RACK1 inhibits fowl adenovirus serotype 4 replication by targeting the viral protein Hexon for ubiquitin-proteasome degradation

Abstract

Fowl adenovirus serotype 4 (FAdV-4), the most pathogenic member of the genus Aviadenovirus, within the family Adenoviridae, primarily induces hepatitis-hydropericardium syndrome (HHS), leading to high morbidity and mortality in chickens. The capsid protein Hexon has been established as a core virulence factor of FAdV-4, but the underlying interaction mechanisms between Hexon and the host remain to be fully elucidated. In this study, we identified that the host protein RACK1 endogenously interacts with the Hexon, and that overexpression of RACK1 significantly inhibited FAdV-4 replication, whereas knockdown of RACK1 markedly promoted this process. Mechanistically, the interaction between RACK1 and Hexon promotes the degradation of Hexon via the ubiquitin-proteasome pathway, thus suppressing FAdV-4 replication. Our results further enrich the understanding of the interaction between Hexon and the host and identifies a novel host factor interaction that may inform future strategies for controlling HHS.

Introduction

Hepatitis-hydropericardium syndrome (HHS), an acute, highly fatal disease of poultry, is caused by fowl adenovirus serotype 4 (FAdV-4) and has achieved global distribution. Characterized by high morbidity and mortality rates, HHS poses a substantial economic threat to the poultry industry worldwide (Ren et al., 2019). Currently, control efforts are centered on the use of inactivated vaccines and yolk antibody; however, these approaches remain insufficient to fully contain the transmission and infection of FAdV-4. Therefore, a deeper understanding of the molecular pathogenesis of FAdV-4 is imperative for developing more effective strategies to control HHS.

Hexon, the major capsid protein of adenovirus, has been identified as one of the core virulence factors of FAdV-4. Studies have shown that replacing the hexon gene of a virulent strain with that of the non-pathogenic strain ON-1 significantly alleviated infection-induced liver damage and hydropericardium while substantially improving survival rates. Further research revealed that the arginine at position 188 (R188) in the Hexon protein of virulent strains is a critical determinant of pathogenicity. Mutation of this residue to isoleucine (I188) markedly reduced the virulence of the strain (Zhang et al., 2021; Wang et al., 2022). Our previous findings demonstrated that the Hexon protein interacts with the host protein BAG3, thereby inducing autophagy to facilitate viral replication (Li et al., 2023). HSP70 can interact with Hexon, subsequently leading to its degradation via the autophagy pathway to inhibit viral replication (Cao et al., 2022). Additionally, it was reported that members of the CCT chaperonin family, such as CCT5 and CCT7, have also been found to interact with Hexon, thereby modulating the replication of FAdV-4 (Zhang et al., 2025; Gao et al., 2019). These results collectively underscore the pivotal role of the Hexon protein in both the replication of FAdV-4 and the induction of liver damage and hydropericardium in chickens, highlighting its potential as a key target for controlling FAdV-4 infections.

Receptor for Activated C Kinase 1 (RACK1, also known as GNB2L1, is a member of the tryptophan-aspartate repeat (WD-repeat) protein family. It interacts with various receptors and kinases, performing diverse functions such as kinase activation, protein scaffold, and signal transduction. Current studies have demonstrated that RACK1 regulates viral replication through diverse mechanisms. For instance, suppressing innate immunity (Zhang et al., 2021), participating in plasma membrane remodeling by inducing autophagosome formation (Lee et al., 2019), facilitating translation through the assembly of protein complexes (Brugier et al., 2022), enhancing viral release by interacting with viral proteins (Demirov et al., 2012) and etc. Taken together, RACK1 may regulate viral replication by engaging with distinct viral proteins and activating different intracellular processes. In our prior research, a preliminary screening of host proteins potentially interacting with the FAdV-4 Hexon protein was performed via mass spectrometry analysis, which identified RACK1 as a candidate interactor. Given the pivotal role of RACK1 in modulating multiple viral infections, it is hypothesized that RACK1 may regulate FAdV-4 replication through the interaction with Hexon. Therefore, the present study aims to characterize the RACK1-Hexon interaction and elucidate the underlying molecular mechanisms by which this interaction influences FAdV-4 replication.

In summary, our study demonstrates an endogenous interaction between RACK1 and Hexon, and further elucidates how this interaction affects Hexon stability and FAdV-4 replication. These findings extend the understanding of the host interaction mechanisms with the Hexon protein.

Materials and methods

Cell and virus

LMH cells were preserved in our laboratory. The cells were cultured in Dulbecco's modified Eagle medium (DMEM)/F12 (Gibco, United States) supplemented with 10% fetal bovine serum (Sunview, China) in 5% CO₂ incubator at 37°C. Before LMH cell treatment, the culture flasks need to be pre-treated with a 0.1% gelatin (Sigma-Aldrich, USA) solution for 20 min at room temperature. The FAdV-4 HuBWH strain was kindly provided by Prof. Shijun J. Zheng (CAU, Beijing, China).

Reagents and antibodies

The pRK5-flag-Hexon and pCMV-myc-RACK1 plasmids were synthesized by Tsingke Biological Technology (Beijing, China). Anti-Flag monoclonal antibody was purchased from ABclonal (China). Anti-c-Myc, anti-RACK1, anti-β-actin monoclonal antibodies and normal mouse IgG was purchased from Santa Cruz Biotechnology (USA). Anti-Hexon monoclonal antibody was kindly provided by Prof. Shijun J. Zheng (CAU, Beijing, China). TRITC/FITC-AffiniPure Goat Anti-Rabbit IgG (H + L) were purchased from FUSHENBIO (China), TRITC/FITC -conjugated goat anti mouse IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG antibodies were purchased from DingGuoShengWu (China). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Solarbio (China). Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific (USA). RIPA lysis buffer was purchased from Beyotime (China). Dimethyl sulfoxide (DMSO), Z-VAD-FMK, MG132, BafA1, NH₄Cl were purchased from MedChemExpress (MCE, China). Glutathione Sepharose 4B was purchased from Cytiva (USA). Protein A/G Plus agarose and anti-Flag Magnetic Beads were purchased from Beyotime (China).

Western blot assay

To determine the protein content, LMH cells were seeded on 12-well plates and cultured for 12 h, followed by transfection with plasmids or infection with FAdV-4. Twenty-four or forty-eight hours after treatment, cell samples were prepared using RIPA lysis buffer on ice for 30 min and then were placed in a metal bath at 100°C for 10 min. The prepared samples were separated by 12% SDS-PAGE protein gel, followed by transfer to PVDF membranes. After sealing with 10% skimmed milk for 2 h, the membranes were washed three times with TBST buffer. After overnight incubation of these membranes with the corresponding antibody, the membranes were incubated with HRP-conjugated secondary antibodies (EASYBIO, Beijing). Protein bands were detected using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) and the band density was quantified using ImageJ software (Version 1.38).

Immunofluorescence assay

pRK5-flag-hexon and pCMV-myc-RACK1 were co-transfected into LMH cells, and then the cells were fixed in 4% PFA fixative. Subsequently, the samples were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 10% skimmed milk for 1 h. Next, the samples were administered with rabbit anti-Flag and mouse anti-Myc antibodies, followed by incubation with FITC-conjugated goat anti-rabbit antibodies (green) and TRITC-conjugated goat anti-mouse antibodies (red). The nuclei were counterstained with DAPI (blue). Ultimately, the samples were observed using a confocal laser scanning microscope (Leica, Germany).

Co-immunoprecipitation (Co-IP) assay

LMH cells were seeded in 6-well plates, followed by co-transfection with the plasmids pRK5-Flag-Hexon and pCMV-myc-RACK1, or pRK5-Flag-Hexon and the empty vector. At 24 h post-transfection, the cell lysates were harvested with IP lysis buffer. Cell debris was removed by centrifugation at 12,000 rpm for 10 min at 4°C. The resulting supernatant was then incubated with Flag magnetic beads at room temperature for 1 h. After incubation, the beads were washed with TBS six times, and protein loading buffer was added to prepare samples for Western blotting.

For the detection of endogenous RACK1 interaction with Hexon protein, LMH cells were cultured in 6- well plates and then infected with FAdV-4. After infection, cell lysates were collected as described above and incubated with Protein A/G Plus agarose beads pre coupled with anti-Hexon antibody at 4 °C for 6 h. After washing, the samples were prepared for Western blot analysis.

GST pull-down assay

LMH cells were transfected with pCMV-myc-RACK1 plasmid or empty vector for 24 h, and then the cell lysates were harvested and incubated with GST-Hexon fusion protein at room temperature for 1 h. After incubation, the samples were incubated with Glutathione Sepharose 4B at 4°C for 6 h. Bead-bound complexes were pelleted by centrifugation at 3,000 rpm at 4°C for 5 min, and then the samples were washed six times and further analyzed by Western blotting using anti-Myc antibody.

RNA interference

The siRNA sequences were synthesized by GenePharma Co. (Shanghai, China), based on our previous research (Lin et al., 2015). LMH cells were cultured in 24-well plates, followed by transfection with RNAi-RACK1 or RNAi-ctrl. After transfection, the cells were infected with FAdV-4 at an MOI of 0.1 for different times (24 and 48 h), and then the samples were collected for further analysis.

Quantitative real-time-PCR

The samples were harvested using TRIzol reagent (Solarbio, China). Total RNA was extracted from the collected samples following the manufacturer’s instructions, and the cDNA was obtained using HiScript II Q RT SuperMix (Vazyme, China). The cDNA samples were employed as templates for qRT-PCR amplification, which was performed using ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, China). The gapdh gene was selected as the internal reference for data normalization, and the relative mRNA expression levels of target genes were calculated using the 2^⁻ΔΔCt method. The relative expression of the hexon gene was determined using specific primers: F-5′-ACAGGTCCTCAGCTACAAGA-3′; R-5′-TGACCCTAACGGTGTCGA-3′.

Animal trial

A total of ten 3-week-old SPF chicks were randomly divided into two groups: the FAdV-4 infection group and the control group. The chicks were housed in negative pressure isolators with ad libitum access to water and feed. The chicks in the infection group were given subcutaneous injections of 0.2 mL of a virus solution containing 10⁶ TCID₅₀ per chick at the breast. Liver tissues were collected on day 3 post-infection for subsequent experiments.

Statistical analysis

The statistical analysis was performed using GraphPad Prism 9. The results were presented as mean + SD, and the significance of the differences between groups was determined by Student's t-test and two-way analysis of variance (ANOVA) accordingly. Statistical significance is indicated as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

Results

FAdV-4 Hexon interacts with host protein RACK1

To determine the interaction between RACK1 and Hexon, pRK5-flag-hexon and pCMV-myc-RACK1 were co-transfected into LMH cells, and the interaction between Flag-Hexon and Myc-RACK1 was examined by co-immunoprecipitation (Co-IP). As shown in Fig.1A, the interaction of Flag-Hexon and Myc-RACK1 was readily detectable. In addition, prokaryotically expressed recombinant GST-Hexon was incubated with cell lysate generated from LMH cells transfected with pCMV-myc-RACK1, followed by GST pull-down, and the result showed that GST-Hexon was bound to Myc-RACK1 (Fig. 1B). To further confirm the interaction between Hexon and RACK1, confocal microscopy was performed to investigate the localization of Hexon and RACK1. As a result, a significant co-localization between Flag-Hexon and Myc-RACK1 was observed (Fig. 1C). As shown by confocal Z-stack analysis, most of the FAdV-4 Hexon were colocalized with RACK1 (Fig. 1D). To validate this interaction under physiological conditions, we performed endogenous Co-IP assays, which detected the interaction between endogenous RACK1 and FAdV-4 Hexon. As a result, endogenous RACK1 showed a robust interaction with Hexon protein in both FAdV-4-infected cells and liver tissues (Fig. 2A&C). Confocal microscopy further confirmed their co-localization in FAdV-4-infected LMH cells (Fig. 2B). These results clearly demonstrate that Hexon interacts with RACK1 in LMH cells.

Fig. 1.

The exogenous interaction between Hexon and RACK1. (A) LMH cells were co-transfected with Flag-Hexon and Myc-RACK1 or Flag-Hexon and empty vector. At 24 h post transfection, cell lysates were prepared and immunoprecipitated with anti-Flag mAb. (B) LMH cells were transfected with Myc-RACK1 or empty vector for 24 h. Cell lysates were then collected and incubated with GST-Hexon at room temperature for 1 h, followed by a pull-down assay using an anti-GST antibody. (C&D) LMH cells were transfected separately with Flag-Hexon, Myc-RACK1, or co-transfected with both Flag-Hexon and Myc-RACK1. At 24 h post transfection, cells were fixed and probed with rabbit anti-Flag and mouse anti-Myc antibodies, followed by incubation with FITC-conjugated goat anti-rabbit antibodies (green) and TRITC-conjugated goat anti-mouse antibodies (red). The nuclei were counterstained with DAPI (blue). The cell samples were observed with a confocal laser scanning microscope (C) or imaged on a three-dimensional laser confocal microscope (D).

Fig. 2.

The endogenous interaction between FAdV-4 Hexon and RACK1 in FAdV-4-infected LMH cells. (A) LMH cells were mock infected or infected with FAdV-4 at an MOI of 0.1. At 24 h post infection, cell lysates were prepared and immunoprecipitated with anti-Hexon mAb. Both the cell lysates and immunoprecipitates were examined by Western blotting. “Isotype” indicates the isotype control antibody (IgG2b) of anti- Hexon mAb. (B) LMH cells were mock infected or infected with FAdV-4 at an MOI of 0.1. At 24 h post infection, cells were fixed and probed with mouse anti-Hexon and rabbit anti-RACK1 antibodies, followed by incubation with FITC-conjugated goat anti-mouse antibodies (green) and TRITC-conjugated goat anti-rabbit antibodies (red). The nuclei were counterstained with DAPI (blue). The cell samples were observed with a confocal laser scanning microscope. Scale bar = 5 μm. (C) Liver tissues were collected from SPF chickens that infected with FAdV-4 or mock infection, followed by homogenization and preparation of tissue protein lysates. And then, the lysates were prepared and immunoprecipitated with anti-Hexon mAb. Both the lysates and immunoprecipitates were examined by Western blotting.

RACK1 inhibits FAdV-4 replication in LMH cells

As Hexon interacted with RACK1, we speculated that RACK1 has an impact on FAdV-4 replication. To verify this hypothesis, we examined the expression of Hexon in LMH cells infected with FAdV-4 after RACK1 expression plasmids transfection. As shown in Fig. 3A and B, RACK1 significantly inhibited the expression of Hexon in FAdV-4-infected LMH cells. The mRNA levels detection results were consistent with the protein levels (Fig. 3C). Similarly, overexpression of RACK1 markedly reduced the viral titers in LMH cells (Fig. 3D). These results suggest that RACK1 inhibits FAdV-4 replication in LMH cells.

Fig. 3.

Overexpression of RACK1 significantly inhibits the replication of FAdV-4. (A) LMH cells were transfected with Myc-RACK1 or empty vector (2 µg) for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (24 and 48 h). After infection, the cell lysates were harvested and examined by Western blotting. (B) The relative expression levels of Hexon in panel A. (C) LMH cells were transfected with Myc-RACK1 or empty vector (2 µg) for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (12 and 24 h). After infection, the mRNA of hexon was examined by qRT-PCR. (D) LMH cells were transfected with Myc-RACK1 or empty vector (2 µg) for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (12, 24, 48, and 72 h). After infection, the viral titers in the cell cultures were determined by TCID₅₀ assay. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

Knockdown of RACK1 promotes FAdV-4 replication in LMH cells

Since RACK1 inhibits FAdV-4 replication, we proposed that depletion of RACK1 would conversely enhance viral replication. To further corroborate the role of RACK1 in FAdV-4 replication, we knocked down RACK1 by RNAi and detected the knockdown efficiency by Western blotting. We observed a significant decrease in RACK1 levels in LMH cells treated with RNAi (Fig. 4A). Upon RNAi-mediated silencing of RACK1, Western Blot analysis revealed that protein levels of Hexon were increased (Fig. 4B and C). Furthermore, hexon mRNA levels also enhanced in RNAi-transfected cells at 12 and 24 h after FAdV-4 infection (Fig. 4D). Consistently, the viral titers in RACK1-RNAi-transfected LMH cells markedly increased at 48 and 72 h compared with those of the controls (Fig. 4E). These results indicate that the knockdown of RACK1 significantly promotes FAdV-4 replication.

Fig. 4.

knockdown of RACK1 increases the replication of FAdV-4. (A) The effect of RACK1 knockdown was determined by Western blotting. (B) LMH cells were transfected with RNAi-RACK1 or RNAi-ctrl for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (24 and 48 h). After infection, the cell lysates were harvested and examined by Western blotting. (C) The relative expression levels of Hexon in panel B. (D) LMH cells were transfected with RNAi-RACK1 or RNAi-ctrl for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (12 and 24 h). After infection, the mRNA of hexon was examined by qRT-PCR. (E) LMH cells were transfected with RNAi-RACK1 or RNAi-ctrl for 12 h, followed by infection with FAdV-4 at an MOI of 0.1 for different time (12, 24, 48, and 72 h). After infection, the viral titers in the cell cultures were determined by TCID₅₀ assay. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

RACK1 induces Hexon degradation via ubiquitin-proteasome pathway

Given that RACK1 inhibits FAdV-4 replication, we sought to investigate the underlying mechanism by which RACK1 exerts this inhibitory effect. The results showed that a significant degradation of Hexon was detected at 24 and 48 h after pCMV-myc-RACK1 transfection (Fig. 5A and C). Furthermore, pronounced degradation of Hexon was detected in LMH cells that were transfected with either 0.4 or 2 μg of pCMV-myc-RACK1 (Fig. 5, Fig. 5). To investigate how RACK1 reduces Hexon expression levels, we measured Hexon degradation by blocking protein synthesis with cycloheximide (CHX). The results showed that Hexon was more markedly degraded in RACK1-transfected cells than in control cells (Fig. 5, Fig. 5). Subsequently, we tested inhibitors of various protein degradation pathways to determine the specific pathway responsible for Hexon degradation. As shown in Fig. 5, Fig. 5, neither the caspase inhibitor Z-VAD-FMK, the autophagy inhibitor bafilomycin A1 (BafA1) nor the lysosomal inhibitor NH₄Cl prevented Hexon degradation following pCMV-myc-RACK1 transfection. However, when the proteasome inhibitor MG132 was employed, RACK1 failed to trigger Hexon degradation. These results indicate that RACK1 induces Hexon degradation through the ubiquitin-proteasome pathway.

Fig. 5.

Interaction with RACK1 targets Hexon for degradation through the ubiquitin-proteasome pathway. (A) LMH cells were co-transfected with Flag-Hexon (2 µg) and Myc-RACK1 (2 µg) for different time (24 and 48 h). After transfection, the cell lysates were harvested and examined by Western blotting. (B) LMH cells were co-transfected with Flag-Hexon and Myc-RACK1 (0.4 or 2 µg) for 24 h. After transfection, the cell lysates were harvested and examined by Western blotting. (C) The relative expression levels of Flag-Hexon in panel A. (D) The relative expression levels of Flag-Hexon in panel B. (E) LMH cells were co-transfected with Flag-Hexon and Myc-RACK1 or Flag-Hexon and empty vector. At 24 h post transfection, cells were treated with cycloheximide (CHX, 50 μg/mL) at different time points (0, 2, 4, 6, and 8 h) before being harvested. Cell lysates were prepared and examined by Western blotting using the indicated antibodies. (F) The relative expression levels of Flag-Hexon in panel E. (G) LMH cells were pretreated with DMSO, Z-VAD-FMK, NH₄Cl for 2 h, followed by co-transfection with Flag-Hexon and Myc-RACK1 for 24 h in the presence of the respective inhibitors. Cell lysates were prepared and examined with Western blotting using the indicated antibodies. For MG132 (10 μM), cells were not pretreated; instead, this inhibitor was added to the culture medium 12 h prior to sample collection. (H) The relative expression levels of Flag-Hexon in panel G. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

The expression of RACK1 is unaffected by Hexon

Since RACK1 is known to induce Hexon degradation via the ubiquitin-proteasome pathway, the potential degradative effect of Hexon on RACK1 has not been elucidated. To further investigate whether Hexon affected RACK1 expression, the expression level of RACK1 was examined in LMH cells infected with FAdV-4 at different time points and different MOIs. As shown in Fig. 6A, RACK1 expression had no significant difference between the different MOIs at 24 h after FAdV-4 infection. Consistently, RACK1 expression showed no change at different times post-infection (Fig. 6B). Overexpression of Hexon, similar to FAdV-4 infection, had no effect on the content of endogenous RACK1 (Fig. 6, Fig. 6). Collectively, these results indicate that Hexon has no effect on RACK1 expression.

Fig. 6.

Both FAdV-4 infection and flag-hexon transfection have no effect on the expression of RACK1. (A&B) FAdV-4 infection had no effect on RACK1 expression. LMH cells were mock-infected or infected with FAdV-4 at an MOI of 0.1, 1, and 10. At 24 h post infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (A). LMH cells were mock-infected or infected with FAdV-4 at an MOI of 0.1 for different times. After infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (B). (C&D) Overexpression of Hexon had no effect on RACK1 expression. LMH cells were transfected with pRK5-flag or pRK5-flag-hexon at 0.2, 1, and 5 μg. At 24 h post infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (C). Endogenous β-actin expression was examined as an internal control and the relative protein levels of RACK1 in (C) were shown in panel D. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

Discussion

Hexon, the most abundant and critical capsid component of FAdV-4, maintains viral particle stability by forming an icosahedral structure. Extensive research has established Hexon as a core virulence factor of FAdV-4 and a key viral protein responsible for liver damage and mortality in infected chickens. The pathogenic mechanisms of viral proteins mainly rely on their interactions with host proteins, making it scientifically significant to elucidate the interactions between Hexon and the host for controlling FAdV-4 infection. To date, several host factors that regulate viral replication have been reported to interact with Hexon, including CCT5, CCT7, and HSP70 (Cao et al., 2022; Zhang et al., 2025; Gao et al., 2019). Our previous mass spectrometry analysis identified BAG3 as a key host factor binding to Hexon and demonstrated that it promotes viral replication by inducing autophagy (Li et al., 2023). In this study, we further confirmed that the host factor RACK1 endogenously interacts with Hexon and suppresses viral replication by promoting Hexon degradation. These results together indicate that various host proteins interact with Hexon and exhibit contrasting roles in FAdV-4 replication. We hypothesize that this functional divergence is closely associated with distinct stages of viral infection. During the early phase of infection, when overall viral replication is limited, Hexon-interacting proteins may predominantly function to suppress viral replication, thereby maintaining cellular homeostasis. In contrast, during late stage of infection, when host antiviral responses become insufficient to counteract viral propagation, these Hexon-interacting proteins may be more likely to be exploited by the virus to promote viral replication or release. This hypothesis, however, remains to be experimentally validated. In addition, the regulatory outcome may also be determined by the expression levels of the host proteins and their respective binding affinities to Hexon, which together shape the efficiency and direction of viral replication control. Taken together, these findings advance our understanding of the mechanisms underlying Hexon-host interactions and may facilitate the development of key targets for combating FAdV-4 infection.

RACK1 modulates viral replication through the interactions with multiple viral proteins. For instance, RACK1 interacts with Chikungunya virus Nsp4 protein, inhibiting the degradation of Nsp4 via the ubiquitin-proteasome pathway and thereby promoting viral replication (Yan et al., 2024). Furthermore, RACK1 binds to the Nsp1 protein of various flaviviruses, facilitating the formation of viral factories (Shue et al., 2021). In the case of infectious bursal disease virus (IBDV), viral protein VP5 forms a complex with VDAC2 and RACK1 to suppress early apoptosis and enhance viral replication. Additionally, hepatitis C virus (HCV) Ns5A interacts with both RACK1 and ATG14L, promoting the formation of HCV replication organelles (Lee et al., 2019). Collectively, these studies demonstrate that RACK1 supports viral replication by modulating multiple intracellular biological processes. Nevertheless, although most studies support a proviral role for RACK1, it also serves as a restriction factor in viral replication. It has been reported that RACK1 interacts with STING, promotes type I interferon (IFN-I) activation, and thereby inhibits pseudorabies virus (PRV) infection (Li et al., 2025). Similarly, our results confirm the important role of RACK1 in restricting FAdV-4 replication by interacting with Hexon. These results clearly demonstrate that RACK1 plays a dual role in viral replication and exert opposite effects during different viral infections. Notably, studies have confirmed that the arginine (R) at position 188 of the Hexon protein is a critical site determining its virulence. Here, we found that RACK1 interacts with Hexon and promotes its degradation. This raises a compelling question: Does RACK1 specifically recognize the R188 site of Hexon to mediate its degradation? This is an issue worthy of further investigation, as it may provide profound insights into whether host defense mechanism has evolved to recognize such "virulence markers." In addition, we only examined the effect of RACK1 on the stability of Hexon. Further studies are required to determine if RACK1 modulates additional signaling pathways through the interaction with Hexon, including autophagy, apoptosis, viral protein translation, viral organelle biogenesis, etc.

The clearance of intracellular proteins is executed by three key mechanisms: caspase-dependent cleavage, the ubiquitin-proteasome pathway, and autophagy. In response to the presence of impaired, misfolded, or extraneous proteins, these systems are mobilized to degrade such components, which is essential for sustaining a stable internal cellular environment. Among them, the ubiquitin-proteasome pathway, the best-characterized mechanism, entails substrate ubiquitination, a process primarily mediated by E3 ubiquitin ligases, followed by degradation within the proteasome (Dikic I., 2017.). During viral infection, this system becomes a two-way battlefield. On the one hand, viral proteins interact with host factors to activate the ubiquitin-proteasome pathway, leading to the degradation of host factors and promoting viral replication (Gao et al., 2006). For example, after PEDV infection, the Nsp2 protein binds to the host protein FBXW7, targeting it for degradation via the ubiquitin-proteasome pathway, thereby suppressing innate immunity (Li et al., 2022). Similarly, the PRRSV E protein facilitates the degradation of pCH25H through the ubiquitin-proteasome pathway, enhancing viral replication (Ke et al., 2019). On the other hand, virus-infected cells can harness the ubiquitin-proteasome pathway to target and degrade key viral proteins, thereby inhibiting viral activity (Zhou et al., 2024; He et al., 2024; Katoh et al., 2015). Regarding FAdV-4, previous studies have demonstrated that CCT7 and HSP70 regulate the stability of the FAdV-4 Hexon protein through the ubiquitin-proteasome pathway (Gao et al., 2019; Cao et al., 2022). In this study, we further identified a novel function of RACK1 in degrading Hexon via the ubiquitin-proteasome pathway. Taken together, following FAdV-4 infection, Hexon protein forms a complex interaction network with host factors. Given that Hexon serves as a core virulence factor of FAdV-4, further elucidation of the specific ubiquitination sites and types holds promise for providing theoretical support for the prevention and control of FAdV-4 infection.

Notably, despite the observed significant inhibitory effect of RACK1 on FAdV-4 replication and the absence of marked downregulation in RACK1 expression after viral infection, FAdV-4 still exhibits efficient infectivity in LMH cells. To explain this observation, we speculate that FAdV-4 infection may significantly suppress the kinase activity of RACK1 or alter its subcellular localization. Such alterations could potentially enable the virus to evade RACK1-mediated degradation of the Hexon protein, thereby facilitating the successful and highly efficient infection of LMH cells.

In addition, the regulatory mechanism of RACK1 extends beyond the post-translational level. Here, we observed that RACK1 overexpression not only promotes the degradation of Hexon protein but also suppresses the mRNA expression of the hexon gene, suggesting that RACK1 may regulate hexon transcription by modulating certain unknown transcription factors and related signaling pathways. Overall, RACK1 may establish a multi-layered regulatory network involving both transcriptional and post-translational modifications to inhibit Hexon expression, providing a new insight for research into FAdV-4-host interactions.

In conclusion, our study reveals a novel mechanism by which RACK1 targets FAdV-4 Hexon for ubiquitin-proteasome degradation. This finding further expands the interaction network between FAdV-4 Hexon and the host, potentially providing a theoretical reference for future strategies to control HHS.

CRediT authorship contribution statement

Wei Li: Writing – original draft, Validation, Investigation, Formal analysis, Data curation, Conceptualization. Hongnuan Wang: Validation, Software, Formal analysis, Data curation. Zhen Yan: Investigation, Data curation. Xiaoge Wang: Investigation, Formal analysis. Yaqi Chen: Investigation, Data curation. Mengyuan Liu: Software, Investigation. Chenyang Wang: Formal analysis, Data curation. Zilong Sun: Supervision, Project administration. Ding Zhang: Supervision, Project administration. Bo Yang: Writing – review & editing, Supervision, Resources, Project administration.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.