In conclusion, our data suggest that MG132 inhibits RABV replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway. MG132 demonstrates excellent antiviral effects both in vitro and in vivo, providing theoretical support for its use in inhibiting RABV replication and treating rabies. Abbreviations 3-MA 3-Methyladenine ANOVA One-way analysis of variance ATM ataxia-telangiectasia mutated ATRA retinoic acid CCK-8 Cell Counting Kit-8 CCCP Carbonyl Cyanide m-Chlorophenylhydrazone CC 50 50% cytotoxic concentration CDDO-Me bardoxolone methyl CSFV classical swine fever virus DFA direct fluorescent antibody DMSO dimethyl sulfoxide dpi days post infection EBV Epstein-Barr virus HBV hepatitis B virus HO-1 heme oxygenase 1 HSV-1 herpes simplex virus type 1 hpi hours post infection HTLV-1 Human T-cell leukemia virus type 1 IC 50 50% inhibitory concentration Keap1 Kelch-like ECH-associated protein 1 LIR LC3-interacting region MEF embryonic fibroblast MOI multiplicity of Infection NDV Newcastle disease virus Nfe2l2/Nrf2 nuclear factor erythroid-derived 2-like 2 NQO1 NAD(P)H: quinone oxidoreductase 1 NS1 nonstructural protein Parkin Parkin RBR E3 ubiquitin ligase PINK1 PTEN-induced kinase 1 RABV Rabies virus RSV respiratory syncytial virus SI 50 50% selective index Declarations Ethics approval and consent to participate: All experiments with mice were performed at the Animal Experiment Center of the Chinese Center for Disease Control and Prevention in accordance with institutional guidelines for the care and use of laboratory animals, upon approval by the Animal Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC (approval number: bdbs20250331045) (Title: Inhibitory effects of small-molecule inhibitors on rabies virus) (Date of approval: 31/03/2025). Consent for publication: All the authors give their consent for publication in Virology Journal. Availability of data and materials: All data associated with this study are included in the paper. Competing interests: The authors declare that they have no conflicts of interest. Funding: This study was supported by the National Natural Science Foundation of China CDC (IVDC-202402) and the Youth Science Foundation of the Chinese Center for Disease Control and Prevention. Authorship contribution statement ： Ying Lin Chi: Writing-original draft, Formal analysis, Data curation, Conceptualization. Nuo Yang: Formal analysis, Data curation. Yuan Xie: Formal analysis, Data curation. Xiaoyan Tao: Resources, Supervision. Pengcheng Yu: Resources, Supervision. Qian Liu: Resources, Supervision. Minghui Zhang: Resources, Supervision. Shu Qing Liu: Writing-review & editing, Supervision, Resources, Funding acquisition, Data curation, Conceptualization. Wu Yang Zhu: Writing-review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Acknowledgments: We sincerely thank colleagues Qi Shi, Liping Gao, Weibang Huo, Rongbao Gao and Fei Ye for their assistance with laser confocal microscopy, and thank Xiaoyan Tao, Pengcheng Yu, Qian Liu, and Minghui Zhang for their help in the experiments. We also thank Shuqing Liu and Wuyang Zhu for their guidance in manuscript revision and conceptualization, as well as Xuan Xie and Nuo Yang for their support in data analysis. We thank Junfeng Hao from the Institute of Biophysics, Chinese Academy of Sciences, for assistance with histopathology and immunohistochemistry assays, and we gratefully acknowledge Jia Chen and Yuezhang Wu for their help with immunohistochemistry. References Jackson AC. Rabies: a medical perspective. Rev Sci Tech. 2018;37(2):569–80. Swinkels HM, Koury R, Warrington SJ. Rabies. [Updated 2025 Mar 28]. StatPearls [Internet]. 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Cell viability was assessed using the CCK-8 assay. (B, C) N2a cells were infected with CVS-11 or CTN (MOI = 1) and then treated with MG132 at the indicated concentrations (1, 2, 5, 10, 20 and 25 μM) or with DMSO as a control. Culture supernatants were collected at 24, 48 and 72 hpi, and viral titers were determined by DFA assay. The EC\u003csub\u003e50\u003c/sub\u003e, CC\u003csub\u003e50\u003c/sub\u003e and SI\u003csub\u003e50\u003c/sub\u003e values were calculated for each condition. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/efb5971c32bdb4d672da9bfe.png"},{"id":98626062,"identity":"43150c97-d15f-4df6-9a58-6215998e2658","added_by":"auto","created_at":"2025-12-19 17:09:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1898461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the inhibitory effects of MG132, ribavirin and T705.\u003c/strong\u003e\u003cbr\u003e\n(A, C) N2a cells were infected with CVS-11 or CTN (MOI = 1) and then treated with MG132 (5 μM), ribavirin (25 μM) or T705 (1000 μM). RABV-infected, DMSO-treated cells served as controls. Viral antigen was detected by DFA assay; apple-green fluorescence indicates FITC-labeled RABV. Scale bar: 100 μm. (B, D) Progeny viral titers in the culture supernatants were measured by focus-forming assay. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/7d72ceb67e20d24a2859f148.png"},{"id":98542046,"identity":"7d61edb9-67af-4096-876f-6d2740a94fbc","added_by":"auto","created_at":"2025-12-18 17:46:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1502345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of the Nrf2/SQSTM1/PINK1/Parkin pathway by different concentrations of MG132 during RABV infection.\u003c/strong\u003e\u003cbr\u003e\n(A, C) N2a cells were infected with CVS-11 (MOI = 1) or CTN (MOI = 1) and then treated with MG132 at the indicated concentrations (1, 2, 5, 10, 20 and 25 μM) or with DMSO as a control. Protein levels of Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3A/B and RABV P were determined by western blot. (B, D) Densitometric quantification of Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3B and RABV P normalized to β-actin. Data represent the mean ± SD from three independent experiments. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/fb36b846cb3dc2d13c70bf63.png"},{"id":98542068,"identity":"6778cfa4-2ff3-4d4b-a62f-282dfaa69be0","added_by":"auto","created_at":"2025-12-18 17:47:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":997976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMG132 inhibits CVS-11 and CTN replication via the Nrf2/SQSTM1/PINK1/Parkin pathway in N2a cells.\u003c/strong\u003e\u003cbr\u003e\n(A, C) N2a cells were infected with CVS-11 or CTN (MOI = 1) and then treated with MG132 (5 μM) or DMSO as a control. Protein levels of Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3A/B and RABV P were determined by western blot. (B, D) Densitometric quantification of Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3B and RABV P normalized to β-actin. (E, F) Viral titers of CVS-11 and CTN in the culture supernatants were measured. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/1ce124ea962a68f8f6c766fd.png"},{"id":98542044,"identity":"e74fee8b-a5c1-42db-83ef-45bb9adbf1b0","added_by":"auto","created_at":"2025-12-18 17:46:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":697013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMG132 inhibits CVS-11 and CTN replication by enhancing the Nrf2 pathway.\u003c/strong\u003e\u003cbr\u003e\n(A, E) N2a cells were infected with CVS-11 (MOI = 1) or CTN (MOI = 1) and then treated with CDDO-Me (0.5 μM), MG132 (5 μM) respectively, or in combination for 48 h. Nrf2, SQSTM1 and RABV P protein levels were examined by western blot. (C, G)N2a cells were pretreated with ATRA (10 μM) for 6 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 h. Nrf2, SQSTM1 and RABV P protein levels were determined by western blot.\u003cbr\u003e\n(B, D, F, H) Densitometric quantification of Nrf2, SQSTM1 and RABV P normalized to β-actin.\u003cbr\u003e\n(I, J) Viral titers in the culture supernatants were determined. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/d8fbffd02e2ad2e72d4b7cca.png"},{"id":98625971,"identity":"51cf52be-0ae3-4288-b961-fc8750b34116","added_by":"auto","created_at":"2025-12-19 17:09:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":744807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMG132 inhibits CVS-11 and CTN replication by modulating the autophagy pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, E) N2a cells were pretreated with rapamycin (2 μM) for 2 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 h. Nrf2, SQSTM1 and RABV P protein levels were determined by western blot. (C, G) N2a cells were pretreated with 3-MA (2 mM) for 2 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 h. Nrf2, SQSTM1 and RABV P protein levels were determined by western blot.\u003cbr\u003e\n(B, D, F, H) Densitometric quantification of Nrf2, SQSTM1 and RABV P normalized to β-actin.\u003cbr\u003e\n(I, J) Viral titers in the culture supernatants were measured. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/c2645e055183d865f254c771.png"},{"id":98625770,"identity":"d28323de-3967-4270-8525-023f77cce52c","added_by":"auto","created_at":"2025-12-19 17:09:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1321912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of SQSTM1 on the mitochondrial autophagy pathway and RABV titer.\u003c/strong\u003e\u003cbr\u003e\n(A, C) N2a cells were transfected with siSQSTM1 or siNC, and then infected with CVS-11 or CTN (MOI = 1) respectively. Protein levels of SQSTM1, PINK1, Parkin, Tomm20, LC3A/B and RABV P were analyzed by western blot. (B, D) Densitometric quantification of SQSTM1, PINK1, Parkin, Tomm20, LC3B and RABV P normalized to β-actin. (E, F) Viral titers in the culture supernatants were determined. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/8cc24deb1c5b9cb9010f7c57.png"},{"id":98625812,"identity":"400edbec-d820-4f40-9c52-6b83e664c1d3","added_by":"auto","created_at":"2025-12-19 17:09:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4862953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MG132 on the colocalization of SQSTM1 with PINK1/Parkin in N2a cells.\u003c/strong\u003e\u003cbr\u003e\n(A) N2a cells were infected with RABV and then treated with MG132 (5 μM). Cells were fixed, stained with mouse anti-PINK1 (red) and rabbit anti-SQSTM1 (green), and examined by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) N2a cells were infected with RABV and then treated with MG132 (5 μM). Cells were stained with mouse anti-Parkin (red) and rabbit anti-SQSTM1 (green), and nuclei were counterstained with DAPI (blue). DMSO-treated cells served as a negative control, and CCCP-treated cells served as a positive control for mitophagy. Scale bar: 50 μm. (C, D) Quantification of the overlap coefficient for MOCK, RABV-infected, CCCP-treated and MG132 + RABV treatment groups. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/ef449d6ea62085b267e1735a.png"},{"id":98542065,"identity":"58f3c191-e6d0-42ba-bec5-6cf66faab866","added_by":"auto","created_at":"2025-12-18 17:47:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":599941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMG132 alleviates weight loss and reduces mortality in RABV-infected mice.\u003c/strong\u003e\u003cbr\u003e\nBALB/c mice were randomly divided into five groups (n = 5 per group). Four groups were injected intramuscularly in both hind legs with 50 LD\u003csub\u003e50\u003c/sub\u003e of SC16 and then treated intraperitoneally 1 h later with saline, MG132 (5 mg/kg), MG132 (10 mg/kg) or ribavirin (30 mg/kg) once daily. RABV single -infected group received saline only. (A) Changes in body weight over 28 dpi. (B) Survival curves over 28 dpi.\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/445ec34d3a940e74ec068a24.png"},{"id":98542062,"identity":"ddf6530f-5d9e-401b-84e4-b8d7a0af7710","added_by":"auto","created_at":"2025-12-18 17:47:00","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":447578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMG132 inhibits RABV in vivo via the Nrf2/SQSTM1/PINK1/Parkin pathway.\u003c/strong\u003e\u003cbr\u003e\n(A) BALB/c mice were infected with SC16 and treated daily with MG132 (10 mg/kg) or saline as a control. Brain tissues were collected, and Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3A/B and RABV P protein levels were determined by western blot. (B) Densitometric quantification of Nrf2, SQSTM1, PINK1, Parkin, Tomm20, LC3B and RABV P normalized to β-actin. Data represent the mean ± SD (n = 5 per group). Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/04b73d6b077b6232f91ab7d1.png"},{"id":98542063,"identity":"7a17cdfe-cd54-4b7e-9c55-717d94da94b7","added_by":"auto","created_at":"2025-12-18 17:47:01","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":4368252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MG132 on the colocalization of SQSTM1 with PINK1/Parkin in vivo.\u003c/strong\u003e\u003cbr\u003e\n(A) BALB/c mice were infected with SC16 and treated daily with MG132 (10 mg/kg) or saline as a control. Brain sections were fixed and stained with mouse anti-PINK1 (red) and rabbit anti-SQSTM1 (green), and nuclei were stained with DAPI (blue). Images were acquired by confocal microscopy. Scale bar: 50 μm. (B) SC16-infected mice were treated with or without MG132 (10 mg/kg), and brain sections were stained with mouse anti-Parkin (red) and rabbit anti-SQSTM1 (green); nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (C, D) Quantification of the overlap coefficient for MOCK, RABV-infected and MG132 + RABV treatment groups. Data represent the mean ± SD from three independent experiments performed in triplicate. Statistical analysis was carried out using one-way ANOVA with Dunnett’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/18190c06b1e869ab20e08e51.png"},{"id":108437952,"identity":"89c80adf-09d7-4516-b2d1-f22932b4b178","added_by":"auto","created_at":"2026-05-04 16:04:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17670682,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/e4fa8087-e0b3-4025-a511-71c6cd177d6d.pdf"},{"id":98542104,"identity":"c7fb85aa-177c-415f-bfb2-c3f94c1241bb","added_by":"auto","created_at":"2025-12-18 17:47:08","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":262579078,"visible":true,"origin":"","legend":"","description":"","filename":"fluorescenceimagesandblots.zip","url":"https://assets-eu.researchsquare.com/files/rs-8283741/v1/5100c98337ad1d3bcb0d8604.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"MG132-mediated inhibition of rabies virus replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRabies is a fatal zoonotic disease caused by the rabies virus (RABV), responsible for approximately 60,000 deaths globally each year, imposing a substantial disease burden worldwide [1, 2]. Post-exposure immunization remains the only effective treatment for rabies [3], and there is still no effective antiviral drug against RABV. Consequently, the search for effective antiviral therapies remains a central focus of rabies research.\u003c/p\u003e\n\u003cp\u003eMG132 is a reversible competitive proteasome inhibitor that is widely utilized in antiviral therapy research. It primarily inhibits the chymotrypsin-like activity of the proteasome, preventing the degradation of misfolded or damaged proteins. Current treatment strategies for virus-induced neurodegenerative diseases focus on maintaining proteasomal balance and stability in the body [4]. Recent studies suggest that MG132 inhibits viral replication by blocking proteasomal degradation of viruses such as classical swine fever virus (CSFV) [5], herpes simplex virus type 1 (HSV-1) [6], and hepatitis B virus (HBV) [7]. However, the antiviral effect of MG132 on RABV remains unclear.\u003c/p\u003e\n\u003cp\u003eMG132 also inhibits the degradation and promotes the accumulation and nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nfe2l2; Nrf2), which suppresses inflammation and oxidative damage [8]. In vitro studies have confirmed that RABV infection induces the accumulation of reactive oxygen species (ROS), leading to a strong oxidative stress response [9]. Nrf2 is a critical transcription factor that regulates several genes related to the oxidative stress response, such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) [10, 11]. Previous studies have indicated that MG132 promotes both the mRNA and protein levels of SQSTM1 in hypothalamic organotypic cultures and induces high SQSTM1 expression in embryonic fibroblast (MEF) cells [12, 13].\u0026nbsp;We previously found that enhancing Nrf2 expression could inhibit RABV replication and was accompanied by an elevation of SQSTM1 expression\u0026nbsp;[14]. Studies have shown that Nrf2 cooperates with SQSTM1 to regulate mitochondrial autophagy and maintain the expression levels of H2AX and ataxia-telangiectasia mutated (ATM), thereby inhibiting Epstein-Barr virus (EBV)-driven B-cell oncogenesis\u0026nbsp;[15]. SQSTM1 directly interacts with Kelch-like ECH-associated protein 1 (Keap1) and disrupts the association between Keap1 and Nrf2, resulting in the stabilization and nuclear accumulation of Nrf2\u0026nbsp;[16]. Meanwhile, SQSTM1 is known as a target of Nrf2, and Nrf2 activation induces SQSTM1 production[17-19]. Moreover, the synergy between SQSTM1 and Nrf2 also regulates NQO1, enhancing antioxidant production and preventing muscle atrophy caused by oxidative stress\u0026nbsp;[20]. These findings underscore the crucial role of the Nrf2–SQSTM1 pathway in modulating disease progression.\u003c/p\u003e\n\u003cp\u003eAs a scaffold protein, SQSTM1 is responsible for recognizing specific organelles and protein aggregates and regulating autophagy, a process that plays important roles in various neurodegenerative diseases [21-23]. The structure of SQSTM1 contains several functional domains, including the LC3-interacting region (LIR), which collaborates with related proteins to maintain cellular activity and homeostasis [24]. Furthermore, SQSTM1 also plays a significant regulatory role in viral infections. Studies have found that GAL9, a member of the galectin-9 family, interacts with the antiviral factor viperin to guide the autophagic degradation of HBV-HBc. Viperin interacts with HBc, inducing its accumulation in cytoplasmic foci, enhancing the self-ubiquitination of RNF13, and promoting the recruitment of SQSTM1 and the formation of LC3-positive autophagosomes, thereby participating in the regulation of viral infection. Targeting SQSTM1 aggregation mediated by viperin and GAL9 may reveal viral infection mechanisms and provide reliable targets for antiviral therapy [25]. Previous studies have shown that RABV can influence autophagy by regulating changes in SQSTM1 and LC3, thereby promoting viral replication [26]. Inhibition of SQSTM1- and LC3-mediated autophagic pathways reduces RABV replication [27]. PGG upregulates SQSTM1 to reduce LC3-mediated autophagy, significantly decreasing the accumulation of RABV M protein in autophagic structures[28]. Multiple lines of evidence suggest that SQSTM1 plays a pivotal regulatory role in autophagy induced by RABV.\u003c/p\u003e\n\u003cp\u003eAmple evidence indicates that RABV infection induces ROS generation, leading to oxidative stress, mitochondrial dysfunction, and neuronal injury [29]. SQSTM1 can be recruited to damaged mitochondria to participate in their clearance, and interactions with the mitochondrial autophagy proteins PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) play a major role in this mitophagy process [30]. During the clearance of damaged mitochondria, PINK1 accumulates on the damaged mitochondrial surface and recruits Parkin, marking the damaged mitochondria for autophagic degradation [31, 32]. Human T-cell leukemia virus type 1 (HTLV-1) can elevate its replication by promoting PINK1–Parkin-dependent mitophagy and recruiting the autophagy receptor SQSTM1 to damaged mitochondria [33]. During respiratory syncytial virus (RSV) infection, its nonstructural protein (NS1) promotes the degradation of SQSTM1 to initiate PINK1/Parkin-independent mitophagy, thereby accelerating disease progression [34]. In addition, Newcastle disease virus (NDV) can regulate host cell metabolism by activating mitophagy through the SQSTM1/PINK1/Parkin pathway [35]. Therefore, SQSTM1 plays an important role in regulating PINK1/Parkin-mediated mitophagy and antiviral infection. However, the role of the autophagic mechanism mediated by the Nrf2/SQSTM1/PINK1/Parkin axis in RABV replication remains unclear.\u003c/p\u003e\n\u003cp\u003eThis study investigated the effects of MG132 on RABV replication both in vitro and in vivo and explored the mechanism by which MG132 influences RABV replication through autophagy mediated by the Nrf2/SQSTM1/PINK1/Parkin pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1. Cell culture and reagents\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mouse neuroblastoma cell line N2a (CCL-131) and Golden Syrian hamster kidney cells BSR were purchased from ATCC and maintained in ATCC-formulated Dulbecco’s modified Eagle medium (DMEM, ATCC) with 10% fetal bovine serum (FBS, Gibco) at 37℃ in a humidified 5% CO₂ atmosphere. MG132, ribavirin, T705, bardoxolone methyl (CDDO-Me), retinoic acid (ATRA), rapamycin (Rapa), 3-methyladenine (3-MA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Med-Chem-Express. Stock solutions were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Each stock solution was freshly diluted with culture medium to a final concentration of 0.05% DMSO (v/v). Control cells were treated with 0.05% DMSO.\u003c/p\u003e\n\u003cp\u003e2.2. Cell viability assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Cell Counting Kit-8 assay (CCK-8; Dojindo, Mashikimachi, Japan) was used to analyze cell viability in the absence or presence of MG132 in culture medium according to the manufacturer’s instructions. Briefly, N2a or BSR cells were seeded and grown to approximately 70-80% confluence, and the culture medium was replaced with freshly prepared MG132 solutions that had been diluted to the appropriate concentrations (1 μM, 2 μM, 5 μM, 10 μM, 20 μM or 25 μM) in culture medium. N2a or BSR cells treated with DMSO served as controls in the experiment. The commercially available CCK-8 assay was used to evaluate the cytotoxic effect of MG132. The absorbance of the supernatants at 450 nm was measured with a FLUOstar Omega instrument (BMG LABTECH, USA). All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e2.3. Virus strains\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo RABV strains were used in vitro, CVS-11 and CTN. These strains were chosen for this study because their isolation backgrounds, genetic characteristics, and genomic information were known and complete. The RABV strains were maintained in our laboratory. CVS-11 was a classic fixed RABV strain adapted from the CVS strain through BHK-21 cells, with strong neurotoxicity. It was used as the standard challenge strain in the rapid fluorescent focus inhibition test and was preserved in our laboratory [36, 37].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4. Viral titration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN2a cells or BSR cells were plated in 96-well culture plates. The cells were incubated with different strains at a multiplicity of infection (MOI) of 1 suspended in test medium for 1 h at 37℃, washed, and incubated with culture medium. At 48 h post infection (hpi), the viral titer in N2a or BSR cells was determined using a focus assay as described previously [38]\u0026nbsp;. Viral foci were detected using a FITC-conjugated anti-RABV N antibody (Fujirebio Diagnostics, Inc, Malvern, PA, USA). The foci were counted to determine the viral titer (FFU/mL).\u003c/p\u003e\n\u003cp\u003e2.5. Antiviral assays\u003c/p\u003e\n\u003cp\u003eN2a or BSR cells were used for antiviral assays, as they are standard models for RABV [39]. Cells were cultured to 70-80% confluence, then infected with RABV (CVS-11 or CTN) at an MOI of 1. MG132 was added at varying concentrations (1 μM, 2 μM, 5 μM, 10 μM, 20 μM or 25 μM) in triplicate. DMSO-treated virus-infected cells served as controls. At 48 h post infection, virus concentrations in the culture supernatants were measured using a direct fluorescent antibody (DFA) assay. Cells were fixed and stained with FITC-conjugated anti-RABV N antibody [40],\u0026nbsp;and fluorescence signals were detected with an Olympus IX70 microscope (Olympus, Tokyo, Japan). Ribavirin and T705 were used as reference drugs. The antiviral effects of MG132, ribavirin, and T705 were assessed in N2a cells at 48 hpi, with virus titers determined. Stored supernatants were titrated as described previously\u0026nbsp;[41]. The 50% cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e) and 50% effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) were calculated via dose–response regression analysis. The 50% selective index (SI\u003csub\u003e50\u003c/sub\u003e) was calculated by dividing the CC\u003csub\u003e50\u003c/sub\u003e for each compound and virus by the corresponding EC\u003csub\u003e50\u003c/sub\u003e as described previously\u0026nbsp;[42]. All experiments were repeated in triplicate.\u003c/p\u003e\n\u003cp\u003e2.6. Western blot\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN2a cells were seeded in six-well plates, infected with CVS-11 or CTN for 1 h, and then cultured with MG132 for 48 hpi. After adding proteinase and phosphatase inhibitors (Roche; Indianapolis, IN, USA), cells were lysed using the M-PER mammalian protein extraction kit (Pierce; Rockford, IL, USA). Proteins were separated by SDS‒PAGE, transferred to PVDF membranes, blocked with 5% nonfat milk in TBST, and incubated with primary antibodies (anti-Nrf2 (#12721; Cell Signaling Technology), anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#6946; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-Tomm20 (#AB186735; Abcam), anti-LC3 (#12741; Cell Signaling Technology)), a mouse monoclonal antibody (mAb) against the RABV P protein (kindly provided by Professor Shoufeng Zhang’s laboratory), and anti-β-actin (#4967; Cell Signaling Technology) as a loading control. Membranes were then incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using Super Signal West Dura and analyzed with Image Lab 3.0 (Bio-Rad).\u003c/p\u003e\n\u003cp\u003eMouse brain tissues were lysed with the T-PER Tissue Protein Extraction kit (Pierce; Rockford, IL, USA) [43]. The brain tissue was ground and prepared as a 10% homogenate and then boiled, and western blotting was performed as described above.\u003c/p\u003e\n\u003cp\u003eN2a cells were seeded in six-well plates and pretreated with 2 µM Rapa or 5 mM 3-MA for 2 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 hpi [27]. Additionally, N2a cells were pretreated with 10 µM ATRA for 6 h and/or treated with MG132 (5 μM) during CVS-11 infection (MOI = 1) or CTN (MOI = 1) for 48 hpi. Another group of N2a cells was infected with CVS-11 or CTN at an MOI of 1 for 1 h, followed by treatment with 0.5 µM CDDO-Me , MG132 (5 μM) respectively, or in combination for 48 hpi [14], and then cell lysates were collected. Antibodies against Nrf2, SQSTM1 and β-actin were used. Western blotting steps were performed as described above. Each experiment was conducted with three independent replicates.\u003c/p\u003e\n\u003cp\u003esiSQSTM1 (Santa Cruz, #A3024) and siNC (Santa Cruz, #SC-37007) were transfected into N2a cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA). After 24 h of culture, the cells were infected with CVS-11 or CTN strains for 1 h, followed by 24 hpi of incubation. Cell lysates were collected for subsequent western blotting, which was performed as described above. Each experiment was conducted with three independent replicates.\u003c/p\u003e\n\u003cp\u003e2.7 Immunofluorescence staining experiment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;N2a cells were seeded into a Millicell® EZ 8-well plate and cultured for 24 h until 60-70% confluence. Then, CVS-11 at an MOI of 1 was added for a 1-h infection. A control group of uninfected cells and a mitochondrial autophagy-positive control (CCCP for 6 h) were prepared. Another set of experiments involved one group supplemented with 5 μM MG132 1 h after CVS-11 infection. After blocking, the cells were incubated with anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#517353; Santa Cruz), and anti-Parkin (#66674-1; Proteintech) primary antibodies overnight at 4℃. After overnight incubation, the cells were then probed with Alexa Fluor 488-labeled goat anti-rabbit (#4412; Cell Signaling Technology) and Alexa Fluor 647-labeled goat-derived anti-mouse secondary antibodies (#4410; Cell Signaling Technology) for 1 h. The cells were stained with the nuclear dye DAPI for 30 min at room temperature and then treated with a fluorescent mounting medium to protect against photobleaching. Finally, the cells were observed under a Leica confocal laser microscope (Leica, TCS SP8, Germany), and the confocal images were analyzed using ImageJ. Each experiment was conducted with three independent replicates.\u003c/p\u003e\n\u003cp\u003eParaffin-embedded brain tissue sections were dewaxed and then subjected to antigen retrieval in a solution prepared by dissolving 0.4 g citric acid and 3 g sodium citrate in 1000 mL distilled water for 30 min. The sections were then permeabilized with 0.3% Triton X-100 at room temperature for 30 min. After removing the permeabilization solution, goat serum blocking solution was applied for 1 h at room temperature. Then, diluted anti-SQSTM1 (#23214; Cell Signaling Technology), anti-PINK1 (#517353; Santa Cruz), and anti-Parkin (#66674-1; Proteintech) antibodies were added for overnight incubation. The subsequent steps were the same as those used for the cell experiments.\u003c/p\u003e\n\u003cp\u003e2.8. In vivo assay of MG132 anti-RABV effects\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Animal experiments were conducted according to the animal ethics review protocol approved by the Virus Disease Prevention and Control Institute of the China CDC (permit number: 20250331045). SC16, the viral strain used for the animal experiments, was isolated from the brain of a rabid dog in Sichuan in 2016. After passaging in mouse brains and adaptation to growth in N2a cells, it reached a high titer and was preserved in our laboratory. BALB/c mice were randomly divided into five groups (n = 5 per group). Four groups of mice were injected with 50 LD50 of SC16 in both hind leg muscles\u0026nbsp;[44], followed by intraperitoneal injections 1 h later with saline, 5 mg/kg MG132, 10 mg/kg MG132, or 30 mg/kg ribavirin once daily. The same volume of saline was used for mock controls. All animals were observed daily for 28 days post infection (dpi). Disease progression was evaluated as described in our previous study [45].\u003c/p\u003e\n\u003cp\u003e2.9. Statistical analysis\u003c/p\u003e\n\u003cp\u003eData were analyzed with SPSS 17.0 and GraphPad Prism software (version 9.0). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the signal densities of western blot bands. One-way analysis of variance (ANOVA) was used, and differences were considered statistically significant at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Cytotoxicity and antiviral activity of MG132 in vitro\u003c/p\u003e\n\u003cp\u003eCytotoxicity assays in N2a cells showed that MG132 did not exhibit significant cytotoxicity at concentrations up to 5 µM (Figure 1A). We also found that MG132 demonstrated inhibitory effects on RABV at concentrations ranging from 1 µM to 25 µM, with the most effective inhibition of viral replication observed at 5 µM (Figure 1B, C). Cytotoxicity and antiviral efficacy in BSR cells were similar to those observed in N2a cells (data not shown). Combining these findings with cytotoxicity experiments, we preliminarily determined that 5 µM was the optimal working concentration of MG132 in this study. MG132 displayed effective inhibitory effects on different viral strains at different stages in vitro. To further evaluate the antiviral activity of MG132, we used 1000 μM T705 and 25 μM ribavirin as positive controls in vitro. We treated CVS-11- or CTN-infected N2a cells with 5 μM MG132 for 48 hpi, and the results showed that 5 μM MG132 exerted stronger antiviral activity against RABV and its progeny than 1000 μM T705 and produced an inhibitory effect similar to that of 25 μM ribavirin (Figure 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2 MG132 regulates the Nrf2/SQSTM1/PINK1/Parkin pathway in N2a cells during RABV infection\u003c/p\u003e\n\u003cp\u003eTo determine and evaluate the effects of MG132 at different concentrations on the Nrf2/SQSTM1/PINK1/Parkin pathway in RABV-infected N2a cells, we detected the levels of pathway components by western blot.\u0026nbsp;RABV-infected cells treated with MG132 showed increased expression of Nrf2, SQSTM1, and Tomm20 and significantly reduced levels of PINK1, Parkin, and LC3B proteins compared with the RABV-infected control group, for both the virulent strain CVS-11 (Figure 3A, B) and the attenuated strain CTN (Figure 3C, D). Both the activity of the Nrf2/SQSTM1/PINK1/Parkin pathway and the expression of the RABV P protein were progressively decreased by MG132 in a dose-dependent manner.\u003c/p\u003e\n\u003cp\u003e3.3 MG132 inhibits RABV replication via accumulation of SQSTM1 and regulation of the Nrf2/SQSTM1/PINK1/Parkin pathway in N2a cells\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; We treated CVS-11- or CTN-infected N2a cells with 5 µM MG132 to evaluate the Nrf2/SQSTM1/PINK1/Parkin pathway and viral replication. MG132 treatment downregulated PINK1, Parkin, LC3B, and RABV P protein while upregulating Nrf2, SQSTM1, and Tomm20, thereby reducing CVS-11 or CTN replication (Figure 4). We further tested the role of Nrf2 by treating cells with Nrf2 activator CDDO-Me or inhibitor ATRA after MG132 treatment. CDDO-Me enhanced the inhibitory effect of MG132, while ATRA blocked it, regardless of the viral strain (Figure 5). The Nrf2/SQSTM1 pathway is essential for antioxidant defense and cell survival [8] [46]. MG132 has been reported to promote SQSTM1 expression, and in our system we confirmed its role in regulating the Nrf2/SQSTM1/PINK1/Parkin pathway. Autophagy activator rapamycin and inhibitor 3-MA were used with MG132 treatment. Combined treatment with rapamycin and MG132 increased the RABV titer and the expression of RABV P protein, whereas combined treatment with 3-MA and MG132 decreased them, indicating that Nrf2 and SQSTM1 significantly influence MG132-mediated regulation of this pathway and RABV progeny (Figure 6).\u003c/p\u003e\n\u003cp\u003e3.4 MG132 enhances the interaction between SQSTM1 and PINK1/Parkin in RABV-infected N2a cells\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;Previous studies have shown that SQSTM1 plays a crucial role in mitochondrial damage and autophagy in neuronal cells through the PINK1/Parkin pathway [30]. We investigated whether suppressing SQSTM1 could enhance the PINK1/Parkin pathway in RABV-infected N2a cells. Knocking down SQSTM1 before infection with CVS-11 or CTN significantly reduced the expression of SQSTM1 and Tomm20, while increasing the levels of PINK1, Parkin, LC3B, and RABV P protein (Figure 7A, C). Furthermore, siSQSTM1 enhanced RABV replication (Figure 7E, F). To confirm the effect of MG132, we treated cells with MG132 and observed colocalization of SQSTM1 and PINK1/Parkin. The results showed that MG132 inhibited SQSTM1 degradation and enhanced colocalization, strengthening the interaction between SQSTM1 and PINK1/Parkin and ultimately affecting the Nrf2/SQSTM1/PINK1/Parkin pathway and RABV replication (Figure 8).\u003c/p\u003e\n\u003cp\u003e3.5 Antiviral effect of MG132 in vivo\u003c/p\u003e\n\u003cp\u003eBALB/c mice were infected with the SC16 strain and injected intraperitoneally with ribavirin (30 mg/kg) or MG132 (5 or 10 mg/kg) for 28 days. Mice were monitored for weight and mortality. As shown in Figure 9, the 10 mg/kg MG132 and 30 mg/kg ribavirin groups showed reduced weight loss and gradual recovery from 11 to 16 dpi. The 5 mg/kg MG132-treated group had a 20% survival rate, the 10 mg/kg group had a 40% survival rate, and the ribavirin group had an 80% survival rate. Western blot analysis showed that MG132 treatment decreased PINK1, Parkin, LC3B, and RABV P protein expression and increased Nrf2, SQSTM1, and Tomm20 in mouse brain tissues, similar to the results observed in vitro (Figure 10). Immunofluorescence staining revealed enhanced co-localization of SQSTM1 and PINK1/Parkin in MG132-treated brain tissues (Figure 11). These findings suggest that MG132 inhibits RABV replication by enhancing the interaction between SQSTM1 and PINK1/Parkin, showing potential for rabies treatment.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDespite the availability of effective vaccines, rabies remains a public health challenge, and there is currently no specific antiviral drug for therapy [39]. Some novel anti-RABV drugs, such as CDDO-Me, BCX4430 and PGG, can inhibit RABV replication by enhancing Nrf2 expression or inhibiting autophagy [14, 27, 28]. Additionally, antiviral drugs like Z-limonene, β-d-N4-hydroxycytidine and λ-carrageenan P32 have demonstrated potent anti-RABV activity [47-49]. Ribavirin and T705 have also shown promising antiviral effects, but they cause mild liver and kidney toxicity [50, 51]. Therefore, current research is focused on identifying new antiviral drugs for rabies.\u003c/p\u003e\n\u003cp\u003eMG132 is a widely used proteasome inhibitor that has been proven effective in antiviral research. Furthermore, MG132 exerts antiviral effects on HSV-1 and PRRSV by inhibiting proteasomal degradation and thereby suppressing viral replication [6, 52]. In this study, our data showed that the SI\u003csub\u003e50\u003c/sub\u003e values of MG132 in CVS-11 infection were 13.094, 7.693, and 6.030 at 24, 48, and 72 hpi, respectively. In CTN infection, the SI\u003csub\u003e50\u003c/sub\u003e values were 7.837, 5.224, and 3.778 at 24, 48, and 72 hpi, with no cytotoxicity observed in N2a cells at concentrations below 5 μM. These results are consistent with previous studies showing that MG132 inhibits the replication of HSV-1 [6] or HCV [53]. We also investigated the antiviral activity of MG132 against RABV in N2a cells. The results showed that MG132 exhibited antiviral activity similar to ribavirin and superior to T705 in N2a cells. These results suggest that MG132 has anti-RABV potential.\u003c/p\u003e\n\u003cp\u003eMG132 leads to the accumulation of oxidized proteins and activates the Nrf2 pathway. Nrf2 is a critical antioxidant transcription factor that regulates the expression of genes associated with preventing oxidative stress to help cells cope with oxidative damage. MG132 can enhance cellular resistance to viral infection and oxidative damage by activating Nrf2 [54]. Previously, we found that enhancing Nrf2 could effectively reduce RABV replication [14]. In this study, we demonstrated that MG132 treatment increased Nrf2 expression and reduced RABV replication by regulating the Nrf2/SQSTM1/PINK1/Parkin pathway. Nrf2 is essential for antiviral defense by controlling cellular antioxidant mechanisms and facilitating the elimination of viral particles [55]. Furthermore, we utilized Nrf2 activator CDDO-Me and Nrf2 inhibitor ATRA to investigate the regulatory effects of MG132 on Nrf2 and SQSTM1 during RABV infection. Indeed, our data showed that the inhibitory effect of MG132 on RABV replication was enhanced when Nrf2 expression was induced, whereas this inhibition was abolished by the Nrf2 inhibitor for both the virulent strain CVS-11 and the attenuated strain CTN. We propose that Nrf2 plays a crucial role in the MG132-mediated Nrf2/SQSTM1/PINK1/Parkin pathway and in the regulation of RABV replication in vitro.\u003c/p\u003e\n\u003cp\u003ePrevious studies indicate that activating the Nrf2 pathway can enhance the protective effects of antioxidant defenses on neurons by upregulating autophagy-related genes [56]. Moreover, RABV infection induces different degrees of increase in autophagy and reduces the level of SQSTM1 in SK cells [26], with ROS accumulation leading to oxidative damage and mitochondrial dysfunction [29]. Nrf2-mediated induction of SQSTM1 activates the noncanonical Keap1-Nrf2 pathway under lipotoxic conditions [57]. Our previous research also suggested that the accumulation of Nrf2 reduced the degradation of SQSTM1 during RABV infection and effectively inhibited RABV replication [14]. MG132 increased both SQSTM1 mRNA and protein expression in mouse hypothalamic organotypic cultures in a dose dependent manner [12]. MG132 has been described as a proteasome inhibitor that induces autophagy in tumor cells in response to proteotoxic and ER stress, supporting a role for MG132 as an autophagy inducer in cancer cells [58, 59]. Other studies have shown that exposure to the proteasome inhibitor MG132 inhibits both basal and rapamycin-induced autophagy when polyubiquitinated proteins markedly accumulate in Bombyx cells, indicating that the attenuation of autophagy in these cells results from a distinct suppression of autophagosome formation after MG132 treatment [60]. These observations are consistent with our findings in MG132-treated, RABV-infected neuronal cells. In this study, we showed that autophagy was enhanced when Nrf2 and SQSTM1 were knocked down, and the RABV titer increased following combined MG132 and rapamycin treatment during RABV infection. This increase was reversed by MG132 and 3-MA treatment in RABV-infected cells. These data also imply that SQSTM1 acts as a regulator of the MG132-mediated Nrf2/SQSTM1 autophagy pathway and of RABV replication in vitro.\u003c/p\u003e\n\u003cp\u003eNotably, we observed that the accumulation of SQSTM1 negatively regulated the expression of the mitophagy-related factors PINK1 and Parkin, leading to increased RABV replication. Besides, MG132 effectively inhibited the degradation of SQSTM1 and enhanced the colocalization between SQSTM1 and PINK1/Parkin. These results are in line with previous research. Parkin-mediated degradation of outer mitochondrial membrane proteins and mitofusins is prevented by the proteasome inhibitor MG132 [61-63]. Meanwhile, SQSTM1 and Parkin possibly have overlapping functions in mitochondrial ubiquitination [64], and the ubiquitination of Parkin-dependent mitophagy requires the ubiquitin-autophagy adaptor protein SQSTM1[30]. Here, we confirmed that MG132 increased the level of SQSTM1 and strengthened the interaction of SQSTM1 with PINK1 and Parkin. Therefore, our results suggest that MG132 treatment of RABV-infected cells increases Nrf2 accumulation, alleviates oxidative stress, inhibits SQSTM1 degradation, modulates SQSTM1-mediated mitophagy, enhances mitochondrial function, and ultimately reduces RABV replication in vitro.\u003c/p\u003e\n\u003cp\u003eFurthermore, we investigated the effect of MG132 on RABV infection in vivo. The survival rate of mice in the MG132 (10 mg/kg)-treated group increased by 40% during RABV infection, and these mice also showed significant improvements in body weight during the treatment period. These results suggest that MG132 holds therapeutic potential for rabies treatmentin vivo. Similarly, we found that MG132 enhanced the expression of Nrf2 and SQSTM1, reduced PINK1 and Parkin levels, and significantly increased the colocalization of SQSTM1 with PINK1/Parkin in vivo.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our data suggest that MG132 inhibits RABV replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway. MG132 demonstrates excellent antiviral effects both in vitro and in vivo, providing theoretical support for its use in inhibiting RABV replication and treating rabies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3-MA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3-Methyladenine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOne-way analysis of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eATM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eataxia-telangiectasia mutated\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eATRA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eretinoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCCK-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Counting Kit-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCCCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCarbonyl Cyanide m-Chlorophenylhydrazone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50% cytotoxic concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCDDO-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebardoxolone methyl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCSFV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eclassical swine fever virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edirect fluorescent antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edimethyl sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edpi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edays post infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEBV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEpstein-Barr virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHBV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehepatitis B virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHO-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eheme oxygenase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHSV-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eherpes simplex virus type 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehpi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehours post infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHTLV-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHuman T-cell leukemia virus type 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50% inhibitory concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKeap1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKelch-like ECH-associated protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLIR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLC3-interacting region\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMEF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eembryonic fibroblast\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMOI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emultiplicity of Infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNDV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNewcastle disease virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNfe2l2/Nrf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enuclear factor erythroid-derived 2-like 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNQO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNAD(P)H: quinone oxidoreductase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enonstructural protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParkin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParkin RBR E3 ubiquitin ligase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePINK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePTEN-induced kinase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRABV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabies virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRSV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003erespiratory syncytial virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50% selective index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e All experiments with mice were performed at the Animal Experiment Center of the Chinese Center for Disease Control and Prevention in accordance with institutional guidelines for the care and use of laboratory animals, upon approval by the Animal Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC (approval number: bdbs20250331045) (Title: Inhibitory effects of small-molecule inhibitors on rabies virus) (Date of approval: 31/03/2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All the authors give their consent for publication in Virology Journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All data associated with this study are included in the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by the National Natural Science Foundation of China CDC (IVDC-202402) and the Youth Science Foundation of the Chinese Center for Disease Control and Prevention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eYing Lin Chi: Writing-original draft, Formal analysis, Data curation, Conceptualization. Nuo Yang: Formal analysis, Data curation. Yuan Xie: Formal analysis, Data curation.\u0026nbsp;Xiaoyan Tao: Resources, Supervision. Pengcheng Yu: Resources, Supervision. Qian Liu: Resources, Supervision. Minghui Zhang: Resources, Supervision. Shu Qing Liu: Writing-review \u0026amp; editing, Supervision, Resources, Funding acquisition, Data curation, Conceptualization. \u0026nbsp; \u0026nbsp; Wu Yang Zhu: Writing-review \u0026amp; editing, Supervision, Resources, Funding acquisition, Conceptualization. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe sincerely thank colleagues Qi Shi, Liping Gao, Weibang Huo, Rongbao Gao and Fei Ye for their assistance with laser confocal microscopy, and thank Xiaoyan Tao, Pengcheng Yu, Qian Liu, and Minghui Zhang for their help in the experiments. We also thank Shuqing Liu and Wuyang Zhu for their guidance in manuscript revision and conceptualization, as well as Xuan Xie and Nuo Yang for their support in data analysis.\u0026nbsp;We thank Junfeng Hao from the Institute of Biophysics, Chinese Academy of Sciences, for assistance with histopathology and immunohistochemistry assays, and we gratefully acknowledge Jia Chen and Yuezhang Wu for their help with immunohistochemistry.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJackson AC. Rabies: a medical perspective. Rev Sci Tech. 2018;37(2):569\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwinkels HM, Koury R, Warrington SJ. Rabies. [Updated 2025 Mar 28]. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArsuaga M, de Miguel Buckley R, Diaz-Menendez M. Rabies: Epidemiological update and pre- and post-exposure management. 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Autophagy. 2019;15(11):2012\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MG132, RABV, Nrf2, SQSTM1, PINK1, Parkin","lastPublishedDoi":"10.21203/rs.3.rs-8283741/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8283741/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRabies virus (RABV), a fatal neurotropic virus, still lacks effective antiviral drugs for treatment once symptoms appear. MG132 acts as a novel therapeutic agent against viral infection, inhibits the degradation of nuclear factor erythroid-derived 2-like 2 (Nfe2l2; Nrf2), and enhances SQSTM1 expression in mouse hypothalamic organotypic cultures. Rapamycin-induced autophagy and Parkin-mediated mitophagy could also be inhibited by MG132. Autophagy signaling was triggered by RABV replication, but the antiviral effects of MG132 against RABV remained unclear. Here, we showed that MG132 exerted potent inhibitory activity against different viral strains, with an efficacy comparable to that of ribavirin and higher than that of T705 in vitro. We further demonstrated that MG132 inhibited RABV replication by disrupting the Nrf2/SQSTM1/PINK1/Parkin pathway, with SQSTM1 acting as a key mediator. MG132 also prevented SQSTM1 degradation and enhanced colocalization between SQSTM1 and PINK1/Parkin. In vivo, MG132 (10 mg/kg) attenuated body weight loss and prolonged survival of RABV-infected mice by 40%. Overall, our findings indicated that MG132 inhibited RABV replication via the Nrf2/SQSTM1/PINK1/Parkin-related autophagy pathway and highlighted MG132 as a potential therapeutic agent for rabies.\u003c/p\u003e","manuscriptTitle":"MG132-mediated inhibition of rabies virus replication via the Nrf2/SQSTM1/PINK1/Parkin autophagy pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 17:46:54","doi":"10.21203/rs.3.rs-8283741/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-27T09:34:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T07:16:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T18:23:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156692174552379118710526410246091011147","date":"2025-12-17T17:30:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125828887931545329474203231784580183773","date":"2025-12-16T04:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-15T11:09:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T17:35:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T15:13:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2025-12-05T03:07:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c589973-c47f-4df5-84c1-c9a04e9a7a89","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:04:25+00:00","versionOfRecord":{"articleIdentity":"rs-8283741","link":"https://doi.org/10.1186/s12985-026-03164-8","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2026-04-29 15:58:03","publishedOnDateReadable":"April 29th, 2026"},"versionCreatedAt":"2025-12-18 17:46:54","video":"","vorDoi":"10.1186/s12985-026-03164-8","vorDoiUrl":"https://doi.org/10.1186/s12985-026-03164-8","workflowStages":[]},"version":"v1","identity":"rs-8283741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8283741","identity":"rs-8283741","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}