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. 2025 Oct 17;16(1):2563925. doi: 10.1080/21505594.2025.2563925

Eukaryotic translation elongation factor 1 alpha 1 facilitates coxsackievirus B replication through interacting with PABP, viral protein 3 CD and viral RNA

Shuoxuan Zhao a,b, Yanyan Dong a, Yao Wang a, Hong Yang a, Tian Luan a, Yingxia He a, Lexun Lin c, Yang Chen c, Yanru Fei d, Guangtian Wang c, Yan Wang d, Zhaohua Zhong d, Nan Qi b,e,✉, Wenran Zhao a,✉
PMCID: PMC12539751  PMID: 41108062

ABSTRACT

Viral myocarditis, often caused by Coxsackievirus B (CVB), is the leading cause of dilated cardiomyopathy and heart failure. Despite extensive research, the pathogenesis of CVB infection remains incompletely understood. Our previous study found that Anisomycin inhibits CVB replication by promoting the degradation of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). However, the precise mechanism through which eEF1A1 facilitates the replication of CVB remains to be fully elucidated. Here, we demonstrated that upregulated eEF1A1 is required for CVB3 replication. In vitro transcription and RNA pulldown assay demonstrated that eEF1A1 binds to the 5’ cloverleaf region of CVB3 RNA. We observed that eEF1A1 interacts with double-stranded viral RNA, viral precursor protein 3 CD, and poly(A)-binding protein (PABP), which enhances its interaction with 3 CD. We show that CVB3 upregulates eEF1A1 expression by activating NF-κB. Chromosome-immunoprecipitation assay confirmed that NF-κB p65 binds to the EEF1A1 promoter. Luciferase reporter assays validated that NF-κB up-regulates EEF1A1 transcription. We further showed that eEF1A1 promotes CVB3 replication through interacting with viral RNA, 3 CD, and cellular protein PABP. This study highlights that eEF1A1, which is essential for cellular translation, is manipulated by CVB3 to promote viral replication. These findings suggest that targeting eEF1A1 could be a potential antiviral strategy against CVB infection.

KEYWORDS: Coxsackievirus B, eukaryotic translation elongation factor 1 alpha 1, viral myocarditis, 5’-untranslated region, Poly(A)-binding protein, nuclear factor kappa B

GRAPHICAL ABSTRACT

graphic file with name KVIR_A_2563925_UF0001_OC.jpg

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Introduction

Viral myocarditis, a common disease worldwide, has long been recognized as one of the primary causes of dilated cardiomyopathy and cardiac failure [1,2]. Coxsackievirus B (CVB) is one of the main pathogens of viral myocarditis [3]. Children and young adults are vulnerable to CVB infection [4]. Specific antiviral treatment for viral myocarditis is not available primarily due to that the pathogenesis of CVB infection is still not completely understood [5].

CVB are a group of positive-sensed, single-stranded RNA viruses belonging to the Enterovirus genus in the Picornaviridae family [6]. There are six serotypes of CVB (CVB 1–6), of which three (CVB1, 3 and 5) exhibit pronounced cardiotropism. The enterovirus genome consists of two open reading frames (ORFs) (one big and one short) flanked by untranslated region (UTR) at the 5’and 3’ ends [7]. CVB encodes four capsid proteins (VP1–VP4) and seven non-structural proteins (2Apro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol), which are generated by the autocleavage of the polyprotein translated from the big ORF of the virus [8]. Viral protein 3 CD, the precursor of the mature 3Cpro and 3Dpol with full protease activity, lacks polymerase function [3]. The 5’UTR of enteroviruses contains complex secondary structures organized into two parts, a 5’-end cloverleaf and an internal ribosome entry site (IRES), while 3’UTR contains a short poly(A) tail [9]. The 5’cloverleaf (5’CL) structure is believed to function as a platform to bind viral and cellular proteins to initiate viral RNA replication [10], and the IRES structure enables enterovirus to translate viral RNA in cap-independent manner [11].

Enterovirus RNA replication involves two stages. First, a complementary negative strand RNA is synthesized from the positive-stranded RNA template. Second, daughter RNA is synthesized using the negative-stranded RNA as a template [9]. Subsequently, +ssRNA serves as the genome of the progeny viruses and as the template for viral protein translation. Ultimately, +ssRNA and viral structural proteins are packaged into virions [12].

Accumulating evidence shows that the 5’CL of enterovirus plays a critical role for viral RNA replication. The 5’CL is highly conserved among all species of the Enterovirus genus [12,13]. The sequence and structure conservation of 5’CL implicates its functional importance [9]. Structure study with nuclear magnetic resonance revealed that enterovirus 5’CL contains four highly organized subdomains (A-D). Each of these subdomains folds into distinct stem-loop structure [9]. Subdomain B of 5’CL binds poly(C)-binding protein (PCBP) to help viral RNA circularization through interacting with poly(A)-binding protein (PABP), which binds the 3’ poly(A) tail of viral genome [14,15]. Subdomain D interacts with viral 3 CDpro precursor through its 3C region which allows 3Dpol, the RNA-dependent RNA polymerase (RdRp) to initiate RNA replication [9]. In addition, a stem-loop structure in 2C-coding region of the enterovirus genome also functions as cis-replication element through interacting with viral 3 CD and facilitates the uridylation of viral VPg. The latter (VPg) serves as the primer for the synthesis of +ssRNA [16].

Enteroviruses modulate multiple eukaryotic initiation factors (eIFs) to reprogram host cell translation machinery, thereby ensuring the preferential translation of viral mRNA. Viral infections can induce the phosphorylation of eukaryotic initiation factor 4E (eIF4E), leading to alterations in its activity. In parallel, phosphorylation of eIF4G1—such as through the PKC-Raf-ERK1/2 signaling pathway – is directly linked to the 5’ cap-independent translation of enteroviruses. This mechanism enables the virus to maintain translational activity despite the global suppression of host protein synthesis [17,18]. As a scaffold protein, eukaryotic initiation factor 3b (eIF3b) plays an essential role in translation initiation. Its regulatory modifications, particularly through phosphorylation, can modulate the efficiency of viral translation [19]. During the late phase of infection, enteroviruses modulate the host translational machinery through viral proteins, including the protease 3Cpro, thereby facilitating the preferential utilization of translational resources for viral RNA [20,21].

Our previous research has demonstrated that Anisomycin, an inhibitor of Eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), exhibits effective antiviral effect against CVB3 infection. Anisomycin treatment also significantly relieved CVB3-induced myocarditis. Eukaryotic translation elongation factor 1 α (eEF1A), a GTP-binding protein and a subunit of the eukaryotic translation elongation 1 complex (eEF1), plays important role in translation elongation through delivering aminoacylated tRNAs (aa-tRNAs) to the A site of the ribosome [22]. There are two isoforms of eEF1A, eEF1A1 and eEF1A2, which share more than 90% of amino acid identity [23]. As one of the most abundant proteins in eukaryotic cells, eEF1A makes up 3% in total cellular proteins. Studies have shown that eEF1A [24], beside functioning as translation elongation factor, plays a pivotal role in multiple cellular activities such as nuclear export, protein quality control during translation, organization of cytoskeleton, and apoptosis [24]. Moreover, eEF1A is hijacked by both DNA and RNA viruses, including positive-stranded RNA viruses such as West Nile virus (WNV) [25] and SARS-Cov 2 [26], to promote viral infection [27,28]. However, the precise role played by eEF1A in viral replication remains unclear.

Since eEF1A is involved in the infection of a variety of viruses, we asked whether eEF1A plays a role in CVB infection. Inspired by the potent antiviral effect of plitidepsin against SARS-Cov 2, an eEF1A inhibitor which prevents the release of GTP-bound eEF1A from the A site of ribosome and blocks polypeptide elongation [26], we previously studied the anti-CVB3 effect of anisomycin, a translation inhibitor. We found that anisomycin inhibits CVB3 replication through promoting eEF1A1 degradation [29], supporting the favorable role of eEF1A1 in CVB3 infection. In this study, we investigated the mechanism underlying the involvement of eEF1A1 in CVB3 replication. Our data show that eEF1A1 promotes CVB3 RNA replication through binding to the 5’CL of viral RNA, viral precursor protein 3 CD, and poly(A)-binding protein (PABP). We also reveal that the increased expression of eEF1A1 during CVB3 infection is regulated by NF-&#954B. This study provides novel understanding on how cellular proteins are utilized by CVB, and also suggests that targeting eEF1A1 might be a potential therapeutic strategy for the diseases caused by CVB infection.

Results

eEF1A1 expression is upregulated in CVB3-infected cells

Our previous study demonstrated that anisomycin, which promotes the degradation of eEF1A1, shows anti-CVB3 effect. These findings indicate that eEF1A1 facilitates CVB infection. To reveal the molecular mechanism underlying the involvement of eEF1A1 in viral replication, we began with determining the expression of eEF1A1 in the context of CVB3 infection. As shown in Figure 1, the expression of eEF1A1 was increased at both protein and mRNA levels in the cells infected with CVB3 for 12 h (Figure 1(A-C)), while the expression of eEF1A2, the highly homologous isoform of eEF1A1, remained unchanged (Figure 1(D-F)). These data demonstrated that eEF1A1 expression was upregulated in CVB3-infected cells.

Figure 1.

Figure 1.

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CVB upregulates eEF1A1 expression. (A-F)HeLa cells were infected with CVB3 (MOI = 1) for 12 h. Cells were collected and analyzed by RT-qPCR and immunoblotting. Quantifications of Western blots (C and F) were performed with ImageJ. *p < 0.05; **p < 0.01; ns: non-significant. Data represent means ± s.D. Of n = 3 biologically independent experiments.

CVB-induced myocarditis is primarily common among children and young adults [30]. Moreover, suckling mice have been used as the murine model of CVB3-induced myocarditis due to that newborn mice are susceptible to CVB infection [31]. It has been reported that eEF1A1 is expressed ubiquitously, while the expression of eEF1A2 is tissue-specific. eEF1A1 is also expressed during embryonic development and in postnatal period [23,32]. To understand to correlation between the tissue-specific expression of eEF1A1 and myocarditis, we determined eEF1A1 expression in the myocardium of newborn and adult mice. In agreement with the previous report, eEF1A1 was highly expressed in the myocardium of sucking mice, while eEF1A2 is only expressed in the myocardium of adult mice (Fig. S1). These data suggest that eEF1A1 might play an important role in CVB3-induced myocarditis.

eEF1A1 is required for CVB3 replication

With the observation that eEF1A1 expression was upregulated in CVB3-infected cells, we wondered whether eEF1A1 is required for CVB3 replication. To this end, we determined how eEF1A1 overexpression or knockdown would impact viral replication. HEK293T cells were transfected with pEGFP-eEF1A1 or si-eEF1A1, followed by CVB3 infection. We show that eEF1A1 overexpression increased viral protein 3Dpol (Figure 2A,B) and RNA levels (Figure 2C). However, overexpression of eEF1A2, which is highly homologous to eEF1A1, did not show promoting effect on viral replication (Figure 2D-F). In contrast, eEF1A1 knockdown significantly decreased viral protein 3Dpol (Figure 2G,H) and viral RNA (Figure 2I) levels. Similar to eEF1A2 overexpression, eEF1A2 knockdown did not change viral 3Dpol and RNA (Figure 2J-L) levels. These data indicate that it is eEF1A1, but not eEF1A2, that is required for CVB3 replication.

Figure 2.

Figure 2.

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CVB replication requires eEF1A1 participation. (A-F) HeLa cells were transfected with either pEGFP-eEF1A1 or pEGFP-eEF1A2. Control cells were transfected with empty vector. Cells were infected by CVB3 (MOI = 1) at 24 h of post-transfection. Cells were collected at 12 h of post-infection and analyzed by RT-qPCR (C and F) and immunoblotting (A and D). (G-L) HeLa cells were transfected with either si-eEF1A1 or si-eEF1A2 for 24 h. Control cells were transfected with scramble RNA (si-control). Cells were then infected with CVB3 (MOI = 1) for 12 h and analyzed by RT-qPCR (I and L) and immunoblotting (G and J). Quantifications of Western blots (B, E, H, K) were performed with ImageJ. Ctrl: control; *p < 0.05; **p < 0.01; ****p < 0.0001; ns: non-significant. Data represent means ± s.D. Of n = 3 biologically independent experiments.

eEF1A1 promotes CVB3 genome replication

We next asked how eEF1A1 is used by CVB3 to promote viral replication. Previous studies show that eEF1A1 interacts with the genomic RNA of a variety of viruses [33,34], indicating that eEF1A1 can function as an RNA-binding protein. Therefore, we hypothesized that eEF1A1 supports CVB3 infection by promoting viral genome amplification. To this end, we determined how eEF1A1 would impact the accumulation of viral double-strand RNA (dsRNA), the essential intermediate of viral RNA replication and the hallmark feature of viral replicative capacity [35,36]. Cells were infected with CVB3, viral dsRNA was observed at various time points of post-infection with specific anti-dsRNA antibody J2 (Figure 3). We show that the abundance of viral dsRNA was significantly increased at 12 h and 24 h of post-infection in the cells with eEF1A1 overexpression (Figure 3A-C). In contrast, viral dsRNA abundance was markedly reduced in the cells with eEF1A1 knockdown (Figure 3B-D). These observations indicate that eEF1A1 favors CVB3 genome replication.

Figure 3.

Figure 3.

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eEF1A1 facilitates CVB3 genome replication. (A and C) HeLa cells were transfected with plasmid expressing GFP or GFP-EEF1A1 for 24 h. Cells were then infected with CVB3 (MOI = 1). Cells were fixed at 6, 9, 12, and 24 h of post-infection and subjected to immunofluorescence analysis (A). The fluorescence intensity of J2 (red) and DAPI (blue) was measured and calculated by operetta CLS high content imaging analysis system (C). (B and D) HEK293T were transfected with si-eEF1A1 for 24 h, followed by CVB3 (MOI = 1) infection for 24 h. Cells were fixed and subjected to immunofluorescence assays using anti-dsRNA antibody (J2) and DAPI. The fluorescence intensity of J2 (green) and DAPI (blue) was measured and calculated by operetta CLS high content imaging analysis system. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data represent means ± s.d. of n = 3 biologically independent experiments.

eEF1A1 interacts with the 5 ‘UTR of CVB3 genomic RNA

As an RNA-binding protein, eEF1A1 has been reported to interact with the genomic RNA of some viruses such as West Nile virus (WNV) [25], Dengue virus [34] and Chinese wheat mosaic virus [37]. With the finding that eEF1A1 promotes CVB3 RNA level, we proposed that it might be the result of interaction of eEF1A with viral RNA.

To confirm the interaction of eEF1A1 with viral RNA, RNA-immunoprecipitation (RIP) assay (Figure 4A) and Dual-luciferase reporter assay was performed (Fig. S2). HeLa cells were infected with CVB3 (MOI = 1) for 12 h, and cells were harvested to collect RNA-protein complexes. RIP was carried out using RIP-grade anti-eEF1A1 antibody. IgG served as control. The precipitated RNAs were determined by RT-qPCR. We show that CVB3 sequence was highly enriched in the RNA-protein complex which contains eEF1A1 (Figure 4A), demonstrating that eEF1A1 binds CVB3 RNA.

Figure 4.

Figure 4.

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eEF1A1 interacts with the 5 “UTR of CVB3 genomic RNA. (A) RNA-immunoprecipitation (RIP) assay was performed to determine the interaction between eEF1A1 and viral RNA. HeLa cells were infected with CVB3 (MOI = 1) for 12 h. Cells were harvested to extract protein-RNA complexes. Immunoprecipitation was carried out using RIP grade eEF1A1 antibody. IgG served as the negative control. The precipitated RNA in RIP complex was extracted, and CVB3 RNA was determined by RT-qPCR. (B) diagram of the RNA-protein pull-down assay. pMKS1, the construct containing the cDNA of the entire CVB3 genome, was used as the template in PCR to amplify the various regions of viral genome, 5’UTR, 5’CL, IRES, and 3’UTR. During PCR amplification, T7 promoter sequence was added to upstream of the different segments of CVB3 cDNA. In vitro transcription was carried out to obtain viral RNA corresponding to the various regions of CVB3 genome labeled with dethiobiotin. Cells were infected with CVB3 (MOI = 1) for 12 h. Cell lysate was collected and incubated with in vitro transcribed, biotin-labeled viral RNA and streptavidin-conjugated magnetic beads. The eluted proteins were analyzed by immunoblotting. (C) schematic diagram of CVB genome. (D) immunoblot analysis of the eluted proteins obtained from RNA-pulldown. The poly(A)25 RNA was used as control RNA in RNA-pulldown assay. 5’CL: 5” cloverleaf; IRES: internal ribosome entry site. ****p < 0.0001. Data represent means ± s.d. of n = 3 biologically independent experiments.

To ensure efficient viral replication, a variety of cellular RNA-binding proteins are utilized by CVB3 [38,39]. So far, we demonstrated that eEF1A1 binds to CVB3 RNA, 5’UTR in particular. We next asked which region in the CVB3 5’UTR eEF1A1 interacts with. To this end, the two important cis-acting RNA elements of CVB3 5’UTR, 5’CL and IRES, were amplified from pMKS1, which contains the cDNA of the entire CVB3 genome (Figure 4B,C). As positive and negative control, the 5’UTR and 3’UTR of CVB3 were also obtained, respectively. During PCR amplification, T7 promoter sequence was added to the 5’-end of each of these RNA elements in order to carry out in vitro transcription (Figure 4B,C). Biotin-labeled RNA elements of CVB3, which were obtained by in vitro transcription, were immobilized on streptavidin-conjugated magnetic beads. RNA-pulldown was carried out by incubating the RNA trapped on the magnetic beads with the cell lysate containing total cellular proteins. The pulldown products were analyzed by immunoblotting with anti-eEF1A1 antibody (Figure 4D), demonstrating that eEF1A1 interacts with 5’CL element of CVB3 5’UTR.

eEF1A1 interacts with 3 CD of CVB3 mediated by viral RNA

It has been demonstrated that to initiation of the negative strand RNA synthesis of Poliovirus, viral protease 3 CDpro, the precursor of viral RNA polymerase 3Dpol, and cellular protein PCBP bind viral 5’UTR [38,40]. To further clarify the role of eEF1A1 in CVB3 RNA synthesis, we examined its interaction with the viral protease 3 CDpro. To ensure the abundant expression of 3 CD, a plasmid expressing a mutated 3 CD (3 CDm), which lacks protease activity, was constructed. Cells were transfected with pHA-3 CDm for 24 h, followed by CVB3 infection for 12 h. Viral dsRNA was determined by J2 antibody (Figure 5A, panel a and b). We show that 3 CDm was co-localized with viral dsRNA, demonstrating that 3 CD interacts with viral RNA of CVB3. In mock-infected cells, neglectable co-localization between eEF1A1 and viral 3 CDm was also observed (Figure 5A, panel c). In contrast, intense co-localization between eEF1A1 and 3 CDm was observed in the cells infected with CVB3 (Figure 5A, panel d), demonstrating that the interaction between eEF1A1 and viral 3 CD is mediated by viral RNA.

Figure 5.

Figure 5.

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eEF1A1 interacts with 3 CD of CVB3 mediated by viral dsRNA. (A) HeLa cells were co-transfected with the pHA-3 CDm, in which the active site of 3C protease activity was mutated, and pEGFP-eEF1A1 or empty vector for 48 h. Cells were infected with CVB3 for an additional 12 h. Immunofluorescence observation was conducted using J2 and anti-HA antibody. The images were captured using the ZEISS LSM880 NLO laser confocal microscope. (B) HEK293T cells were transfected with pFlag-eEF1A1 and pHA-3CDm for 24 h. Cells were harvested and subjected to co-IP analysis. (C) HEK293T cells were transfected with pFlag-eEF1A1 and pHA-3 CDm for 24 h. Cells were then infected with CVB3 (MOI = 1) for 12 h. Cells were harvested and subjected to co-IP analysis.

To further confirm the interaction between eEF1A1 and viral 3 CD, IP assays were carried out in the context with or without CVB3 infection (Figure 5B, C). We show that eEF1A1 did not interact with viral 3 CD in the cells without CVB3 infection (Figure 5B). On the other hand, strong interaction between eEF1A1 and viral 3 CD was found in the cells infected with CVB3 (Figure 5C). These observations support that the interaction between eEF1A1 and viral 3 CD is mediated by viral RNA.

eEF1A1 interacts with CVB3 dsRNA and RNA binding protein PABP

Previous studies have shown that the 5’CL element of 5’UTR of enteroviruses is essential for efficient viral RNA replication [12]. To validate the role of eEF1A1 in viral RNA replication, the interaction between eEF1A1 and viral dsRNA was observed with anti-dsRNA antibody J2 in the cells overexpressing EGFP-eEF1A1 (Figure 6A). It has been demonstrated that to initiate viral RNA synthesis and translation, CVB3 genome circularizes. This process is mediated by a variety of cellular proteins including PCBP1/2 and PABP, which bind to 5’UTR and 3’UTR, respectively [41,42]. Thus, we hypothesized that eEF1A1, if it promotes viral RNA synthesis by binding to the 5’CL region of the viral genome, may also interact with RNA-binding proteins involved in viral RNA circularization. Thus, we also observed the co-localization of eEF1A1 and PABP (Figure 6A). Cells were co-transfected with pEGFP-eEF1A1 and pHA-PABP for 48 h, followed by the infection of CVB3 for 12 h. Viral dsRNA was determined with J2 antibody. As anticipated, viral dsRNA was colocalized with PABP (Figure 6A, panel b). Compared to mock-infected cells, the co-localization of eEF1A1 and PABP was dramatically enhanced (Figure 6A, panel c and d). Importantly, it is also obvious that eEF1A1 and PABP also co-localize with viral dsRNA (Figure 6A, panel d). These observations indicate that the enhanced interaction of eEF1A1 and PABP is mediated by the presence of CVB3 dsRNA (Figure 6D).

Figure 6.

Figure 6.

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eEF1A1 interacts with PABP and the dsRNA of CVB3. (A) HeLa cells were co-transfected with HA-PABP and pGFP-eEF1A1 or empty vector for 48 h. Cells were infected with CVB3 for an additional 12 h. Immunofluorescence analysis was performed with J2 and anti-HA antibody. The images were taken with the ZEISS LSM880 NLO laser confocal microscope. (B) HeLa cells were co-transfected with pEGFP-eEF1A1 and HA-PABP for 48 h. Cells were then infected with CVB3 (MOI = 0.5, 1, 5, 10, 20) for 8 h. Immunoprecipitation was performed with anti-HA antibody. (C) quantifications of Western blots were performed with ImageJ. *p < 0.05; **p < 0.01; ns: non-significant. Data represent means ± s.d. of n = 3 biologically independent experiments. (D) the illustration which interprets how eEF1A1 interacts with PABP and CVB3 RNA.

To validate that interaction between eEF1A1 and PABP is mediated by CVB3 RNA, IP assay was carried out in the cells overexpressing EGFP-eEF1A and HA-PAPB, followed by CVB3 infection at increasing MOI (Figure 6B,C). We show that the interaction between eEF1A1 and PABP was markedly enhanced in the cells infected with CVB3 at higher MOI (MOI = 10 or 20). These data support that the interaction between eEF1A1 and PABP is enhanced along with CVB3 replication.

PABP facilitates the interaction of eEF1A1 with viral 3 CD

Since eEF1A1 shows intrinsic binding feature with PABP, we determined how the interaction between eEF1A1 and viral 3 CD is influenced by the PABP. Cells were co-transfected with pFlag-eEF1A1, pHA-3 CDm, and si-PABP, and the interaction between eEF1A1 and 3 CD was determined by co-IP (Fig. S3A and B). We show that the interaction between eEF1A1 and viral 3 CD was indeed hampered in the cells with PABP knockdown, suggesting that PABP facilitates the role played by eEF1A1, which enhances viral RNA replication.

eEF1A1 expression is upregulated by NF-κB in CVB3-infected cells

To reveal the role of eEF1A1 in CVB3 infection, we next investigated how eEF1A1 expression is upregulated during CVB3 infection. To this end, we first determined the mRNA level of eEF1A1 in CVB3-infected cells (Figure 7A-C). We show that the mRNA level of eEF1A1, but not eEF1A2, was increased in time-dependent manner in CVB3-infected cells (Figure 7B,C).

Figure 7.

Figure 7.

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eEF1A1 expression is upregulated by NF-kB in CVB3-infected cells. (A-C) HeLa cells were infected with CVB3 (MOI = 1). Cells were collected at 0, 3, 6, 9, and 24 h of post-infection. The RNA levels of CVB, eEF1A1, and eEF1A2 were detected by RT-qPCR. (D and E) HeLa cells were infected with CVB3 (MOI = 1) for 12 h. Cells were collected and subjected to immunoblotting to determine the expression levels of phospho-NF-κB p65, NF-κB p65, and 3D of CVB3. (F-I) HEK293T cells were transfected with pmCherry-p65 or empty vector (pmCherry-HA) for 24 h. Cells then were infected with CVB3 (MOI = 1) for 12 h. The expression of eEF1A1 was determined by RT-qPCR and immunoblotting. Quantification of the blots was performed with ImageJ. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: non-significant. Data represent means ± s.d. of n = 3 biologically independent experiments.

One of the hallmark features of CVB3 infection is the inflammatory response of the cells, which is primarily induced by the transcription factor NF-&#954B [43]. Moreover, eEF1A1 has been reported to correlate with the increased production of inflammatory cytokine IL-6 [44], while treatment with Didemin B, the eEF1A1 inhibitor, improved the inflammatory infiltration of the liver caused by hepatic toxicity [45]. These data indicate that eEF1A1 is correlated with inflammatory response. Therefore, we hypothesized that the increased expression of eEF1A1 is the result of NF-&#954B activation during CVB3 infection. To this end, the effect of NF-&#954B on eEF1A1 expression was determined in CVB3-infected cells (Figure 7D-I). Our data show that the phosphorylated NF-&#954B p65 was significantly increased in the cells infected with CVB3 (Figure 7D,E), demonstrating that NF-&#954B is activated during CVB3 infection. We further investigated whether eEF1A1 expression is regulated by NF-&#954B during CVB3 infection. To this end, cells were transfected with pmCherry-p65 for 24 h and infected with CVB3 for 12 h. The expression of eEF1A1 was determined at both mRNA (Figure 7F,G) and protein (Figure 7H,I) levels. We show that NF-&#954B p65 overexpression upregulated eEF1A1 expression in CVB3-infected cells (Figure 7F,I). Moreover, p65 overexpression also increased the levels of viral RNA (Figure 7G) and viral protein 3D (Figure 7H). Collectively, these data show that NF-&#954B, which is activated by CVB3 infection, up-regulates eEF1A1 expression.

NF-&#954B binds to the promoter region of EEF1A1 gene and promotes its transcription

We next validated that the transcription of EEF1A1 was regulated by NF-&#954B. To this end, ChIP assay was used to demonstrate the binding of NF-&#954B p65 with the putative promoter region of EEF1A1. Cells were infected with CVB3 for 12 h, and cell lysates were collected. ChIP was carried out with anti-NF-&#954B p65 antibody (Figure 8A). The ChIP products were analyzed with quantitative PCR using the primers covering the different sites in the putative promoter region of EEF1A1 gene (Figure 8B). The information of EEF1A1 gene was obtained from the NCBI database (Gene ID: 1915). The upstream sequence from −2000 bp to + 100 bp (NC_000006.12:c73523032–73520933) was predicted as the promoter region of EEF1A1 gene using UCSC Genome Browser. ChIP results show that NF-_4B p65 binds to the up-stream sequence of EEF1A1 gene within its putative promoter region (Figure 8C).

Figure 8.

Figure 8.

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NF-κB binds to the promoter region of EEF1A1 gene and promotes its transcription. (A) the schematic diagram to show the process of chromatin-immunoprecipitation (ChIP) in order to determine that NF-kB controls eEF1A1 expression. HeLa cells were infected with CVB3 (MOI = 1) for 12 h. Cells were harvested, and the crosslinking of cell lysate was carried out with the addition of formaldehyde. ChIP was carried out with anti-NF-kB p65 antibody through incubating with ChIP grade protein A/G plus agarose. The eluted DNA was analyzed by real-time PCR using primers covering the different regions of the upstream sequence of EEF1A1 gene (from -c73523032 to -c73520933). (B) the diagram which shows the upstream region of EEF1A1 gene (from -c73523032 to -c73520933) and the nine regions which are covered by the PCR products of ChIP. (C) the enrichment of the ChIP PCR products generated by the primers covering the regions upstream of EEF1A1 gene illustrated in (B). Anti-RNA polymerase II was used as positive control. IgG was used as negative control. (D, E) reporter pGL4.11-A1 promoter expressing firefly luciferase which is controlled by the predicted eEF1A1 promoter was generated (E). HEK293T cells were co-transfected with pGL4.11-A1 promoter and the control vector pGL4.74 which constitutively expresses renilla luciferase for 24 h. Cells were harvested and luciferase activity was determined. A1 promoter: eEF1A1 promoter. (F) HEK293T cells were co-transfected with pmcherry-p65, pGL4.11-A1 promoter (or pGL4.11-A1 promoter), and pGL4.74 for 24 h. Renilla luciferase expressed by pGL4.74 was used as internal control. Cells were harvested and luciferase activity was determined. p65: pmCherry-p65. A1 promoter: pGL4.11-A1 promoter. (G) the diagram shows the report pGL4.11-A1 which contains the predicted promoter sequence of EEF1A1 without NF-kB binding site. (H) HEK293T cells were co-transfected with pGL4.74, pmcherry-p65, and pGL4.11-A1 promoter or pGL4.11-A1 promoterΔ for 24 h. Luciferase activity was determined. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns: non-significant. Data represent means ± s.D. Of n = 3 biologically independent experiments.

To confirm that NF-&#954B controls eEF1A1 transcription, a reporter plasmid was generated to express firefly luciferase which was controlled by the predicted EEF1A1 promoter (Figure 8D). Cells were transfected with pGL4.11-A1 promoter which contains the predicted EEF1A1 promoter sequence, and firefly luciferase activity was determined (Figure 8E). Our results show that the predicted EEF1A1 promoter sequence showed promoter activity (Figure 8E).

To further validate that EEF1A1 expression was controlled by NF-&#954B, a reporter construct (pGL4.11-A1 promoterΔ) which is lacking the NF-&#954B binding site was generated (Figure 8G). Cells were co-transfected with pGL4.11-A1 promoter or pGL4.11-A1 promoterΔ, and luciferase activity was determined (Figure 8F,H). We show that without NF-&#954B binding site, the luciferase activity was a significant reduction in the luciferase activity (Figure 8H). Taken together, these data demonstrate that NF-&#954B binds to the promoter region of EEF1A1 and promotes its transcription.

Discussion

CVB is one of the primary etiological pathogens of viral myocarditis and dilated cardiomyopathy, which may progress to in heart failure [46]. Viral myocarditis is also one of the critical causes for sudden cardiac death among young adults [47]. Like many positive-stranded RNA viruses, CVB replication requires the participation of viral RdRp and cellular proteins which assemble into viral replication complex [48]. In this study, we aim to elucidate how cellular protein eEF1A1, one of the most abundantly expressed RNA-binding proteins, is manipulated to promote CVB3 RNA replication. We demonstrated that CVB3 infection upregulates eEF1A1 expression through the activation of NF-&#954B, which binds to the promoter sequence of EEF1A1 and upregulates its expression. eEF1A1 enhances CVB3 RNA replication by interacting with viral RNA at 5’UTR and recruiting viral precursor 3 CDpro and cellular protein PABP. This study identified a novel constituent in the RNA replication complex of CVB3.

It has been reported that, as RNA-binding protein, eEF1A (eEF1A1 or eEF1A2) interacts with the genome of a variety of RNA viruses [49,50]. An early study by Harris et al. showed that Poliovirus 3 CDpro binds viral RNA only in the presence of elongation factor 1 alpha (EF-1a) with molecular weight of 50 kDa (p50) and its N-terminal cleavage fragment p36 [51]. Our previous study demonstrated that upregulated degradation of eEF1A1 (also named as EF1-a1) significantly inhibited CVB3 replication [29]. Based on these data, we proposed that eEF1A1 is the cellular factor EF-1a referred in the study of Harris [51]. Although CVB and Poliovirus share highly similar secondary structure in viral 5’UTR, experimental evidence is still needed to understand the role played by eEF1A1 in CVB infection.

In this study, we first demonstrated that eEF1A1, which is upregulated during CVB3 infection, is required for virus replication. To understand how eEF1A1 promotes virus replication, we first observed whether eEF1A1 affects the abundance of viral dsRNA, which represents viral RNA replication capacity. We show that the abundance of CVB3 dsRNA was significantly increased in the cells overexpressing eEF1A1, while it was obviously decreased by eEF1A1 knockdown. These observations demonstrate that eEF1A1 enhances viral RNA replication.

The interactions between eEF1A1 and viral components carry significant biological implications. During HIV-1 infection, eEF1A1 directly interacts with the viral reverse transcriptase, thereby enhancing the efficiency of reverse transcription and facilitating the conversion of viral genomic RNA into DNA [52,53]. Furthermore, eEF1A1 contributes to the assembly of viral particles through the modulation of the cytoskeletal network [54]. eEF1A1 is found to regulate STAT3/IL-6 pathway in coordination with viral proteins, thereby modulating inflammatory responses [44,55]. The interaction between eEF1A1 and viral components constitutes a key mechanistic basis through which viruses co-opt the host translation machinery to promote their replication. These interactions provide potential avenues for the development of broad-spectrum antiviral strategies that target host factors. Therefore, elucidating the interaction between eEF1A1 and CVB viral components is of significant importance. We demonstrated that eEF1A1 participates in the replication of CVB3 RNA through interacting with 5’ CL and cellular protein PABP and recruiting viral protein 3 CDpro. Upon the infection of picornaviruses, positive-sense RNA genome sequentially functions as mRNA for viral protein synthesis and then as template for viral negative strand RNA synthesis [56]. Early studies on Poliovirus showed that the initiation of viral RNA synthesis, including both negative-strand and positive-strand, requires the assembly of ribonucleoprotein complex containing viral precursors 3 CDpro, cellular protein PCBP1/2 [39] and PABP [38], as well as the first 100 nucleotides of viral genomic RNA, which forms a cloverleaf structure (also named stem-loop I) [49]. Since 3 CDpro itself has no RNA-binding capacity, cellular factors are required to mediate the interaction between viral RNA cloverleaf and 3 CDpro. An early study demonstrated that EF-1a or its cleavage product p36 promotes the binding of 3 CDpro to the cloverleaf of poliovirus RNA [51]. In the present study, we found that eEF1A1 was fully colocalized with 3 CDpro and viral dsRNA in CVB3-infected cells, while no interaction of these two proteins was identified in mock-infected cells. These observations suggest that eEF1A1 mediates the interaction of 3 CDpro to CVB3 RNA.

Our data show that eEF1A1 also interact with PABP, and this interaction facilitates the binding between eEF1A1 and viral 3 CDpro. For the initiation of Poliovirus RNA replication, viral RNA circularizes through a long range of binding between 5’ and 3’UTR mediated by cellular RNA-binding proteins such as PCBP1/2 and PABP [38]. Moreover, mRNA translation, including that of viruses, also requires RNA circularization by joining 5’ and 3’UTR based on protein-protein and RNA-protein interaction [57]. Most mRNA circularization involves the interaction of eIF4E-eIF4G-PABP [58]. During enterovirus infection, viral 2Apro cleaves eIF4G and PABP [59]. The cleavage of these proteins leads to the shutoff of the cap-dependent translation [60], while IRES-mediated translation is not affected [59]. However, the proteolytic cleavage of PABP would also prevent the circularization of virus RNA [61]. Therefore, it remains unclear whether other cellular proteins are involved to establish RNA circularization of CVB and picornaviruses alike.

In this study, interaction of eEF1A1 and PABP was identified at the foci of CVB3 replication, where viral dsRNA was localized (Figure 6D). Moreover, weak interaction between eEF1A1 and PABP was also observed in mock-infected cells, implicating the intrinsic interacting feature between these two protein molecules. Since PABP directly binds the 3’ poly(A) of mRNA, our study indicates that eEF1A1 mediates the circularization and enhances the replication of viral RNA through binding to 5’CL of CVB3 and PABP.

To further elucidate the mechanism underlying the involvement of eEF1A1 in viral replication, we determined whether PABP influences the interaction between eEF1A1 and viral protein 3 CDpro. We show that PABP knockdown moderately inhibited the interaction between eEF1A1 and 3 CDpro, implicating that interacting with PABP favors the recruitment of 3 CDpol to eEF1A1. Similarly, a previous study shows that PABP mediates the genome circularization of CVB3 and facilitates viral translation through binding to viral 3’UTR and PCBP1/2 [41], Based on the previous data and our observations, we propose that eEF1A1 binds PABP and the 5’CL of CVB to facilitate the circularization of viral RNA, and binding PABP enhances the capacity of eEF1A1 to recruit 3 CDpro to initiate viral RNA replication. Enhanced viral RNA replication certainly increases viral translation.

Previous studies found that PABP was cleaved by viral proteases during poliovirus infection, and it is full length PABP that binds viral RNA [60]. Although PABP cleavage in CVB3-infected cells was not investigated in the present study, we proposed that the abundant expression of PABP may conceal the negative influence caused by PABP cleavage. Nonetheless, CVB replication cycle was not interrupted in spite of the cleavage of PABP, suggesting that intact PABP in CVB3-infected cells is sufficient to support viral replication. In agreement with our hypothesis, it has been demonstrated that only half of PABP was cleaved during poliovirus infection [56].

We also found that eEF1A1 was cleaved at D332 during CVB3 infection, generating an N-terminal fragment (Fig. S4 A-C), which presumably corresponds to the p36 of eEF-1αshown previously [51]. However, we found that the overexpression of the N-terminal fragment of eEF1A1 (p36) did not promote CVB3 replication (Fig. S4 D and E), while overexpression of eEF1A1 did. Therefore, whether the cleaved eEF1A1 plays role in CVB3 replication remains to be investigated.

In this study, we show that NF-&#954B upregulates the eEF1A1 expression during CVB3 infection. eEF1A1 protein shares high homology with eEF1A2 protein at the nucleotide level (75%) and amino acid level (96%). It is unclear how CVB3 infection exclusively enhances the expression of eEF1A1, while eEF1A2 expression remains unchanged. We hypothesized that the gene of EEF1A1 and EEF1A2, which is localized in distinct chromosomes, is controlled by different transcription factor. This hypothesis might be partly supported by the fact that the physiological expression pattern of eEF1A1 and eEF1A2 is very different. eEF1A1 is expressed in most cell types, while eEF1A2 is only expressed in neuron and muscle cells (including cardiomyocytes) [23,62]. One of the most prominent features of CVB infection is the rapid and long-lasting inflammation response, which primarily induced by the activation of NF-&#954B [43,63]. Since NF-&#954B controls a wide range of gene expression except inflammatory cytokines [64], we proposed that eEF1A1 might be regulated by NF-&#954B during CVB3 infection. As expected, we found that it is eEF1A1, but not eEF1A2, was upregulated by NF-&#954B p65, during CVB3 infection. Moreover, we demonstrated that NF-&#954B p65, likely in the form of p65/p50 dimer, interacted with the promoter region of EEF1A1.

It remains to be elucidated concerning the unique regulatory mechanism of NF-&#954B on EEF1A1 expression. It is likely that epigenetic alterations such as DNA methylation, histone modification, and nucleosome position are involved. Increasing evidence shows that histone modification plays a critical role in the infection of a variety of viruses such as human immunodeficiency virus (HIV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), and hepatitis B and C viruses (HBV and HCV) [65,66]. It was reported that histone deacetylase (HDAC) 2 activity was increased in CVB3-infected cells [67]. This finding could be a clue that epigenetic alterations are involved in the pathogenesis of CVB infection, although comprehensive investigation is needed.

eEF1A1 is highly expressed across a broad spectrum of cancers, such as hepatocellular carcinoma, prostate cancer, and pancreatic cancer. Its overexpression is significantly associated with adverse clinical outcomes in patients [68]. eEF1A1 inhibitors have exhibited antitumor efficacy in both cellular and animal models [69]. Our previous study demonstrated that eEF1A1 inhibitor anisomycin effectively inhibited CVB3 replication and alleviated viral myocarditis [29], suggesting the potential application for eEF1A1 inhibitor for the treatment of CVB infection. Since eEF1A1 plays critical role in CVB replication, further studies are deserved to find effective antiviral agents targeting eEF1A1 with minimal in vivo side effect.

Taken together, data of this study demonstrated that eEF1A1, which is upregulated by NF-&#954B during CVB3 infection, promotes virus replication through mediating the interaction of 5’ UTR and 3’ poly(A) tail of the virus genome. These data provide further understanding that cellular proteins are manipulated to promote CVB replication. This study also suggests that targeting eEF1A1 could be a potential antiviral strategy.

Materials and methods

Ethics statement

Experiments involving laboratory mice were approved by the Ethics Committee of Harbin Medical University under project license HMUIRB2025003 and carried out in accordance with the Regulation on the Use and Care of Laboratory Animals for Research of Harbin Medical University. The animal experiments in this study complied with the ARRIVE guidelines.

Cell culture

The HeLa and HEK 293 cell line was kindly provided by the Department of Microbiology at Harbin Medical University. HeLa and HEK 293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (CLARK), 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cells were grown at 37°C with 5% CO2 in a incubator. After virus infection, cells were maintained in DMEM containing 2% FBS.

Mice

Animal experiments were approved by Harbin Medical University Ethics Committee. Experimental procedures were carried out in accordance to the regulation on the use of laboratory animal of Harbin Medical University. Balb/c suckling mice were obtained from Harbin Medical University Laboratory Animal Institute (Harbin, China). Mice were kept in pathogen-free setting with a temperature of 25°C and a humidity level of 40–50%. Food and drink were available ad libitum. Select healthy adult and suckling mice as study subjects. After euthanizing the mice with carbon dioxide, myocardial tissues were extracted for detection.

Virus

CVB3 Woodruff was kindly provided by Scrips Institute (San Diego, California) [70]. The virus was propagated in HeLa cells and titrated using 50% tissue culture infectious dose (TCID50) assay using the Reed-Muench formula [71].

Western blot

Cells were collected and lysed with RIPA buffer. Protein concentration was measured using Bradford assay. Cell lysates were separated by PAGE and transferred to nitrocellulose membranes. Blots were incubated with first and second antibodies and visualized with chemiluminescent imaging system (Tanon 5200, China). The antibodies used in this study were listed below: eEF1A1 polyclonal antibody (11402–1-AP, Proteintech, Wuhan, China), eEF1A2 polyclonal antibody (16091–1-AP, Proteintech, Wuhan, China), GAPDH monoclonal antibody (60004–1-AP, Proteintech, Wuhan, China), EGFP tag polyclonal antibody (50430–2-AP, Proteintech, Wuhan, China), DYKDDDDK tag polyclonal antibody (Binds to Flag tag epitope; 20543–1-AP, Proteintech, Wuhan, China), NF-κB p65 polyclonal antibody (10745–1-AP, Proteintech, Wuhan, China), Phospho-NF-κB p65 (Ser536) antibody (3031, Cell Signaling, Boston, MA), MYC tag polyclonal antibody (16286–1-AP, Proteintech, Wuhan, China), PABP polyclonal antibody (10970–1-AP, Proteintech, Wuhan, China), PCBP2 polyclonal antibody (15070–1-AP, Proteintech, Wuhan, China). 3Dpol polyclonal antibody of CVB3 was prepared by the Department of Microbiology, College of Basic Medical Sciences, Harbin Medical University.

Immunofluorescence

HEK293T cells were transfected with pEGFP-eEF1A1, followed by CVB3 infection. Cell culture was harvested and fixed for 15 min with 4% paraformaldehyde. Cells were treated with 0.5% Triton X-100 for 5 min at room temperature to achieve membrane permeabilization. Cells were blocked with 5% bovine serum albumin (BSA) for 30 min. Cells were then treated with the primary antibody at 4°C overnight, followed by the incubation with the corresponding fluorescence-conjugated secondary antibody at room temperature for 1 h. Nuclei were stained with Hoechst 33,258 (Beyotime, Shanghai, China). Cells were visualized with a confocal microscope (LSM880 NLO With Fast Airyscan, Carl Zeiss).

Reverse transcription and quantitative real-time PCR

RNA was extracted with TRIzol. Reverse transcription and quantitative real-time PCR (qRT-PCR) was carried out using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (TansGen, Beijing, China) according to the instruction of the supplier. The thermocycling condition was used as this: denaturation at 94°C for 30 s, followed by 40 cycles of denaturation at 94°C for 5 s, annealing at 55°C for 15 s, and extension at 72°C for 10 s.

Coimmunoprecipitation

Cell lysates were prepared with immunoprecipitation (IP) lysis buffer [50 mM Tris, 150 mM NaCl, 0.1%~0.5% detergent (TritonX-100, Tween 20 or NP40)] containing protease inhibitor PMSF. Incubated with the Anti-DYKDDDDK Tag magnetic beads (B26102, Selleck, Texas, USA) or Anti-HA Tag magnetic beads (B26202, Selleck, Texas, USA) at 4°C for 2 h. The magnetic beads with bound antibody-antigen complex were washed three times with wash buffer (50 mM Tris, 150 mM NaCl, 0.1%~0.5% detergent). The antibody-antigen complex bound on the magnetic beads was dissolved in loading buffer and subjected to SDA-PAGE and immunoblotting.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using Pierce Agarose ChIP Kit (26156, Thermo, IL,USA). Cell lysates were crosslinked with 1% formaldehyde for 10 min. The crosslinking reaction was terminated by the addition of glycine with 5 min incubation. Cell pellets were obtained with the treatment of ice-cold PBS containing Halt Cocktail. Cell pellets were dissolved, and nuclei were collected. The chromatins were digested by MNase (26158, Thermo, IL,USA). IP was performed by incubating the extracted chromatins with ChIP-grade protein A/G plus agarose at 4°C overnight in the presence of 5 μg anti-NF-κB (10745–1-AP, Proteintech, Wuhan, China). Normal rabbit IgG (26158, Thermo, IL,USA) was used as the negative control. Anti-RNA polymerase II (26158, Thermo, IL,USA) was set as for positive control.

RNA immunoprecipitation

RNA Immunoprecipitation (RIP) assay was carried out with Imprint® RNA Immunoprecipitation Kit (Millipore, Billerica, MA). HEK293 cells were infected with CVB3 (MOI = 1) for 12 h. Cells were washed with ice-cold PBS and harvested. Cell lysates were obtained with harsh lysis buffer. Cell lysates were incubated with magnetic beads conjugated with anti-eEF1A1 (11402–1-AP, Proteintech, Wuhan, China) or IgG (negative control). Magnetic beads were washed, and supernatant was removed on a magnetic separator. The magnetic beads were resuspended in IP buffer and incubated at 4°C overnight with rotation. Upon the finish of incubation, magnetic beads were washed five times, and RNA was extracted with TRizol and chloroform, followed by quantitative RT-PCR analysis. Fold enrichment was calculated.

RNA-Protein pulldown

The RNA-protein pulldown was performed with Pierce™ Magnetic RNA-Protein Pull-Down Kit (20164, Thermo, IL, USA). DNA fragments of T7 promoter-5’UTR (designated as T7–5’UTR), T7-IRES, and T7–3’UTR were obtained from the plasmid pMSK1 which contains the cDNA of the entire CVB3 genome sequence by PCR with primers containing T7 promoter sequence. T7–5’CL DNA fragment was synthesized by GENEWIZ. The DNA fragments containing T7 promoter were transcribed with in vitro transcription kit (C11001–1, RIBOBIO, Guangzhou, China). The 3’-end of the transcribed RNA was linked to desulfurobiotin using Pierce RNA 3’Desthiotinylation Kit (20163, Thermo, IL, USA). The biotin-labeled RNA was incubated with magnetic beads conjugated with avidin, followed by the incubation with cell lysate obtained with IP-grade lysis extraction buffer. RNA-protein complex was eluted and analyzed by immunoblotting.

Statistical analysis

All experiments were conducted using small samples of cells and animals. For all experimental data, statistical analyses were performed using t test or ANOVA. The normality of the data was assessed using the Shapiro-Wilk test with GraphPad Prism 10. In all statistical analyses, two-tailed tests were employed, and the significance level was defined as α = 0.05. Experiments using cell culture were carried out with three repeats for each treatment. Animal experiment was carried out with 16 mice in each treatment group. All experiments were repeated at least three times.

Supplementary Material

FIG-S4~1.TIF
KVIR_A_2563925_SM2995.tif (308.5KB, tif)
FIG-S2~1.TIF
KVIR_A_2563925_SM2994.tif (3MB, tif)
FIG-S1~1.TIF
KVIR_A_2563925_SM2993.tif (10.1MB, tif)
FIG-S3~1.TIF
KVIR_A_2563925_SM2992.tif (30.4MB, tif)
supplementary data 250814.doc
KVIR_A_2563925_SM2991.doc (8.7MB, doc)

Acknowledgments

WZ, ZZ and NQ conceived this study and wrote the manuscript. SZ performed the majority of the experiments and analyzed the data. YD and Yao Wang constructed the expression vectors and performed Coimmunoprecipitation. Yao Wang, HY, TL and YH performed part of the RNA-Protein pulldown and immunoblotting. LL, YC, YF participated in the cytotoxicity assay and virus titration assay. GW and Yan Wang provided instruction for the virus experiments. All the authors have read and approved the manuscript.

Funding Statement

The Major Project of Guangzhou National Laboratory and the Guangdong Special Support Plan for Young Top-Notch Talents are institutionally managed funding programs administered by Guangzhou National Laboratory and the Department of Science and Technology of Guangdong Province, respectively.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are openly available in ScienceDB at: https://doi.org/10.57760/sciencedb.19351, reference number 19,351.

Supplementary Information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2563925

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIG-S4~1.TIF
KVIR_A_2563925_SM2995.tif (308.5KB, tif)
FIG-S2~1.TIF
KVIR_A_2563925_SM2994.tif (3MB, tif)
FIG-S1~1.TIF
KVIR_A_2563925_SM2993.tif (10.1MB, tif)
FIG-S3~1.TIF
KVIR_A_2563925_SM2992.tif (30.4MB, tif)
supplementary data 250814.doc
KVIR_A_2563925_SM2991.doc (8.7MB, doc)

Data Availability Statement

The data that support the findings of this study are openly available in ScienceDB at: https://doi.org/10.57760/sciencedb.19351, reference number 19,351.

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