https://orcid.org/0000-0002-5202-7796
Figures
Abstract
Rotavirus (RV) VP8* peptide-induced CLDN3 mislocalization supports the hypothesis that CLDN3 negatively regulates viral binding, while the molecular basis of this inhibitory function remains unresolved. To counteract the CLDN3 defense strategies, RV infection indeed disrupts its localization to the plasma membrane. We also found that RV infection could reduce its protein levels in both in vitro and animal models. Knockdown or knockout of CLDN3 effectively promotes RV binding and entry. Further, we found that CLDN3 EC1 loop could interact with the N-terminal domain of VP7 and structural studies reveal a conserved glutamic acid at position 74 (E74) in VP7 as critical for the CLDN3-VP7 interaction. Mechanistically, VP7 is involved in viral attachment. Binding of the CLDN3 EC1 loop to VP7 reduces viral adsorption, whereas the E74K mutation disrupts the CLDN3-VP7 interaction and consequently enhances viral attachment. More importantly, a single E74K mutation enhances viral pathogenicity in vivo, confirming this interaction’s biological significance. Our results demonstrate for the first time that the tight junction protein CLDN3 acts as a decoy receptor that specifically counters the VP7-mediated viral attachment. This highlights the antiviral mechanisms utilized by CLDN3.
Author summary
Rotavirus (RV) is a major global pathogen, and understanding the intricate battle between the virus and host defenses is critical. While it is known that RV infection disrupts tight junction proteins like claudins (CLDNs) to facilitate its entry, the specific protective mechanisms of these proteins remain unknown. Our study demonstrated that VP7 glycoprotein is involved in RV adsorption, and CLDN3 as a decoy receptor that directly counters its attachment function. VP7 E74 residue is a key site responsible for association with CLDN3 EC1 loop. The conservation and functional essentiality of the VP7 E74 residue, validated by its profound impact on viral binding and pathogenicity, underscore that VP7 is also an important virulence factor. We elucidate a novel mechanism by which CLDN3 inhibits rotavirus adsorption. This work not only reveals a new front in the host-virus arms race but also identifies a potential target for the development of broad-spectrum antiviral strategies.
Citation: Pan Y, Huang J, Guo L, Li Z, Shi H, Zhou Y, et al. (2026) CLDN3 inhibits rotavirus attachment by targeting residue 74 of VP7. PLoS Pathog 22(3): e1014045. https://doi.org/10.1371/journal.ppat.1014045
Editor: Gabriel I. Parra, US Food and Drug Administration, UNITED STATES OF AMERICA
Received: November 3, 2025; Accepted: March 1, 2026; Published: March 20, 2026
Copyright: © 2026 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All study data are included in the article and Supporting information.
Funding: Jin Tian is receiving the funding from Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-BRC-LPDC-2025-01).The funders do not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Alfarotaviridae (RVA) is an important causative agent of acute gastroenteritis in infants and children under 5 years of age, resulting in ~258 million diarrheal cases and ~128,000 deaths worldwide each year [1]. RVA infection also causes diarrhea in the young of avian and many other animals [2]. Rotavirus primarily infects mature enterocytes on the small intestinal villi, leading to cell death and villus shortening. This structural damage results in osmotic diarrhea and impairs nutrient adsorption. In addition to mature enterocytes and enteroendocrine cells of the small intestine, viral antigens have also been detected in multiple extraintestinal tissues (stomach, liver, lungs, spleen, kidneys, pancreas, thymus, and bladder) as well as in the serum [3]. More importantly, RVA also can infect macrophages, which is considered as a potential cause of viremia and extraintestinal viral dissemination [3]. Calcium imbalance induced by RVA infection is considered a key factor. In both infected and non-infected cells, RV induces the release of intercellular calcium via the nonstructural protein 4 (NSP4) and extracellular adenosine 5′-diphosphate (ADP) [4]. Disruption of host calcium homeostasis drives changes in the cytoskeleton and tight junctions, and activates fluid secretion pathways [5]. Finally, intestinal dysfunction would facilitate the development of diarrhea.
Intestinal epithelial cells form a physical and biochemical barrier that maintains segregation between host and commensal bacteria. As the first line of host defense, intestinal epithelial cells and immune cells collectively form the intestinal mucosal barrier, which functions to limit the propagation and entry of both commensal and pathogenic microorganisms [5]. Tight junctions (TJs) between each cell plays an important role in maintaining intestinal barrier integrity. TJs are composed of multiple components, but the claudins (CLDNs) family members are essential for TJ formation and function [6]. In mammals, a total of 27 CLDNs have been identified which can form homodimer or heterodimer. CLDNs function either by sealing the paracellular space or by forming pores or channels for charged ions and water. CLDN3 is characterized as a classical claudin that mediates heterotypic trans interactions with other claudins, including claudin-1, claudin-2, and claudin-5, in addition to homotypical interactions [7].
To establish an effective infection in the intestine, enteric pathogens have evolved many strategies to destroy intestinal TJ function, overcome the intestinal mucosal barrier and efficiently infect the gut mucosa [8]. In the porcine infection model, RVA infection could induce shorter small intestine villi and desquamation of the gut villi epithelium, which destroys intestinal normal structure [9]. RVA infection may destroy small intestine TJ structure, which accelerates gut pathology and diarrhea magnitude. Loss or disruption of barrier integrity by changing the expression of these proteins is considered to result in intestinal inflammatory diseases and metabolic disorders [10]. However, some virus can also exploit CLDNs to benefit their invasion and virulence. Claudin-1, highly expressed in the liver, is an important co-receptor for hepatitis C virus entry [11]. In the rat RVA infection model, Claudin-1 likely acts as a channel protein. The upregulation of Claudin-1 may enhance interepithelial permeability, which in turn contributes to the elevated intestinal permeability observed in the group of rats [12]. Thus, each CLDN plays a distinct role in viral infections.
Several reports have found that most CLDNs function as physical barriers that restrict viral access to receptors—such as in human rhinovirus and respiratory syncytial virus infections [13], but the underlying mechanisms remained unclear. Rotavirus VP8* peptide was previously shown to cause changes in the distribution of CLDN3 and occludin, as well as the tight junction associated protein ZO-1 in the Caco-2 monolayers, but did not alter the protein content [14]. The disruption of TJ distribution may open the paracellular space, thereby allowing the virus to reach receptors located on the basolateral membrane. But the precise mechanisms remained unclear.
The entry of rotavirus into cells is a complex, multi-step process involving interactions between its surface proteins (VP4 and VP7) and cell surface molecules that serve as attachment and entry factors [15]. VP4 forms trimers on the virion surface. With treatment with trypsin, VP4 protein is cleaved into VP8* and VP5* domains, both of which can exhibit apparent dimeric organization due to structural polymorphism and play a distinct role in the cell entry process [16,17]. This initial adsorption of the virus is mediated by the interactions between VP8* and glycans such as sialic acids, histo-blood group antigens and mucin [18–20]. This interaction may induce subtle conformational changes in the VP4 protein. Concurrently, the ongoing binding of VP8* to glycans further enables the virus to interact with the integrin α2 subunit domain I and HSC70 via the DGE domain on VP5* [21–23]. During this process, the conformational adjustment of VP4 and the VP5*-mediated interactions overlap temporally, collectively paving the way for subsequent entry steps. Subsequently, the VP7 protein binds to the integrin β2 or β3 subunits through its distinct domains, a step that partially overlaps in timing with the aforementioned molecular interactions, ultimately working in concert to mediate viral entry into the cell [24,25]. The current references support the association of VP7-integrin occurs at a post-attachment step, and a role for VP7 after virus binding. However, some studies also revealed that rotavirus VP7 is associated with virus attachment. Antiserum and neutralizing monoclonal antibodies targeting the VP7 glycoprotein could block bovine rotavirus-cell interaction [26]. The VP7 glycoprotein may play a dual role in viral entry, facilitating both initial binding and subsequent post-attachment steps.
So, targeting VP7-mediated viral attachment represents a potential alternative strategy for host defense. In this study, we demonstrate that CLDN3 functions as a decoy receptor and could interact with the outer capsid glycoprotein VP7 as a potential host attachment factor. This host-pathogen interaction sterically hinders virion attachment, thereby blocking the initial step of viral entry. Our study uncovers a novel mechanism by which CLDN3 restricts viral access to receptors.
Results
Rotavirus infection induces significant reduction of tight junction proteins in vitro and in vivo
Rotavirus infection mainly leads to diarrhea in the young animals and children under 5 years of age. Following oral inoculation with porcine rotavirus, one-day-old piglets developed significant intestinal pathology, including prominent submucosal edema, villous atrophy with epithelial sloughing, and degeneration of villous epithelial cells (Fig 1A). These pathological changes may disrupt the intestinal epithelial barrier function. In our previous studies, rotavirus infection mainly occurred in the jejunum segment, and we employed IPEC-J2 (IPEC) cells, porcine enterocytes isolated from the jejunum of a neonatal unsuckled piglet to explore the molecular basis in the following studies. IPEC cells were infected with rotavirus at an MOI of 1, and total cellular proteins were extracted for proteomic analysis at 24 hours post-infection (hpi). Focusing on tight junction (TJ) proteins, we found that rotavirus infection resulted in the decrease of several tight junction proteins, including CLDN3, JAM-A, ZO-3 (Fig 1B).
(A) Effect of virus infection on small intestine villi structure. One-day-old piglets were orally administered 5 ml of DMEM (control group) or strain 923H (n = 3). After 48 hours of infection, the piglets were euthanized, and histopathological lesions in the jejunum of both infected and uninfected piglets were examined. The sections were stained with hematoxylin and eosin. Scale bars: 200 μm and 100 μm. (B) Analysis of TJ proteins using proteomics assay. Total protein was extracted from IPEC cells infected with strain 923H for 24 h and analyzed using an in vitro iTRAQ labeling-based quantitative proteomics approach. The dataset was analyzed to screen and statistically evaluate changes in junction proteins. Samples 1 to 3 are designated as S1, S2, and S3. (C) Protein levels assay of CLDN3 in virus infected jejunal tissues by IHC assay. The expression levels of CLDN3 and VP7 in tissues pre- and post-challenge with strain 923H was detected. Immunohistochemical staining were performed on jejunal tissues from both groups of piglets. Scale bars: 100 μm. Average OD value with positive CLDN3 and VP7 expression levels were calculated according to the area of brown yellow particles using ImageJ software. (D) Protein levels assay of CLDN3 in virus infected IPEC cells by WB assay. IPEC cells infected with strain 923H at an MOI of 1 were harvested at 12 h and 24 h, and immunoblotting was performed using antibodies against CLDN3, VP7, occludin, and tubulin. (E) Protein levels assay of CLDN3 in IPEC cells infected with different strains and doses. Western blot analysis was performed to detect the expression levels of CLDN3 and VP7 in IPEC cells infected with 923H, SA11, and WI61 strains for 24 h. The relative intensity of each band was calculated using ImageJ software (D, E). The data are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
According to the previous report, the paracellular space is sealed by the TJ, which prevents virus access to its receptors [27]. RV VP8* peptide treatment could change the membrane localization of CLDN3, and do not affect its content [27]. But we also found that RV infection also reduces CLDN3 protein level (Fig 1B). To confirm whether rotavirus infection in vivo also reduced CLDN3 protein level, immunohistochemical assay was performed on intestinal tissues from control and infected piglets using specific antibodies against VP7 and CLDN3. Higher levels of CLDN3 protein were detected in the villus basal layer of jejunum, but rotavirus infection resulted in a marked reduction in this region (Fig 1C). In vitro analysis also revealed that RV infection specially decreased CLDN3 protein levels in a time-dependent manner at 12 and 24 hpi in the IPEC-J2 cells, while the protein levels of occludin showed no significant change (Fig 1D).
To assess whether CLDN3 protein reduction by rotavirus infection depended on the virus strain and cells type, IPEC-J2 (Fig 1E), Caco-2 (S1A Fig), and HT-29 cells (S1B Fig) were challenged with a panel of rotavirus strains: porcine 923H, simian SA11, and human WI61. At 24 hpi, Western blot analysis demonstrated that all three strains could infect the three cell lines and the infection significantly reduced CLDN3 protein levels.
These findings demonstrate that rotavirus infection reduce CLDN3 protein levels in both in vitro and in vivo infection models.
CLDN3 as a restriction factor that inhibits RV adsorption
To investigate the role of CLDN3 in RV infection, siRNAs targeting the CLDN3 gene were designed and transfected into IPEC cells for 36 h. Western blot analysis showed a significant decrease in CLDN3 protein levels after transfection with specific siRNA-1 and siRNA-2 (S2A Fig). Knockdown of CLDN3 enhanced viral replication in IPEC cells (Fig 2A) and the viral titer in the supernatant increased approximately 10-fold at 24 hpi (Fig 2B). Interferons (IFNs) are crucial cytokines in the host defense against viral infections, playing roles in antiviral, immune regulation, and antitumor effects. To exclude the possibility that CLDN3 reduction might lead to decreased IFN-β expression, thereby promoting viral replication, we stimulated the IFN pathway with different activators after the knockdown of CLDN3. Results showed that knockdown of CLDN3 did not affect the transcription of IFN-β in response to various stimuli (S2B Fig).
(A, B) Effect of CLDN3 gene knockdown on virus infection. IPEC cells transfected with CLDN3 gene siRNA were inoculated with strain 923H at an MOI of 1 for 24 h, then viral RNA levels in the cells (A) and viral titer in the cell supernatant (B) were determined by qRT-PCR and TCID50 assay, respectively. (C) Effect of CLDN3 gene knockdown on virus binding. IPEC cells transfected with CLDN3 gene siRNA were inoculated with strain 923H at an MOI of 100 for 1 h at 4 ℃, then viral RNA levels were evaluated by qRT-PCR. (D, E, F) Viral adsorption assay by confocal laser scanning microscopy. IPEC cells transfected with CLDN3 gene siRNA were inoculated with strain 923H at an MOI of 100 for 1 h at 4 ℃, then the cells were fixed. The multi-layer image was generated by overlaying 20 confocal Z-stack slices acquired at evenly spaced intervals (D). Green fluorescence in Fig D was quantified using ImageJ, and the average number of puncta per cell was determined (E). To reconstruct 3D image, thirty evenly spaced confocal slices were used using Zeiss software (F). CLDN3 (red), viral VP7 protein (green), and cell nucleus (blue). Scale bar: 5 μm in E. (G) Effect of overexpression of CLDN3 on viral binding. MA104 cells transfected with a CLDN3-encoding plasmid for 24 h were inoculated with strain 923H (MOI = 20) at 4 ℃ for 1 h, prior to the quantification of intracellular viral RNA levels via qRT-PCR. (H) Effect of overexpression of CLDN3 on viral internalization. Following 24 h transfection with a CLDN3-encoding plasmid, MA104 cells were inoculated with strain 923H at an MOI of 20. Virus adsorption was carried out at 4 ℃ for 1 h, after which the temperature was shifted to 37℃ for 1 h to allow internalization. Intracellular viral RNA levels were subsequently determined by qRT-PCR. (I) Effect of overexpression of CLDN3 on viral repliation. MA104 cells transfected with a CLDN3-encoding plasmid for 24 h were inoculated with strain 923H at an MOI of 0.1 at 37 ℃ for 18 h, then viral RNA levels were determined in the cells by qRT-PCR. The data shown are representative of three independent experiments, with the mean ± SD. Statistical significance was calculated by Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Next, we examined whether the CLDN3 decrease affected viral entry. IPEC cells were transfected with siRNA targeting CLDN3, then the virus was inoculated for 1 h at 4℃. qRT-PCR analysis demonstrated that knockdown of CLDN3 significantly enhanced viral adsorption (approximate 4-fold increase) (Fig 2C). To further evaluate the role of CLDN3 in viral adsorption, confocal microscopy was performed (Fig 2D). Both single-plane and Z-stack images revealed a greater number of virions attached to the cell surface upon CLDN3 knockdown. Quantification using ImageJ confirmed a significant increase in the average number of fluorescence puncta per cell (Fig 2E). To investigate how CLDN3 decrease enhances early viral adsorption, we further performed confocal microscopy to generate 3D images with CLDN3 knockdown. The results revealed that CLDN3-knockdown expanded the longitudinal range of virus-infected cells (Fig 2F), indicating that CLDN3 affects viral entry.
To further investigate the effect of CLDN3 on RV infection, MA104 cells were transfected with a CLDN3-Myc expression plasmid for 24 h and then inoculated with virus at the indicated condition. CLDN3 overexpression significantly suppressed viral adsorption and internalization, resulting in a 50% and 70% reduction, respectively (Fig 2G and 2H). At 16 h post-inoculation, viral titers in the supernatant were measured by qRT-PCR (Fig 2I), and infected cells were examined by IFA (S2C Fig). CLDN3 overexpression markedly inhibited viral infection, resulting in a 10-fold and 80% reduction, respectively.
These data reveal that CLDN3 effectively blocking RV binding and thereby preventing viral entry and replication.
CLDN3-knockout promotes virus adsorption and infection
To further evaluate whether CLDN3 functions as a viral restriction factor, we constructed a CLDN3 gene knockout cell line using CRISPR-Cas9 technology. Western blotting and confocal microscopy confirmed the successful knockout of CLDN3 gene, and the expression of occludin protein was not affected (Fig 3A and 3B). Additionally, CCK-8 assays demonstrated that the viability of the knockout cell line was not compromised (S3A Fig). First, we used confocal microscopy to assess the effect of CLDN3-knockout on viral adsorption. Both single-plane and Z-stack images revealed that viral adsorption was significantly increased in the CLDN3-knockout cell clones (Fig 3C), and the average number of fluorescence puncta per cell was quantified using ImageJ (Fig 3D). qRT-PCR analysis also confirmed that CLDN3-knockout led to a significant increase in viral binding to the cell surface (Fig 3E). Next, CLDN3-knockout cells were infected with strain 923H at an MOI of 1. At 24 hpi, the supernatant was collected for virus titration, and the cells were fixed and subjected to IFA using antibodies targeting CLDN3 and VP7. A 5–10-fold increase in viral infection was observed in CLDN3-knockout cells compared to the control (Fig 3F). In line with this data, IFA further confirmed the pronounced enhancement of infection upon CLDN3 knockout (S3B Fig). To rule out potential off-target effects of the sgRNA, we performed a rescue experiment. To this end, CLDN3-knockout cells were transfected with a CLDN3-Myc plasmid then infected with the 923H strain. At 24 hpi, Western blot analysis showed that CLDN3 expression was partially restored, which consequently led to a reduction in viral infection. This observation was further confirmed by a virus titer assay (Fig 3G).
(A, B) Establishment of CLDN3-knockout IPEC cells. IPEC cells were transfected with either the CLDN3-targeting sgRNA plasmids (sgRNA1, sgRNA2) or an empty vector (sgCtrl) for 24 h. Following selection with puromycin and validation by gene sequencing, the CLDN3 expression in the cell clone was detected by Western blot (A) and confocal immunofluorescence (B). Scale bar: 5 μm. (C) Viral adsorption analysis in the CLDN3 gene knockout cells by IFA. Control and the CLDN3 gene knockout IPEC cells were inoculated with strain 923H at an MOI of 100 for 1 h at 4℃ and the cells were fixed. Virus binding was analyzed through confocal laser scanning microscopy. The multi-layer image was generated by overlaying 20 confocal Z-stack slices acquired at evenly spaced intervals CLDN3 (red), viral VP7 protein (green), and cell nucleus (blue). Scale bar: 5 μm. (D) Green fluorescence in Fig C was quantified using ImageJ, and the average number of puncta per cell was determined. (E, F) Binding and infection analysis with knockout of CLDN3 gene. Wild-type and CLDN3-knockout IPEC cells were inoculated with strain 923H at an MOI of 100 for 1 h at 4℃ and viral RNA levels were evaluated by qRT-PCR (E), or inoculated with strain 923H at an MOI of 1 for 24 h and the cell supernatant was harvested for virus titer assay (F). (G) Recovering assay of CLDN3 expression in CLDN3-knockout IPEC cells. Myc-CLDN3 plasmid was transfected into CLDN3-knockout IPEC cells, followed by viral infection with strain 923H at an MOI of 1 for 24 h. The cell supernatant was harvested for virus titer assay. CLDN3 and VP7 expression in the cell were detected by Western blot. The results are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. *, P < 0.05; **, P < 0.01.
These findings indicate that CLDN3 plays a negative role in virus adsorption, suggesting that CLDN3 is an important host restriction factor for RV infection.
CLDN3 interacts with RV VP7 directly
To investigate whether CLDN3 could bind to VP4 or VP7 directly, then inhibit RV attachment, we investigated those interactions. The RV virion surface contains two major outer capsid proteins: VP4, which forms the spikes and mediates cell attachment and membrane penetration [28], and VP7, which cooperates with VP4 during viral attachment and post-attachment steps [29]. VP6 constitutes the intermediate capsid layer, maintaining virion integrity and interacting with both the outer proteins and host factors such as HSC70 [29]. To assess potential interactions, we co-expressed Myc-tagged CLDN3 with Flag-tagged VP4, VP6, or VP7 in HEK-293T cells for co-immunoprecipitation (Co-IP) assays. Immunoblotting revealed that CLDN3 specifically associates with VP7, but not with VP4 or VP6 (Figs 4A, S4A and S4B). We further confirmed the interaction between VP7 and endogenous CLDN3 in RV-infected IPEC cells by Co-IP (Fig 4B). The subcellular colocalization analysis revealed that, during the viral binding stage, viral particles were co-localized with CLDN3 (S4C Fig).
(A) Analysis of the interaction between CLDN3 and VP7. HEK-293T cells were co-transfected with Flag-tagged VP7 and Myc-tagged CLDN3 for 24 h. Cell lysates were subjected to Co-IP, and protein interactions were verified by Western blot. (B) Co-immunoprecipitation assay for association of viral VP7 with endogenous CLDN3 in IPEC cells. IPEC cells were infected with strain 923H at an MOI of 1 for 36 h, then Co-IP assay was performed using anti-CLDN3 antibody. (C) Analysis of the interaction between Flag-tagged VP7 and GFP-tagged EC1 or EC2 on HEK-293T cells. (D) Pull-down assay using purified Fc-EC1 and Fc-EC2 proteins. (E) Schematic diagram of VP7 truncation mutants. (F, G) Flag-tagged VP7 truncation mutants were co-transfected with Myc-CLDN3 into HEK-293T cells. Cell lysates were subjected to Co-IP analysis. The results are representative of three independent experiments.
CLDN3 is a transmembrane protein with two extracellular loops, EC1 and EC2 [30]. To identify the loop mediating the CLDN3-VP7 interaction, HEK-293T cells were co-transfected with Flag-VP7 and each loop for Co-IP analysis. The results indicated that EC1, but not EC2, specifically interacts with VP7 (Fig 4C). Given this specificity, we performed a pull-down assay using purified His-tagged VP7 and Fc-tagged EC1 or EC2 peptides, which confirmed a direct interaction between EC1 and VP7 (Fig 4D). Furthermore, an ELISA-based binding assays for RV virion and fusion protein revealed that EC1 peptide exhibited significant binding to RV (S4D Fig).
To identify the specific domain of VP7 required for its interaction with CLDN3, VP7 was divided into three segments (aa 44–185, aa 115–256, aa 186–327) (Fig 4E). The aa 44–185 and aa 186–327 fragments interacted with CLDN3, whereas aa 115–256 did not (Fig 4F). Further, the N-terminal and C-terminal regions of VP7 were deleted to generate fragments encompassing aa 44–114 and aa 257–327, respectively. Only aa 44–114 could interact with CLDN3 (Fig 4G). These results confirm that the EC1 loop of CLDN3 directly interacts with the N-terminal of the VP7 protein.
CLDN3 serves as a molecular decoy to inhibit VP7 glycoprotein-mediated adsorption
Most research focuses on the role of VP8* protein in virus attachment [15]. However, previous study had also demonstrated that rotavirus VP7 glycoprotein is also associated with virus attachment [26]. Since knockdown or knockout of CLDN3 promoted viral binding, we speculated that CLDN3 may be a decoy receptor and the binding of CLDN3 to VP7 may inhibit viral attachment to cell surface.
To verify the role of VP7 in RV adsorption, we conducted an antibody blockade with an anti-VP7 polyclonal antibody. As shown in Fig 5A, the incubation with VP7 polyclonal antibody significantly inhibits virus binding efficiency, as reported previously. To further confirm the role of VP7-mediated adsorption, we conducted a protein blocking assay using the extracellular domain of CLDN3 (Fig 5B). Purified EC1 or EC2 peptides at different concentrations were incubated with virus at 4°C for 1 h, followed by inoculation into IPEC cells. qRT-PCR results showed that pretreatment with EC1 reduced viral adsorption in a dose-dependent manner (Fig 5B). So, the VP7-CLDN3 association blocked virus binding, suggest that VP7 possessed attachment function during virus entry process. If CLDN3 inhibits VP7 attachment function, its blockade with an anti-CLDN3 antibody should promote viral binding and infection. Consequently, we assessed viral binding efficiency following treatment with anti-CLDN3 polyclonal antibodies. The results confirmed that this pretreatment significantly enhanced both viral adsorption (Fig 5C) and infection (Fig 5D).
(A) Anti-VP7 antibody treatment prevented viral attachment. The strain 923H at an MOI of 100 was incubated with the polyclonal antibody against 923H VP7 for 1 h at 4℃ and then inoculated into IPEC cells for 1 h at 4℃. Viral RNA levels were subsequently analyzed by qRT-PCR. (B) EC1 protein blocked RV adsorption in a dose-dependent manner. After virus was incubated with different dose of recombinant EC1 or EC2 protein for 1 h at 4℃, the mixture was inoculated into IPEC cells at 4°C for 1 h, then viral RNA levels were evaluated by qRT-PCR. (C, D) Anti-CLDN3 antibody treatment promotes viral attachment (C) and infection (D). IPEC cells pretreated with indicated dose of antibody for 1 h at 4℃ were incubated with strain 923H at an MOI of 100 for 1 h at 4℃ (C) or at an MOI of 1 for 16 h at 37℃. Viral RNA levels were determined by qRT-PCR for binding assay (C), and cells infected were fixed for IFA assay (D). The results are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
These data suggest that VP7 is associated with RV attachment and CLDN3 as a molecular decoy could inhibit VP7 glycoprotein-mediated adsorption.
The 74th amino acid of VP7 is a key residue for binding to CLDN3
To identify the key VP7 residues that interact with CLDN3, we used AlphaFold prediction tool. A total of five amino acid residues—located at positions 74, 80, 82, 109, and 113—were identified (Fig 6A). Each of these residues was individually mutated in the VP7 gene according to the following principle: acidic amino acids were converted to basic ones, basic ones to acidic, and neutral ones to either acidic or basic. Co-IP assays showed that when the 74th amino acid was mutated from glutamic acid to lysine (E74K), the interaction between VP7 and CLDN3 was significantly weakened (Fig 6B). Sequence alignment of VP7 proteins including mainly prevalent genotypes strains of human and porcine rotavirus from the NCBI GenBank database, revealed that the majority of strains harbor a glutamate (E) at residue 74, whereas a subset of strains possess aspartic acid (D), Glycine (G), asparagine (N), lysine (K) or serine (S) at this position (S2 Table). Next, we evaluated the effect of VP7 mutations (E74D, E74G, E74N, and E74S) on the VP7-CLDN3 interaction. As shown in the S5 Fig, the interaction was not altered by any of these mutations except E74K.
(A) Schematic diagram shows predicted key VP7 residues interact with CLDN3 by AlphaFold. (B) Identification of key site responsible for the VP7-CLDN3 association. HEK-293T cells were co-transfected with Flag-tagged VP744-327 mutant constructs together with Myc-tagged CLDN3. Co-IP assay was analyzed using anti-Flag antibodies. The relative intensity of each band was quantified using Image J software. (C, D) E74K mutation in the rSA11 (C) and NMTL (D) abolishes EC1-mediated RV adsorption inhibition. After virus (MOI = 100) was incubated with 200 µg/ml of EC1 peptide, the mixture was inoculated into IPEC cells at 4°C for 1 h, then viral RNA levels were evaluated by qRT-PCR. The results are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Since the VP7 E74K mutation disrupted the VP7-CLDN3 interaction, this point mutation may abolish EC1-mediated inhibition of viral adsorption. To validate this speculation, we rescued a mutant strain, rSA11-E74K, using a SA11 reverse genetics system. The wild type and mutant viruses were incubated with recombinant EC1 protein at 4°C for 1 h, followed by inoculation into IPEC cells. qRT-PCR results showed that pretreatment with EC1 significantly reduced the adsorption of wild type SA11, but did not significantly affect the adsorption of rSA11-E74K (Fig 6C). Next, we further confirm the hypothesis in a porcine strain, NMTL and rNMTL-E74K were rescued using a reverse genetics method. Similar to the wild type and mutant NMTL viruses, incubation with recombinant EC1 protein led to decreased adsorption of NMTL and rNMTL, but the same treatment did not reduce the adsorption of rNMTL-E74K (Fig 6D). So, blocking the interaction between VP7 and CLDN3 led to the loss of EC1-mediated inhibition of viral adsorption.
These results suggest that CLDN3 specially binds to the 74th amino acid of viral protein VP7, and the VP7 E74K mutation disrupts the VP7-CLDN3 interaction, which is associated with CLDN3-mediated inhibition of viral adsorption.
A single VP7 E74K mutation promotes virus attachment ability
Due to the role of VP7 in virus attachment and the effect of VP7 E74K mutation on the VP7-CLDN3 interaction, virus attachment ability would be increased in the recombinant virus possessing VP7 E74K mutation. So, we evaluated the binding efficiency of the wild-type and recombinant viruses by qRT-PCR. The results confirmed that rSA11-E74K showed a marked increase in viral adsorption assay (Fig 7A). To determine that this effect was associated with CLDN3, we repeated the experiment in the CLDN3 knockout cell line. In this case, no significant increase in viral attachment was observed for either strain (Fig 7A), Furthermore, Confocal laser scanning microscopy also indicated that the E74K mutation increased viral attachment in wild-type cells (Fig 7B, top panels), but not in CLDN3-KO cells (Fig 7B, bottom panels). Comparing to rSA11, the replication ability of rSA11-E74K was significantly increased in IPEC cells (Fig 7C).
(A, D) Binding assay in wild-type and CLDN3-knockout IPEC cells between wild type and E74K mutant SA11 (A) or NMTL (D). Wild-type and CLDN3-knockout IPEC cells were inoculated with the indicated virus at an MOI of 100 at 4°C for 1 h and viral RNA levels were evaluated by qRT-PCR. (B, E) Viral adsorption analysis by confocal laser scanning microscopy. Wild-type and CLDN3-knockout IPEC cells were infected as in A, D, then the cells were fixed for IFA assay. Viral VP7 protein (green) and nuclei (blue) were visualized by Zeiss confocal microscopy. Top: representative images. Bottom: green fluorescence quantification using Image J. Scale bar: 5 μm. (C, F) Growth-curve assay for rSA11 (C) and NMTL (F) as well as their mutant viruses. IPEC cells were inoculated with the indicated virus at an MOI of 1, then the supernatant was harvested for titer assay on the MA104 cells. The results are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Further we confirmed the result in a porcine origin rotavirus, NMTL. The virus attachment and replication ability were compared among strain NMTL, rNMTL and rNMTL-E74K. Binding efficiency assays in both wild-type and CLDN3-KO cells showed that rNMTL-E74K exhibited significant increase in the wild-type cells, but no differences were observed in the CLDN3-KO cells (Fig 7D). Confocal laser scanning microscopy results also confirmed similar results (Fig 7E). Comparing to rNMTL, the replication ability of rNMTL-E74K was significantly increased (Fig 7F).
Taken together, these data further demonstrate that VP7 glycoprotein could affect RV attachment, and the VP7 E74K mutation enhances viral attachment and replication.
The E74K mutation in VP7 enhances rotavirus pathogenicity
The above findings indicated that the mutation of VP7 E74K increases RV adsorption capacity. To further investigate the impact of this mutation on viral virulence, we conducted an in vivo challenge experiment using neonatal mice. Nine litters of healthy neonatal mice (five pups per litter) were randomly assigned into three groups. Each group was orally inoculated with rSA11, rSA11-E74K, or DMEM, and the mice were monitored for diarrhea symptoms and weight changes. By day 3 post-infection, pups infected with rSA11-E74K exhibited the most severe diarrhea, with an average clinical diarrhea score of 1.88. The symptoms showed progressive improvement, with complete resolution by day 8. In contrast, diarrhea in the rSA11-infected group peaked on day 1 with a mean score of 1.28, showed near-complete resolution by day 5, and achieved full recovery by day 7. No clinical symptoms were observed in the control group (Fig 8A). The percentage of diarrheal pups was also recorded for each group. Throughout the diarrhea period, the rSA11-E74K-infected group consistently showed a higher diarrhea rate compared to the rSA11 group (Fig 8B). Daily weight measurements revealed that, compared to the control group, rSA11-infected mice did not experience significantly weight loss, while rSA11-E74K-infected mice showed significantly weight loss (Fig 8C). By day 9, these pups were visibly smaller than those in the control and rSA11 groups (Fig 8G).
Six to seven days suckling mice (n = 15/group) were orally administered rSA11 and rNMTL as well as their mutant viruses or DMEM (control) at a dose of 1 × 107 TCID50/100 μL. (A, D) Diarrhea scores were recorded every 24 hours. (B, E) The proportion of diarrheic mice was calculated every 24 hours after infection. (C, F) Mouse body weights were recorded every 24 hours after infection. Weight gain ratios were calculated. (G, J) At day 9 post-inoculation, body size differences among mice were recorded. (H, K) Viral load in the jejunum at 72 hours post-inoculation. (I, L) Histopathological changes in the jejunum from infected and uninfected mice were analyzed. The results are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
On day 3 post-infection, mice were euthanized and dissected for virus titration and histopathological analysis of the intestine. In rSA11-infected mice, the virus loading was significantly lower than rSA11-E74K-infected mice (Fig 8H), and the intestine exhibited mild mucosal vacuolization and swelling (Fig 8I). In contrast, rSA11-E74K-infected mice showed higher virus loading (Fig 8H) and extensive vacuolization of epithelial cells at the tips of the intestine villi, with signs of nuclear condensation or disappearance (Fig 8I).
Consistent with the results for rNMTL and rNMTL-E74K, the E74K mutation enhanced the virulence of recombinant virus rNMTL-E74K, as evidenced by higher diarrhea scores and prolonged duration of diarrhea in neonatal mice (Fig 8D), an increased percentage of diarrheic mice (Fig 8E), slower weight gain (Fig 8F), and smaller body size (Fig 8J). Virus loading assay showed that virus RNA copies were significantly higher in rNMTL-E74K group (Fig 8K). Histopathological analysis further demonstrated more extensive vacuolation of the small intestinal villous epithelial cells in rNMTL-E74K group (Fig 8L).
These results demonstrate that the VP7 E74K mutation significantly enhances the virulence of rotavirus.
Discussion
Tight junctions (TJs) are essential structural elements of epithelial cells that establish polarized cell layers with distinct apical and basolateral domains [31]. As epithelial cells serve as primary targets for rotavirus infection, these TJs structures may inhibit viral infection and spread. To counteract this, rotavirus infection could alter the CLDN3 localization and reduce its protein level. We further uncovered CLDN3’s regulatory function in RVA infection. VP7 is involved in RV adsorption, and CLDN3 as a decoy receptor inhibits viral attachment via the CLDN3-VP7 association. Importantly, an E74K mutation in VP7 abolished the interaction, and also enhanced both viral binding capacity in vitro and pathogenicity in a suckling mouse model.
The gastrointestinal mucosa serves as a critical barrier, preventing millions of microbes from infiltrating the intestinal tissue. Defects in the intestinal barrier have been linked to a wide range of disorders, including inflammatory bowel disease (IBD), colon carcinoma, ulcerative colitis, and even extra-intestinal conditions [32]. TJs located at the apical side of epithelial cells, regulate the paracellular transport of small molecules and ions while maintaining epithelial barrier integrity [32]. We observed that RVA infection reduced CLDN3 protein level, which may disrupt ion balance, impair water adsorption, and contribute to diarrhea. Given that these TJ proteins are markers of intestinal integrity in IBD [33], in vivo findings suggest that RVA infection compromises gut barrier function, leading to villous atrophy.
Intestinal barrier integrity is crucial for defense against enteric viruses. A recent study demonstrated that gut microbiota enhances barrier function, thereby inhibiting porcine deltacoronavirus infection in piglets [34]. Conversely, TJ disruption can facilitate enteric bacterial invasion and deeper tissue penetration [35]. Moreover, several viruses have evolved mechanisms to degrade TJ proteins, thereby facilitating viral invasion and dissemination. Examples include West Nile virus, Japanese encephalitis virus, norovirus, human rhinovirus, and porcine reproductive and respiratory syndrome virus [13]. In this study, we speculated that RV infection could antagonize the antiviral function of CLDN3 through altering its localization pattern and reducing its protein level. Before our study, Porfirio Nava et al., found that RV VP8* peptide treatment does not alter the protein content of the TJ proteins ZO-1, occludin and claudin-3, but affects the distribution pattern, which leads to the open of the paracellular space sealed by the TJ and the exposure of cell receptors. However, we found that RV infection could also decrease the protein content of CLDN3, which is another antagonistic strategy.
Several reports have found that most CLDNs restrict viral access to receptors—such as in human rhinovirus and respiratory syncytial virus infections [13], but the underlying mechanisms remain unclear. It has been demonstrated about the role of VP8* protein in virus attachment [15]. But Marta Sabara et al., had also demonstrated another role of bovine rotavirus VP7 glycoprotein in virus attachment, but the specific host factors involved remain unknown [26]. In this study, we demonstrated that VP7 is associated with RV adsorption using anti-VP7 antibody blocking, EC1 blocking, and recombinant VP7 E74K virus binding assays. However, the specific host factors involved in VP7 adsorption remain unknown.
In this study, CLDN3 is used by the virus as a binding molecule. However, this does not increase viral attachment and entry, and instead functions as a decoy receptor. We propose two models. One is the model of spatial segregation and non-productive retention. CLDN3 may reside primarily in membrane microdomains distinct from lipid rafts or may be spatially segregated from key co-receptors (e.g., sialic acid receptors) necessary for downstream endocytic signaling and productive entry. Thus, while the virions bind to CLDN3, they are effectively “trapped” and cannot initiate further internalization phase. Another model is competitive inhibition and endocytic blockade. CLDN3 may bind RV with high affinity, but this binding competitively inhibits the virus from engaging another, as-yet-unidentified, “functional receptor” responsible for triggering productive endocytosis. Our data (e.g., internalization assays and confocal imaging showing virus co-localization with CLDN3 but lack of productive uptake) support the view that this binding does not lead to efficient infection.
In this study, we found that CLDN3 could bind to viral VP7 protein, but not VP4 and VP6 proteins. However, given the structural complexity of the VP4 and VP6 proteins, the Flag tag may potentially interfere with their proper folding, and interactions between VP4 or VP6 and CLDN3 cannot be completely ruled out. We employed AlphaFold to predict key interaction residues between CLDN3 and the viral VP7 protein. Given that VP7 forms functional trimers [36], predictions based solely on the VP7 monomer proved unreliable. As shown in Fig 6A, the EC1 peptide, which forms part of the extracellular loops of CLDN3, may penetrate into the inner part of the hexameric channels on the virion and subsequently bind to the N-terminal region of VP7. While our data suggest that the E74 residue represents one critical interaction site, we anticipate that additional contact residues may be identified once the precise structural interaction between CLDN3 and the VP7 trimer is resolved. Previous studies have suggested that NSP4 and NSP1 contribute to the virulence of rotavirus [37,38]. In this study, we report for the first time that VP7 plays a critical role in rotavirus pathogenicity. Specifically, the mutation of E to K at position 74 in VP7 significantly enhances viral replication and virulence. Notably, a porcine rotavirus strain with VP7 K74 was identified in Denmark in 2021 and its sequence has been uploaded to the NCBI database, indicating that rotavirus continues to evolve strategies to counteract host antiviral defenses. However, it is not prevalent in the population. To better adapt to the host, the virulence of highly pathogenic viruses tends to attenuate after multiple rounds of infection, as observed in viruses like ASFV (African swine fever virus) and SARS-CoV-2. The strains with VP7 74K displays high pathogenicity, which may be not benefit for virus adaption and evolution. Moreover, the interaction between the E74 residue and CLDN3 may be not the sole determinant of virulence and other factors as well as underlying mechanisms require further investigation.
In summary, our study demonstrates that CLDN3 acts as a decoy receptor that anchors the virus, thus preventing it from accessing its receptors and ultimately inhibiting RV attachment. These results reveal a novel mechanism through which the CLDN3 inhibits RV entry. Moreover, RVs have evolved diverse strategies to counteract host antiviral defenses. In addition to altering CLDN3 distribution, our data show that RV infection reduces CLDN3 protein level in the in vitro cell and in vivo small intestine infection models, suggesting this may represent a key immune evasion pathway. Collectively, this work advances our understanding of the regulatory interplay between RV and TJs.
Materials and methods
Ethics statement
This study was conducted in strict compliance with animal welfare regulations under the approval of the Harbin Veterinary Research Institute Animal Care and Use Committee (Protocol No. 250228–03-GR).
Cells and viruses
MA104, HEK 293T and IPEC-J2 (IPEC) cells, porcine enterocytes isolated from the jejunum of a neonatal unsuckled piglet [39] were cultured in DMEM containing 10% FBS (Fetal bovine serum, Clark). HEK-293 suspension cells were grown in 293 Pro CD 293 M Medium. The cells were maintained in 125 mL polycarbonate shake flasks on an orbital shaker platform rotating at 120 rpm, and passaged every 3 days to maintain a density below 4 × 105 cells/mL. Caco-2 cells were cultured in MEM containing 15% FBS and 1% NEAA and HT-29 cells were cultured in McCoy’s 5A medium (Sigma-Aldrich) containing 15% FBS. Rotavirus strains NMTL and 923H (G9P[23], porcine origin; Genbank accession number: PQ117785.1, PQ141611.1-PQ141620.1), WI61 (G9P[8], human origin), and SA11 (G3P[2], simian origin) rescued by reverse genetic method were used in this study.
Plasmids
To prepare secreted proteins, Human IgG Fc gene fragment was inserted into the C-terminus of target genes to facilitate protein purification and the sequence of secreted signaling peptide, TPA, was inserted into the N-terminus of target genes, which would promote the target protein secreting to culture medium. The expression cassette was cloned into pCAGGs vector. The plasmids encoding Flag-VP7, Flag-VP4, Flag-VP6 and CLDN3-Myc as well as their truncation were prepared by cloning each ORF into pCAGGs vector or pEGFP-N1 vector.
Reagents and antibodies
Anti-viral VP7 monoclonal and polyclonal antibodies were prepared by our lab [40]. Rabbit anti-CLDN3 (A2946), mouse anti-Myc (AE010), rabbit anti-Myc (AE070), rabbit anti-His (AE086) were purchased from Abclonal. Rabbit anti-GFP (ab183734), rabbit anti-Occludin (ab31721) were purchased from Abcam. Rabbit anti-Fc (16402–1-AP) were purchased from Proteintech. Mouse anti-Flag (F1804) was purchased from Sigma Aldrich. Mouse anti-Tubulin (A01410) was purchased from GenScript. Rabbit anti-IgG (A7016), Mouse anti-IgG (7028) were purchased from Beyotime. Alexa Fluor 488 goat anti-mouse IgG [H + L] (ab150113), Alexa Fluor 594 goat anti-rabbit IgG [H + L] (ab150080) were purchased from Abcam.
Proteomics assay about the expression of TJ proteins
The IPEC cells were inoculated with strain 923H with an MOI of 1 pretreated with trypsin (10 μg/ml) for 24 h, then the cells were harvested through centrifuge (1,000 × g, 5 min, RT). Through cell lysis and protein extraction, 200 μg of proteins for each sample was digested by trypsin. The digest peptides of each sample were desalted on C18 Cartridges (Empore SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid. 100 μg of peptide mixture of each sample was labeled using iTRAQ reagent according to the manufacturer’s instructions (Applied Biosystems). LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific). The MS raw data for each sample were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis.
In vivo CLDN3 assay upon RVA challenge
All piglets were negative for PEDV, TGEV, and PoRV. One-day-old piglets (n = 3) was orally inoculated with 106 TCID50 of strain 923H, and mock group (n = 3) was orally inoculated with 5 ml of DMEM. Upon challenge, the piglets were raised in separate negative-pressure isolators and were fed milk every two hours. At 48 hours post-infection, piglets from each group were euthanized, and then small intestine samples were collected. Each tissue was placed in 10% buffered formalin for histopathological and immunohistochemical examinations analysis.
Protein expression and purification
Plasmids encoding VP7-His, EC1-hFc and EC2-hFc were transfected into 293 suspension cells, respectively. The culture medium was collected at 72 h post transfection, then the target fusion proteins were purified with Ni NTA 6FF Beads (Millipore, 70666–4) for His-tagged protein purification and Protein A (Genscript) for hFc tagged protein purification according to the instruction manual. The protein concentration was measured by Protein Quantification Kit (Thermo).
Western blotting and co-immunoprecipitation
Briefly, the cultured cells were lysed using RIPA lysis buffer (Beyotime, P0013B, P0013C) containing protease inhibitor, PMSF (Beyotime, ST506). After centrifuge to remove the cell debris, total protein concentration was determined. A total of 20–25 μg of protein sample was separated by using 10%-12% SDS-PAGE and then transferred to nitrocellulose membranes (Thermo) and subjected to immunoblotting.
For the Co-IP assay, cells transfected with plasmids or infected with the 923H strain were lysed using ice-cold RIPA buffer and centrifuged. The lysates were incubated overnight at 4°C with the appropriate amount of anti-Flag, anti-Myc, or anti-CLDN3 antibody, or with immunoglobulin G (IgG) as a control. Then, after washing 3 times with PBS, 50 μl of protein G agarose (Genscript, L00210-10) was added to bind the antibody. Following 2 h to 3 h incubation at room temperature, the agarose was collected by centrifugation, then boiled in 5 × sample buffer before analysis by SDS-PAGE.
For pull-down assays, purified recombinant EC1 or EC2 protein was added to Protein A resin and incubated at room temperature for 1 hour. After washing the resin, it was incubated with purified His-tagged VP7 protein at 4°C for 2 hours. The resin was then washed six times with PBST, resuspended in PBS, and mixed with 5 × SDS-loading buffer before boiling. The samples were subjected to SDS-PAGE and Western blot analysis.
RNA interference and overexpression assays
To downregulate CLDN3 expression, cells (1.0-3.0 × 105/well) were transfected with 2.5 μL (20 μM) of target-specific siRNA (RiboBio, Guangzhou, China; sequences in S1 Table) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, #13778150). After 36 h, cells were lysed with RIPA buffer (Beyotime, China) for Western blot analysis or subjected to qRT-PCR to assess knockdown efficiency. Total RNA was extracted, and cDNA was synthesized using Reverse Transcriptase M-MLV (TAKARA, China) following the manufacturer’s instructions. qRT-PCR was performed using ChamQ Universal SYBR qRT-PCR Master Mix (Vazyme, China). Relative mRNA expression levels were calculated via the 2−ΔΔCt method, with 18S rRNA serving as the internal reference. Primer sequences are provided in S1 Table.
For the effect of CLDN3 overexpression on virus infection, the cells were transfected with a plasmid encoding Myc-CLDN3 using Lipofectamine 2000 transfection (Life technology) for IPEC cells, or Lipofectamine 3000 transfection (Life technology) for MA104 cells, according to the manufacture’s instruction. After 24 hours, cells were inoculated with specific virus strain.
CRISPR–Cas9-mediated CLDN3 gene knockout
To generate the CLDN3-knockout cells, CRISPR-Cas9 gene editing was employed. Two single-guide RNAs (sgRNAs) were designed using the online tool CHOPCHOP (https://chopchop.cbu.uib.no/) with the following sequences: sgRNA-1: 5′-CCTCTGTTGTGGGCAAGATCAAC-3′; sgRNA-2: 5′-GACGCGCAAAAGCGCGAGAT-3′. The DNA fragments encoding these sgRNAs were cloned into the lentiCRISPR-v2 plasmid (a Cas9-expressing vector). IPEC cells were transfected with either the CLDN3-targeting sgRNA plasmids or an empty vector control and selected with puromycin (0.5 μg/mL) to establish knockout cell lines. To verify successful CLDN3 knockout, genomic DNA was amplified by PCR and analyzed via Sanger sequencing to detect insertions or deletions. Primer sequences are provided in S1 Table. Additionally, immunofluorescence assay (IFA) confirmed complete ablation of CLDN3 protein expression.
Virus titration
Briefly, confluent MA104 cell monolayers in 96-well plates were washed three times with DMEM (100 μL per wash). Viral supernatant was serially diluted (10-fold) in DMEM containing 1 μg/mL trypsin, and 100 μL aliquots of each dilution were inoculated onto MA104 cells in quadruplicate. Following 72 h incubation at 37°C, cytopathic effects (CPE) were examined by light microscopy, and viral titers (TCID50/mL) were determined using the Reed-Muench method.
Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan). To analyze the effect of knockout of CLDN3 on cell viability, sgCtrl (control) and CLDN3-KO cells were plated in 96-well plates, and cytotoxicity was evaluated 72 h post-seeding. Relative cell viability was normalized to control cells and expressed as a percentage.
Protein neutralization and antibody blocking assays
For the protein blocking assay, viruses (MOI = 5) were pre-incubated with varying concentrations of EC1-Fc or EC2-Fc in 0.1 mL culture medium at 4°C for 1 h. The virus-protein mixtures were then inoculated onto cells. Following a 1 h adsorption period at 4°C, total cellular RNA was extracted for RT-qPCR analysis.
IPEC cells were seeded in 96-well plates. Before infection, cells were incubated with 0.1 mL of medium containing different concentrations of anti-CLDN3 polyclonal antibody or isotype antibody (40 μg/mL) at 4°C for 1 h. After three washes with DMEM, the cells were inoculated with the strain 923H. For the binding assay, infection was performed at an MOI of 100 (4°C, 1 h) to allow viral binding, then total cellular RNA was extracted following three washes with PBS. For the IFA assay, infection was performed at an MOI of 1 for 1 h at 37°C. Following three washes, the cells were incubated with a medium containing the corresponding antibodies at 37°C for 16 h, then the cells were fixed for IFA assay.
For the anti-VP7 antibody blocking, viruses (MOI = 100) were pre-incubated with varying concentrations of anti-VP7 polyclonal antibody diluted in 0.1 mL culture medium at 4°C for 1 h. The virus-antibody mixtures were then inoculated onto cells. Following a 1 h adsorption at 4°C, total cellular RNA was extracted for RT-qPCR analysis. Relative viral RNA levels were quantified using the 2-ΔΔCt method with 18S rRNA as the endogenous control. Primer sequences are provided in S1 Table.
Subcellular localization
After three washes with PBS, cells were fixed with cold methanol (-20°C) for 20 minutes. Following fixation, samples were blocked with 5% BSA in PBS for 1 hour at room temperature. Primary antibodies, diluted in PBS containing 1% BSA, were applied for 2 hours at room temperature. After three additional PBS washes, cells were incubated for 1 hour at 37℃ with fluorescent secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L), both diluted in PBS with 1% BSA. Nuclei were counterstained with 1 μM DAPI (Sigma-Aldrich) for 5 minutes, followed by three final PBS washes. Fluorescence images were acquired using a Leica LSM800 confocal laser scanning microscope with appropriate filter sets for each fluorophore. To better characterize viral binding processes, at least twenty evenly spaced confocal slices were combined to generate an overlay image.
Virus binding and internalization assay
The amount of virion bound to the cell surface and the amount of subsequent virus internalization was based on viral genome quantification. To assess viral binding, differentially treated cells were incubated with virus at an MOI of 100 at 4°C for 1 h to allow surface attachment while preventing internalization. Unbound virions were removed by three washes with ice-cold PBS, followed by total RNA extraction.
For the internalization assay, cells were first incubated with virus at an MOI of 100 at 4°C for 1 h (binding phase), then shifted to 37°C for 1 h to permit entry. After trypsin washing to remove non-internalized virus, total RNA was isolated. Viral genome copy numbers were quantified by qRT-PCR. Binding and internalization levels were calculated as the ratio of viral RNA copies in treated groups relative to controls, normalized to input virus.
ELISA-based EC1-Fc binding assays
Purified RV (100 ng/100 µL) was coated onto ELISA plates (Corning Costar) in carbonate buffer (35 mM NaHCO₃, 15 mM Na₂CO₃, pH 9.6) and incubated overnight at 4 °C. Plates were washed with PBST (PBS containing 0.05% Tween-20) and then blocked with PBS supplemented with 5% BSA for 1 hour at 37 °C. After washing, the plates were incubated for 1 hour at 37 °C with EC1-Fc or Fc control proteins diluted to indicated concentrations in sample dilution buffer (INNOREAGENTS, SD-001). Following additional washes, plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H + L) (Abclonal, AS002) diluted 1:3000 in protein stabilizer buffer (INNOREAGENTS, PR-SS-002) at 37 °C for 1 hour. After washing, plates were incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma, T0440) for 15 minutes at 37 °C. The reaction was stopped with 2 M H₂SO₄, and the optical density was measured at 450 nm using an ELx808 microplate reader (BioTek).
Key interaction residues prediction using AlphaFold3
The amino acid sequences of CLDN3 (UniProt ID: O15551) and VP7 (UniProt ID: A0A059PCE3) were retrieved from UniProt (http://uniprot.org) for structural analysis. Using AlphaFold3 (hosted at https://golgi.sandbox.google.com/), we performed molecular docking studies with: Full-length CLDN3 or CLDN3 extracellular loop 1 (ECL1). Both targets were docked against VP7 in its monomeric and oligomeric states. AlphaFold3 generated four distinct docking models for each CLDN3-VP7 configuration. All structural models were visualized and analyzed using UCSF ChimeraX. The most biologically relevant CLDN3-VP7 interaction complex was selected based on the highest interface predicted Template Modeling (ipTM) score, which predicts the accuracy of protein-protein interfaces in the model.
Generation of recombinant rotaviruses
SA11 rescue plasmids were purchased from Addgene, and NMTL rescue plasmids were constructed through cloning the 11 gene segments containing 5’ and 3’UTR into a vector flanked by a T7 promoter sequence, hepatitis delta virus (HDV) ribozyme and T7 terminal sequence [41]. These regulating elements are from the SA11 rescue plasmid. Recombinant RVs were rescued as described by Liliana et al, [42,43]. Briefly, 1 × 105 BHK-T7 cells were resuspended in 1 ml of DMEM. The transfection mixture was prepared, including the 11 RVA pT7 plasmid (each 0.4 μg and NSP2/5 2 μg), 800 ng of the plasmid pCMVScript-NP868R-(G4S)4-T7RNAP (C3P3-G1) [44] and 12 μl of Lipofectamine 2000, and then incubated at room temperature for 5 min. The cell suspension and transfection mixture were gently mixed by pipetting. At 4–6 hours post-transfection, the medium was replaced with complete DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin. After 16–18 hours of incubation, cells were washed three times with serum-free DMEM, followed by addition of 1 mL serum-free DMEM containing 2 μg/mL trypsin. Twenty-four hours after trypsin treatment, 1 × 10⁵ MA104 cells suspended in 200 μL serum-free DMEM were added to each well, and the co-culture was maintained for an additional 48 hours. The infected cells were then subjected to three freeze-thaw cycles (-80°C/37°C), and the resulting lysate was inoculated onto fresh MA104 cell monolayers until cytopathic effect (CPE) became evident.
Mice infection
Six- to seven-day-old Balb/C suckling mice were orally inoculated with 107 TCID50 of virus suspension. Clinical monitoring was conducted daily from days 1–9 post-infection, with diarrhea severity assessed using a standardized scoring system: 0 = normal stool; 1 = soft stool (mild diarrhea); 2 = loose stool (moderate diarrhea); 3 = watery stool (severe diarrhea). All animals were maintained in individual isolators within our specific pathogen-free facility, with daily weight monitoring to assess disease progression. For pathological analysis, intestinal tissue samples were collected at 48 h post-challenge and fixed in 10% neutral buffered formalin for subsequent histopathological evaluation or weighted for virus titration assay. Briefly, approximately 100 mg of tissue sample was homogenized in liquid nitrogen. The powder was then suspended in PBS buffer at a 1:10 (w/v) ratio. Followed by centrifugation at 12,000 × g for 10 min, the supernatant was subsequently collected for viral RNA extraction.
Supporting information
S1 Fig. Evaluation of CLDN3 expression in Caco-2 and HT-29 cells upon virus challenge.
CLDN3 expression levels in Caco-2 (A) and HT-29 (B) cells were determined after infection with different strains using Western blot assay. The relative intensity of CLDN3 was calculated using ImageJ software. The data are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
https://doi.org/10.1371/journal.ppat.1014045.s001
(TIF)
S2 Fig. Evaluation of siRNA targeting CLDN3, IFN-β mRNA levels and virus replication with the CLDN3 overexpression.
(A) The siRNAs targeting CLDN3 were transfected into IPEC cells for 36 h, then the CLDN3 protein levels were evaluated by Western blot. (B) In CLDN3-knockdown IPEC cells, transfection with poly(I:C) or infection with SeV or VSV was performed. After 12 hours, total RNA was extracted and IFN-β mRNA levels were measured by qRT-PCR. (C) IFA showed that overexpression of CLDN3 gene inhibited viral infection. MA104 cells transfected with a CLDN3-encoding plasmid for 24 h were inoculated with strain 923H at an MOI of 0.1 at 37 ℃ for 18 h, then the cells were fixed for IFA assay. Scale bar: 275 μm. The relative fluorescence intensity of VP7 was calculated using ImageJ software. Data represent three independent experiments. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
https://doi.org/10.1371/journal.ppat.1014045.s002
(TIF)
S3 Fig. Evaluation of cell viability and virus infection on IPEC-KO cells.
(A) Viability assay for knockout cells. Control (sgCtrl) and IPEC-KO cells (sgRNA1, sgRNA2) were seeded in 96-well plates, cultured at 37°C for 48 h, and cell viability was determined using a CCK8 kit. (B) Evaluation of virus infection with knockout of CLDN3 gene. CLDN3-knockout IPEC cells was inoculated with strain 923H at an MOI of 1 for 24 h. Indirect immunofluorescence assay was performed. Scale bar: 275 μm. The relative fluorescence intensity of CLDN3 and VP7 was calculated using ImageJ software. Statistical significance was calculated by Student’s t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
https://doi.org/10.1371/journal.ppat.1014045.s003
(TIF)
S4 Fig. Analysis of CLDN3 association with viral structural proteins.
(A, B) CLDN3 does not interact with VP4 or VP6. CLDN3-Myc was co-transfected with either VP4-Flag (A) or VP6-Flag (B) into HEK-293T cells. After 36 hours, cells were lysed, and Co-IP assay was performed using an anti-Myc antibody. (C) Confocal laser scanning microscopy revealed an interaction between CLDN3 (red) and viral VP7 (green) during the viral adsorption stage. Nuclei were stained with DAPI (blue). Scale bar: 5 μm. The pearson correlation efficient for colocalization was calculated with ImageJ software. (D) Binding assay of EC1 peptide to virions measured by ELISA. The plate was coated with purified RV, and different doses of EC1-Fc fusion protein was added. The OD450 value was obtained. Data represent three independent experiments.
https://doi.org/10.1371/journal.ppat.1014045.s004
(TIF)
S5 Fig. Analysis of the association among CLDN3 and VP7 variants.
The Flag-tagged VP7 variants together with Myc-tagged CLDN3 were co-transfected into HEK-293T cells. Co-immunoprecipitation using anti-Flag antibodies was analyzed. The relative intensity of each band was quantified using Image J software. The results are representative of three independent experiments.
https://doi.org/10.1371/journal.ppat.1014045.s005
(TIF)
S1 Table. Sequences for primers and siRNAs.
All the primers and siRNA are available in this Table.
https://doi.org/10.1371/journal.ppat.1014045.s006
(DOCX)
S2 Table. Distribution of amino acid residues at position 74 of VP7 in porcine and human strains.
Number represents the numbers of strain with a specific acid residues at position 74 of VP7 from GenBank; (-) No detection.
https://doi.org/10.1371/journal.ppat.1014045.s007
(DOCX)
Acknowledgments
We thank Philippe Jais (EUKARŸS SAS) for the plasmid pCMVScript-NP868R-(G4S)4-T7RNAP (C3P3-G1) and Siyuan Ding for his technical assistance in establishing the rotavirus mouse infection model.
References
- 1. Troeger C, Khalil IA, Rao PC, Cao S, Blacker BF, Ahmed T, et al. Rotavirus Vaccination and the Global Burden of Rotavirus Diarrhea Among Children Younger Than 5 Years. JAMA Pediatr. 2018;172(10):958–65. pmid:30105384
- 2. Ciarlet M, Conner ME, Finegold MJ, Estes MK. Group A rotavirus infection and age-dependent diarrheal disease in rats: a new animal model to study the pathophysiology of rotavirus infection. J Virol. 2002;76(1):41–57. pmid:11739670
- 3. Crawford SE, Patel DG, Cheng E, Berkova Z, Hyser JM, Ciarlet M, et al. Rotavirus viremia and extraintestinal viral infection in the neonatal rat model. J Virol. 2006;80(10):4820–32. pmid:16641274
- 4. Chang-Graham AL, Perry JL, Engevik MA, Engevik KA, Scribano FJ, Gebert JT, et al. Rotavirus induces intercellular calcium waves through ADP signaling. Science. 2020;370(6519):eabc3621. pmid:33214249
- 5. Perez-Lopez A, Behnsen J, Nuccio S-P, Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nat Rev Immunol. 2016;16(3):135–48. pmid:26898110
- 6. Tsukita S, Furuse M. Pores in the wall: claudins constitute tight junction strands containing aqueous pores. J Cell Biol. 2000;149(1):13–6. pmid:10747082
- 7. Garcia-Hernandez V, Quiros M, Nusrat A. Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation. Ann N Y Acad Sci. 2017;1397(1):66–79. pmid:28493289
- 8. Ashida H, Ogawa M, Kim M, Mimuro H, Sasakawa C. Bacteria and host interactions in the gut epithelial barrier. Nat Chem Biol. 2011;8(1):36–45. pmid:22173358
- 9. Miao Q, Pan Y, Gong L, Guo L, Wu L, Jing Z, et al. Full genome characterization of a human-porcine reassortment G12P[7] rotavirus and its pathogenicity in piglets. Transbound Emerg Dis. 2022;69(6):3506–17. pmid:36150417
- 10. Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med. 2018;50(8):1–9. pmid:30115904
- 11. Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wölk B, et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature. 2007;446(7137):801–5. pmid:17325668
- 12. Xu R, Lei Y-H, Shi J, Zhou Y-J, Chen Y-W, He Z-J. Effects of lactadherin on plasma D-lactic acid and small intestinal MUC2 and claudin-1 expression levels in rats with rotavirus-induced diarrhea. Exp Ther Med. 2016;11(3):943–50. pmid:26998017
- 13. Colpitts CC, Baumert TF. Claudins in viral infection: from entry to spread. Pflugers Arch. 2017;469(1):27–34. pmid:27885488
- 14. Dickman KG, Hempson SJ, Anderson J, Lippe S, Zhao L, Burakoff R, et al. Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol. 2000;279(4):G757-66. pmid:11005763
- 15. López S, Arias CF. Multistep entry of rotavirus into cells: a Versaillesque dance. Trends Microbiol. 2004;12(6):271–8. pmid:15165605
- 16. Crawford SE, Mukherjee SK, Estes MK, Lawton JA, Shaw AL, Ramig RF, et al. Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol. 2001;75(13):6052–61. pmid:11390607
- 17. Tian J, Sun J, Li D, Wang N, Wang L, Zhang C, et al. Emerging viruses: Cross-species transmission of coronaviruses, filoviruses, henipaviruses, and rotaviruses from bats. Cell Rep. 2022;39(11):110969. pmid:35679864
- 18. Jiang X, Liu Y, Tan M. Histo-blood group antigens as receptors for rotavirus, new understanding on rotavirus epidemiology and vaccine strategy. Emerg Microbes Infect. 2017;6(4):e22. pmid:28400594
- 19. Sun XM, Wang LH, Qi JX, Li DD, Wang MX. Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands. Journal of Virology. 2018;92.
- 20. Liu Y, Huang PW, Tan M, Liu YL, Biesiada J. Rotavirus VP8: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens. J Virol. 2012;86:9899–910.
- 21. Zárate S, Espinosa R, Romero P, Guerrero CA, Arias CF, López S. Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3. Virology. 2000;278(1):50–4. pmid:11112480
- 22. Zárate S, Cuadras MA, Espinosa R, Romero P, Juárez KO, Camacho-Nuez M, et al. Interaction of rotaviruses with Hsc70 during cell entry is mediated by VP5. J Virol. 2003;77(13):7254–60. pmid:12805424
- 23. Zárate S, Espinosa R, Romero P, Méndez E, Arias CF, López S. The VP5 domain of VP4 can mediate attachment of rotaviruses to cells. J Virol. 2000;74(2):593–9. pmid:10623720
- 24. Guerrero CA, Méndez E, Zárate S, Isa P, López S, Arias CF. Integrin alpha(v)beta(3) mediates rotavirus cell entry. Proc Natl Acad Sci U S A. 2000;97(26):14644–9. pmid:11114176
- 25. Graham KL, Halasz P, Tan Y, Hewish MJ, Takada Y, Mackow ER, et al. Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry. J Virol. 2003;77(18):9969–78. pmid:12941907
- 26. Sabara M, Gilchrist JE, Hudson GR, Babiuk LA. Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein. J Virol. 1985;53(1):58–66. pmid:2578197
- 27. Nava P, López S, Arias CF, Islas S, González-Mariscal L. The rotavirus surface protein VP8 modulates the gate and fence function of tight junctions in epithelial cells. J Cell Sci. 2004;117(Pt 23):5509–19. pmid:15494377
- 28. Dormitzer PR, Sun Z-YJ, Wagner G, Harrison SC. The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J. 2002;21(5):885–97. pmid:11867517
- 29. Gualtero DF, Guzmán F, Acosta O, Guerrero CA. Amino acid domains 280-297 of VP6 and 531-554 of VP4 are implicated in heat shock cognate protein hsc70-mediated rotavirus infection. Arch Virol. 2007;152(12):2183–96. pmid:17876681
- 30. Lal-Nag M, Morin PJ. The claudins. Genome Biol. 2009;10(8):235. pmid:19706201
- 31. Gumbiner B. Structure, biochemistry, and assembly of epithelial tight junctions. Am J Physiol. 1987;253(6 Pt 1):C749-58. pmid:3322036
- 32. Rodgers LS, Beam MT, Anderson JM, Fanning AS. Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1. J Cell Sci. 2013;126(Pt 7):1565–75. pmid:23418357
- 33. Fang Z, Yang X, Shang L. Microfluidic-derived montmorillonite composite microparticles for oral codelivery of probiotic biofilm and postbiotics. Sci Adv. 2025;11(12):eadt2131. pmid:40106563
- 34. Zhang Y, Si L, Shu X, Qiu C, Wan X, Li H, et al. Gut microbiota contributes to protection against porcine deltacoronavirus infection in piglets by modulating intestinal barrier and microbiome. Microbiome. 2025;13(1):93. pmid:40189556
- 35. Shifflett DE, Clayburgh DR, Koutsouris A, Turner JR, Hecht GA. Disrupts tight junction barrier function and structure. Lab Invest. 2005;85:1308–24.
- 36. Aoki ST, Settembre EC, Trask SD, Greenberg HB, Harrison SC, Dormitzer PR. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab. Science. 2009;324(5933):1444–7. pmid:19520960
- 37. Hou G, Zeng Q, Matthijnssens J, Greenberg HB, Ding S. Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission. mBio. 2021;12(6):e0320821. pmid:34903043
- 38. Gebert JT, Scribano FJ, Engevik KA, Huleatt EM, Eledge MR, Dorn LE, et al. Viroporin activity is necessary for intercellular calcium signals that contribute to viral pathogenesis. Sci Adv. 2025;11(3):eadq8115. pmid:39823322
- 39. Brosnahan AJ, Brown DR. Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations. Vet Microbiol. 2012;156(3–4):229–37. pmid:22074860
- 40. Wu L, Jing Z, Pan Y, Guo L, Li Z, Feng L, et al. Emergence of a novel pathogenic porcine G1P[7] rotavirus in China. Virology. 2024;598:110185. pmid:39096775
- 41. Kanai Y, Komoto S, Kawagishi T, Nouda R, Nagasawa N, Onishi M, et al. Entirely plasmid-based reverse genetics system for rotaviruses. Proc Natl Acad Sci U S A. 2017;114(9):2349–54. pmid:28137864
- 42. Sánchez-Tacuba L, Feng N, Meade NJ, Mellits KH, Jaïs PH, Yasukawa LL, et al. An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses. J Virol. 2020;94(18):e01294-20. pmid:32759316
- 43. Philip AA, Perry JL, Eaton HE, Shmulevitz M, Hyser JM, Patton JT. Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System. J Virol. 2019;93(24):e01616-19. pmid:31597761
- 44. Jaïs PH, Decroly E, Jacquet E, Le Boulch M, Jaïs A, Jean-Jean O, et al. C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system. Nucleic Acids Res. 2019;47(5):2681–98. pmid:30726994