LncSMIM14 Hijacks Rab3a-Mediated Endocytosis to Promote Bovine Viral Diarrhea Virus Replication

TL;DR

This study shows how a host long non-coding RNA, lncSMIM14, helps BVDV replicate by controlling Rab3a-mediated endocytosis in cattle cells.

Contribution

The study identifies lncSMIM14 as a novel host factor that promotes BVDV replication via Rab3a-mediated endocytosis.

Findings

lncSMIM14 overexpression increases BVDV replication, while its knockdown reduces it.

Rab3a is essential for BVDV replication and is regulated by lncSMIM14.

lncSMIM14 and Rab3a promote endocytic vesicle formation after BVDV infection.

Abstract

Bovine Viral Diarrhea Virus (BVDV) poses a significant threat to the global cattle industry, causing substantial economic losses. Long non-coding RNAs (lncRNAs) play crucial regulatory roles in various biological processes, including viral infections. However, the specific lncRNAs influencing BVDV replication remain poorly characterized. This study identified lncSMIM14 as a key host factor upregulated during BVDV infection in MDBK cells. Functional analyses demonstrated that lncSMIM14 overexpression significantly enhanced BVDV replication, evidenced by increased viral mRNA levels, progeny virus titers, cytopathic effects, and dsRNA abundance, while its knockdown exerted the opposite effect. Mechanistically, we revealed that lncSMIM14 specifically targets and positively regulates the expression of the endocytosis-related GTPase Rab3a. Importantly, Rab3a itself was shown to be essential for efficient BVDV replication, as its overexpression promoted viral replication, and its knockdown inhibited it. Furthermore, Rab3a co-localized with key endocytic regulators Rab5a and Rab7a, and both lncSMIM14 overexpression and Rab3a overexpression promoted the formation of endocytic vesicles, particularly post-BVDV infection. Our findings unveil a novel mechanism wherein BVDV exploits the host lncRNA lncSMIM14 to hijack Rab3a-mediated endocytosis, facilitating its own replication. This study identifies the lncSMIM14-Rab3a axis as a critical host pathway subverted by BVDV, providing new potential targets for antiviral intervention.

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1. Introduction

Bovine viral diarrhea virus (BVDV) is the primary etiological agent responsible for viral diarrhea and mucosal disease in cattle, conditions collectively referred to as bovine viral diarrhea–mucosal disease (BVD/MD) [1]. As a member of the genus Pestivirus within the family Flaviviridae, BVDV infection manifests clinically through inflammatory diarrhea, enteritis, and mucosal necrosis [2]. BVDV represents a significant global threat to livestock, imposing a substantial economic burden on the cattle industry worldwide [3]. Although BVDV has evolved sophisticated strategies to establish and maintain host infection, the underlying molecular pathogenic mechanisms remain only partially understood.

Long non-coding RNAs (lncRNAs), once dismissed as mere “transcriptional noise,” are now defined as transcripts exceeding 200 nucleotides in length that lack protein-coding potential [4]. In recent years, lncRNAs have emerged as pivotal regulators of diverse biological processes, including cell proliferation, differentiation, and development [5,6]. Accumulating evidence underscores the essential roles of lncRNAs in the initiation, modulation, and progression of various viral infections, such as influenza [7], hepatitis [8], coronaviruses [9], and human immunodeficiency virus [10]. Consequently, identifying key host lncRNAs is crucial for elucidating their functional roles, infection-related targets, and the complex regulatory networks they govern [11].

Endocytosis is a fundamental cellular process for internalizing membrane lipids, proteins, and extracellular material, thereby playing a vital role in modulating intracellular signaling cascades [11,12]. Pathogens frequently hijack endocytic pathways to facilitate their entry and internalization within host cells [13]. Central to these processes are Rab proteins, a family of small GTP-binding proteins that serve as master regulators of intracellular membrane trafficking [14]. The mammalian genome encodes over 60 Rab genes, among which Rab3a traditionally acts as a molecular switch—binding GTP and GDP—to regulate synaptic vesicle exocytosis and hormone secretion [15]. However, emerging evidence suggests that Rab GTPases possess remarkable functional versatility, coordinating multiple stages of vesicular trafficking through “Rab cascades” or cross-talk between endocytic and exocytic pathways [16]. Other Rab family members, such as Rab5a, contribute to signal transduction, receptor downregulation, and the phagocytosis of pathogens [17], with studies indicating that Rab5a upregulation can suppress antiviral immune responses in epithelial cells [18]. Similarly, Rab7a plays a critical role in the intracellular transport of viruses [19]. Primarily localized to late endosomes, Rab7a regulates viral spread [20] and exerts bidirectional regulatory functions throughout the viral replication cycle, facilitating host–pathogen interactions that promote infection [21,22].

Despite the scarcity of research regarding the interplay between lncRNAs and BVDV replication, our study provides novel insights into this relationship. Through lncRNA sequencing (accession number: [to be provided]), we discovered that lncSMIM14 is significantly upregulated during BVDV infection. We identified lncSMIM14 as a key proviral lncRNA that is strongly induced by BVDV and essential for efficient viral replication. Mechanistically, lncSMIM14 enhances the expression of Rab3a, which in turn co-localizes with key endocytic regulators Rab5a and Rab7a to facilitate the formation of endocytic vesicles. Our findings reveal a novel mechanism wherein BVDV exploits the host lncRNA lncSMIM14 to hijack Rab3a-mediated endocytosis, thereby optimizing its own replication. This study identifies the lncSMIM14-Rab3a axis as a critical host pathway subverted by BVDV, offering a new molecular perspective on BVDV intracellular replication and providing potential targets for future antiviral interventions.

2. Results

2.1. The Expression of lncSMIM14 Is Significantly Elevated in MDBK Cells After BVDV Infection

To explore the involvement of long non-coding RNAs (lncRNAs) in bovine viral diarrhea virus (BVDV) infection, we performed RNA sequencing (RNA-seq) to identify differentially expressed lncRNAs in MDBK cells at 6, 12 and 24 h post-infection (hpi) (Figure 1A). The top 13 upregulated lncRNAs identified by RNA-seq were further validated using quantitative real-time PCR (qRT-PCR). The results confirmed that the expression patterns of these 13 candidates were highly consistent with the sequencing data (Figure 1B). Notably, lncSMIM14, lncWIPF3, and lncGPC6 exhibited significant upregulated across all three time points (Figure 1C). Furthermore, Gene Ontology (GO) and KEGG pathway enrichment analyses revealed that these differentially expressed lncRNAs were predominantly associated with the endocytosis pathway (Figure 1D). We used lncLocator to predict the subcellular localizations of lncSMIM14, lncWIPF3, and lncGPC6, which suggested that lncSMIM14 and lncGPC6 are primarily localized in the cytoplasm, while lncWIPF3 is situated within the cytosol (Table 1).

Given that lncSMIM14 expression demonstrated a stable, time-dependent increase throughout the course of BVDV infection (Figure 1C), it was selected as the primary candidate for further functional investigation.

2.2. LncSMIM14 Influences Intracellular Replication of BVDV

To elucidate the role of lncSMIM14 in intracellular BVDV replication, we first performed qRT-PCR to assess its expression in MDBK cells following BVDV infection. The results revealed that lncSMIM14 expression increased in a time-dependent manner during the course of infection (Figure 2A). Subsequently, we established lncSMIM14-overexpressing (lncSMIM14 + MDBK) cells (Figure 2B,C) and lncSMIM14-knockdown (lncSMIM14 − MDBK) cells to evaluate its functional impact. LncSMIM14-overexpressing cells exhibited significantly elevated BVDV mRNA levels, higher progeny virus titers, more pronounced cytopathic effects (CPE), and enhanced fluorescence intensity and broader distribution of BVDV dsRNA (Figure 2D–G). Conversely, lncSMIM14 knockdown led to a marked reduction in BVDV mRNA levels, lower progeny virus titers, attenuated CPE, and diminished dsRNA fluorescence (Figure 3B–E). Collectively, our findings demonstrate that lncSMIM14 acts as a proviral factor that promotes intracellular BVDV replication.

2.3. LncSMIM14 Targets and Regulates the Endocytosis-Related Protein Rab3a

To explore the molecular mechanism underlying lncSMIM14-mediated BVDV replication, we first predicted its potential target genes through bioinformatics analysis (Figure 4A). Subsequently, the binding probabilities between lncSMIM14 and candidate proteins were assessed using the RNA–protein interaction prediction (RPISeq) tool based on Random Forest (RF) and Support Vector Machine (SVM) scores. This analysis identified Rab3a, an endocytosis-related GTPase, as a high-confidence binding target of lncSMIM14 (Figure 4B). To determine whether lncSMIM14 modulates Rab3a expression, MDBK cells were transfected with Myc, Myc–lncSMIM14, shNC, or sh–lncSMIM14. Overexpression of lncSMIM14 significantly upregulated both Rab3a mRNA and protein levels, whereas its knockdown resulted in a marked reduction in Rab3a expression (Figure 4C–F). Following BVDV infection of these modified cells, Rab3a protein levels showed a gradual increase in lncSMIM14-overexpressing cells, peaking at 24–36 h post-infection (hpi) before slightly declining at 48 hpi. Conversely, Rab3a levels progressively decreased in lncSMIM14-knockdown cells post-infection (Figure 4G–H). Finally, RNA immunoprecipitation (RIP) assays demonstrated that Strep–Rab3a pecifically enriched lncSMIM14 compared to the control group (Figure 4I). Taken together, these results confirm a direct regulatory interaction between lncSMIM14 and Rab3a.

2.4. The Endocytosis-Related Protein Rab3a Promotes Intracellular Replication of BVDV

To evaluate the involvement of Rab3a in BVDV replication, we first monitored its expression levels in MDBK cells following BVDV infection. qRT-PCR analysis revealed that Rab3a mRNA levels were significantly upregulated at 48 h post-infection (hpi) (p < 0.05) (Figure 5A). These findings were corroborated by Western blot analysis, which showed a consistent trend in Rab3a protein expression (Figure 5B). Subsequently, we established Rab3a-overexpressing MDBK cells (mCherry–Rab3a) (Figure 5C–G). Upon BVDV infection, these cells exhibited significantly elevated BVDV mRNA levels, increased progeny virus titers, more pronounced cytopathic effects (CPE), and enhanced fluorescence intensity and broader distribution of BVDV dsRNA (Figure 5H). Conversely, Rab3a-knockdown cells (Rab3a–sg1) (Figure 6A–D) displayed markedly reduced BVDV mRNA levels, lower progeny virus titers, attenuated CPE, and diminished dsRNA fluorescence (Figure 6E–G). Collectively, these results demonstrate that Rab3a acts as a critical host factor that facilitates the intracellular replication of BVDV.

2.5. Rab3a Co-Localizes with Key Endocytic Regulators Rab5a and Rab7a and Promotes Endocytic Vesicle Formation

To further delineate the role of Rab3a in BVDV replication, we examined its spatial relationship with the key endocytic GTPases, Rab5a and Rab7a. Following the co-transfection of mCherry-tagged Rab3a with either mCherry-tagged Rab5a or Rab7a plasmids, confocal laser scanning microscopy revealed distinct spatial co-localization among these proteins in HEK-293T cells (Figure 7A), confirming the close association of Rab3a with the endocytic pathway.

We next investigated whether Rab3a modulates endocytic vesicle formation during BVDV infection. Cells transfected with mCherry–Rab3a or Myc–lncSMIM14 were subjected to mock or BVDV infection, and endocytic vesicles were visualized using transmission electron microscopy (TEM). While BVDV infection alone significantly increased the number of endocytic vesicles compared to the mock control, the overexpression of either Rab3a or lncSMIM14 further stimulated intracellular endocytic vesicle formation in the context of infection (Figure 7B).

Collectively, these findings indicate that lncSMIM14 acts directly on Rab3a, which in turn coordinates with Rab5a and Rab7a to promote endosome maturation and formation, thereby facilitating efficient intracellular BVDV replication.

3. Discussion

Endocytosis is a fundamental process for numerous intracellular signaling pathways [23]. Pathogens frequently exploit these pathways to facilitate host cell entry, often leading to severe or persistent infections [24]. In this study, we identified lncSMIM14—a host long non-coding RNA significantly induced by BVDV in MDBK cells—as a novel proviral factor that augments intracellular BVDV replication by modulating host endocytic dynamics. Mechanistically, lncSMIM14 specifically interacts with Rab3a, which in turn orchestrates with key endocytic regulators Rab5a and Rab7a to promote endosome formation.

Our findings are concordant with the expanding paradigm implicating lncRNAs in the complex orchestration of viral life cycles. For instance, lncRNA-CMPK2 and lncRNA NRAV have been shown to promote the replication of HCV and IAV, respectively [25,26], while lncRNA GAS5 acts as an antiviral effector against HCV [27]. In the context of BVDV, previous research suggested that host lncRNAs modulate replication primarily through apoptotic pathways [28]. Our study expands this landscape by demonstrating that lncSMIM14 facilitates the assembly or coordination of endocytosis-related proteins (Rab3a, Rab5a, and Rab7a), thereby catalyzing endocytic vesicle formation. While our current data do not fully delineate the boundaries between initial viral entry and post-entry trafficking, the overall enhancement of the endocytic machinery clearly bolsters the broader BVDV life cycle.

A pivotal discovery of this study is the positive regulatory role of Rab3a in BVDV replication. Traditionally associated with exocytosis and synaptic vesicle trafficking, Rab3a is shown here to be “hijacked” by BVDV via lncSMIM14 induction. By directly binding to Rab3a, lncSMIM14 leverages Rab3a’s spatial association with Rab5a and Rab7a to accelerate endosome maturation, thereby optimizing early viral transport. This non-canonical function is supported by the observed co-localization of Rab3a with endocytic markers (Figure 7A) and is consistent with the “fluid” functional boundaries of Rab proteins in non-neuronal cells. Indeed, the involvement of the Rab3 family in endocytosis is corroborated by findings that Rab3D associates with early endosomes in specific epithelial models [29,30].

The roles of Rab5a and Rab7a in viral infections are multifaceted, ranging from inhibiting rabies virus nucleoprotein expression to facilitating HIV-1 transmission and HBV endocytosis [31,32]. Our observation that Rab3a co-localizes with these proteins suggests a sophisticated reciprocal regulatory relationship among BVDV, lncSMIM14, and the Rab3a-mediated pathway. However, several questions remain. Further studies are required to elucidate the precise interaction sites between lncSMIM14 and Rab3a, as well as the specific molecular triggers for endosome production. Additionally, while we focus on the overall promotion of replication, future research employing direct viral entry assays (e.g., virus attachment and internalization tracking) will be essential to dissect whether the lncSMIM14–Rab3a axis primarily triggers initial entry or governs subsequent intracellular trafficking.

In summary, we describe a novel molecular mechanism wherein BVDV induces host lncSMIM14 to subvert the Rab3a-mediated endocytic pathway, ultimately facilitating efficient viral replication. This lncSMIM14–Rab3a axis represents a potential target for future antiviral interventions against Pestivirus infections.

4. Materials and Methods

4.1. Cell Culture and Virus

Madin–Darby bovine kidney (MDBK) cells and human embryonic kidney (HEK-293T) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NE, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO_2_. Transfections were performed using a DNA transfection reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. The bovine viral diarrhea virus (BVDV) TC strain was preserved in our laboratory (Xinjiang University, Urumqi, China).

4.2. Plasmids

The plasmids pLVX-Myc-IRES-Puro, pLVX-Myc-IRES-Puro-SMIM14, pLKO.1-copGFP-PURO-SMIM14-1/-2/-3/-4, pLKO.1-copGFP-PURO-NC, pCDH-CMV-MCS-EF1-Blast-mCherry-puro, pCDH-CMV-MCS-EF1-Blast-mCherry-Rab3a-puro, pCDH-CMV-MCS-EF1-Blast-mCherry-Rab5a-puro, and pCDH-CMV-MCS-EF1-Blast-mCherry-Rab7a-puro were purchased from QIAGEN. Plasmids pLentiCRISPR v2-Rab3a-1-sgRNA, pLentiCRISPR v2-Rab3a-2-sgRNA, and pLentiCRISPR v2-Scramble-sgRNA were synthesized by GenScript (Nanjing, China). Plasmids psPAX2 and pMD2.G were obtained from Hunan Fenghui Biotechnology Co., Ltd., Hunan, China. Competent Escherichia coli Stbl3 cells were prepared and stored in our laboratory.

4.3. LncRNA Sequencing Analysis

Total RNA was extracted using TRIzol reagent (TIANGEN, Beijing, China) according to the manufacturer’s protocol. RNA quality and integrity were assessed by agarose gel electrophoresis to ensure suitability for library construction. Sequencing libraries were prepared with the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA) and sequenced on the Illumina HiSeq 4000 platform by Novogene Company, Ltd. (Beijing, China). Raw reads were quality-filtered to remove low-quality sequences. Differentially expressed lncRNAs and mRNAs (p-value < 0.05) were subjected to GO term enrichment (GOseq) and KEGG pathway analyses. Selected transcripts were validated by qRT-PCR to confirm concordance with RNA-Seq results.

4.4. Bioinformatics Analysis and Target Prediction

Bioinformatics analysis and target prediction: The potential target genes of lncSMIM14 were predicted using the LncTar and RNAplex databases to identify potential RNA-RNA interactions. To further evaluate the binding probability between lncSMIM14 and candidate proteins, we employed the RPISeq (RNA-Protein Interaction Prediction) web server. Predictions were based on two machine learning algorithms: Random Forest (RF) and Support Vector Machine (SVM). A threshold of >0.5 for both RF and SVM scores was used to identify high-confidence interactions. Among the top-ranked candidates, several proteins involved in vesicular trafficking and immune regulation were identified, including Rab3a, Rab11, and Annexin A2.

4.5. Virus Infection

MDBK cells were seeded at 5 × 10^4^ cells/dish in 10 cm dishes. When cells reached ~70% confluence, the medium was replaced with serum-free DMEM, and BVDV was inoculated at 1000 TCID_50_/mL. After 2 h adsorption, the inoculum was replaced with DMEM containing 2% horse serum. Cells were washed with PBS and collected at 6, 12, and 24 h post-infection (hpi).

4.6. Western Blotting

Cells were lysed in RIPA buffer (Roche Diagnostics, Basel, Switzerland) containing protease inhibitors. Protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA), and blocked with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA). Membranes were incubated with primary antibodies against dsRNA (J2, Scicons, Szirák, Hungary), GAPDH (Wuhan Sanying Biotechnology, Wuhan, China), or Rab3a (Wuhan Sanying Biotechnology, Wuhan, China), followed by HRP-conjugated secondary antibodies (Wuhan Sanying Biotechnology, Wuhan, China). Blots were visualized using BeyoECL Plus (Biosharp Biotechnology, Hefei, China).

4.7. Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted using TRNzol reagent (TIANGEN Biotech, Beijing, China) and reverse transcribed with the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). qRT-PCR was performed using SYBR Green qPCR Master Mix (GenStar, Beijing, China) on a QuantStudio system (Applied Biosystems, Foster City, CA, USA).

4.8. Lentiviral Packaging

HEK-293T cells were seeded in 10 cm dishes one day prior to transfection to reach 80–90% confluence. Packaging was performed by co-transfecting the transfer plasmid (e.g., pLV-EF1α-GFP, 10 μg), psPAX2 (7.5 μg), and pMD2.G (2.5 μg) using polyethyleneimine (PEI). Medium was replaced after 6 h. Viral supernatants were harvested at 48 h and 72 h post-transfection, clarified by centrifugation (3000× g, 10 min, 4 °C), concentrated by ultracentrifugation (70,000× g, 2 h, 4 °C), and resuspended in PBS. Viral titers were determined by fluorescence reporter assay (TU/mL).

4.9. Immunofluorescence Microscopy

MDBK cells on coverslips were fixed in 4% paraformaldehyde (15 min), permeabilized with 0.25% Triton X-100 (5 min), and blocked with 5% BSA. Cells were incubated with primary antibodies, followed by fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired using a Zeiss LSM 780 confocal microscope.

4.10. RNA Immunoprecipitation (RIP)

Cells were crosslinked with 2% formaldehyde and lysed in RIPA buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% NP-40; 0.5% sodium deoxycholate; 0.5 mM DTT; 1 mM PMSF). Lysates were incubated with antibodies and Protein A/G magnetic beads (Thermo Fisher Scientific) at 4 °C. Beads were washed sequentially with RIPA buffer and 1 M RIPA buffer, then treated with proteinase K. RNA was extracted using TRIzol and analyzed by qRT-PCR.

4.11. Statistical Analysis

All experiments were independently repeated at least three times. Data are presented as mean ± SEM. Statistical significance was determined using the Mann–Whitney U test in SPSS 19.0 software. Differences were considered significant at p < 0.05 (p < 0.05; * p < 0.01; ns, not significant).

5. Conclusions

In conclusion, this study provides the first evidence that the long non-coding RNA lncSMIM14 acts as a pivotal modulator of Bovine Viral Diarrhea Virus (BVDV) replication by orchestrating the endocytic pathway. Our findings demonstrate that the BVDV-induced upregulation of lncSMIM14 augments viral replication, a process coupled with the elevated expression of the GTPase Rab3a. Conversely, lncSMIM14 knockdown attenuates BVDV replication and concomitantly reduces Rab3a levels, suggesting that Rab3a expression is a critical determinant of BVDV replication dynamics. Furthermore, the spatial co-localization of Rab3a with key endocytic regulators Rab5a and Rab7a supports a mechanistic model in which the lncSMIM14–Rab3a axis facilitates viral entry or intracellular trafficking via the endocytic pathway. Overall, these insights expand our understanding of host lncRNA functions during Pestivirus infection and identify the lncSMIM14–Rab3a axis as a promising antiviral target for the development of future therapeutic strategies.