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

LncSMIM14 Hijacks Rab3a-Mediated Endocytosis to Promote Bovine Viral Diarrhea Virus Replication | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article LncSMIM14 Hijacks Rab3a-Mediated Endocytosis to Promote Bovine Viral Diarrhea Virus Replication Zhiran Shao, Siqi Ma, FengSiyue Gao, Yang Lou, Rulong Chen, Xinyi Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7956623/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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. Bovine Viral Diarrhea Virus Long non-coding RNA lncSMIM14 Rab3a Viral eplication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Novelty statement We identified lncSMIM14 as a previously unknown host factor that significantly promotes BVDV replication. lncSMIM14 enhances viral propagation by upregulating Rab3a, an endocytosis-related GTPase essential for efficient infection. The lncSMIM14–Rab3a axis facilitates endocytic vesicle formation, revealing a new mechanism exploited by BVDV and potential antiviral targets. Introduction Bovine viral diarrhea virus (BVDV) is the main etiological agent causing viral diarrhea and mucosal disease in cattle, collectively known as bovine viral diarrhea–mucosal disease (BVD/MD or BVD-MD [1] . It belongs to the genus Pestivirus within the family Flaviviridae, and its major clinical manifestations include inflammatory diarrhea, enteritis, and mucosal necrosis [2] . BVDV poses a global threat to cattle populations and imposes a significant economic burden, resulting in substantial losses to the cattle industry worldwide [3] . To date, BVDV has evolved multiple strategies to establish infection in its host; however, the underlying pathogenic mechanisms remain incompletely understood. Long non-coding RNAs (lncRNAs), once dismissed as 'transcriptional noise', are defined as transcripts longer than 200 nucleotides that lack protein-coding capabilities [4] . In recent years, lncRNAs have attracted significant attention as emerging and potentially crucial regulators of biological processes, including cell proliferation, differentiation, and development [5-6] . Accumulating evidence underscores the vital role of lncRNAs in the initiation, modulation, and progression of various viral infections, including influenza [7] , hepatitis [8] , coronaviruses [9] , and human immunodeficiency virus [10] . Therefore, identifying key lncRNAs will enable targeted analysis of their functional roles, thereby elucidating their infection-related targets and regulatory mechanisms [11] . Endocytosis is the process through which cells internalize membrane lipids, membrane proteins, and extracellular material, thereby playing a vital role in regulating numerous intracellular signaling cascades [11-12] . Pathogens often exploit the endocytic pathway to facilitate their internalization within host cells [13] . Rab proteins, a family of small GTP-binding proteins involved in vesicular transport, are crucial for intracellular membrane vesicle trafficking [14] . he mammalian genome contains over 60 Rab genes [15] among which Rab3a acts as a molecular switch by binding GTP and GDP, thus participating in the regulation of synaptic vesicle exocytosis and hormone secretion [15] . Rab5a, another member of the Rab family, contributes to signal transduction, receptor downregulation, and the phagocytosis of pathogens [16] . Studies indicate that Rab5a upregulation can inhibit the antiviral immune response in epithelial cells [17] . Similarly, Rab7a, another small GTP-binding protein, plays a significant role in the intracellular transport of viruses [18] . Located primarily in late endosomes, Rab7a regulates viral spread [19] and serves a bidirectional regulatory function throughout the viral replication cycle, participating in host-pathogen interactions to promote viral infection [20-21] . Despite limited research on the relationship between lncRNA and BVDV replication, our study provides significant insights. We conducted lncRNA sequencing (accession number: [to be provided]) and discovered that lncSMIM14 was significantly upregulated. We identified lncSMIM14 as an lncRNA that is significantly induced by BVDV and plays a crucial role in viral replication. LncSMIM14 enhances the expression of Rab3a, thereby promoting BVDV replication. Moreover, Rab3a co-localizes with key endocytic regulatory factors Rab5a and Rab7a, facilitating the formation of endocytic vesicles, particularly following BVDV infection. Our findings reveal a novel mechanism through which BVDV exploits the host lncRNA lncSMIM14 to hijack Rab3a-mediated endocytosis, thereby enhancing viral replication. This study identifies the lncSMIM14-Rab3a axis as a critical host pathway subverted by BVDV, providing a molecular mechanism that influences BVDV intracellular replication and offering new potential targets for antiviral intervention. Materials and Methods 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) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. Transfections were performed using a DNA transfection reagent (Roche) according to the manufacturer’s protocol. The bovine viral diarrhea virus (BVDV) TC strain was preserved in our laboratory. 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. Competent Escherichia coli Stbl3 cells were prepared and stored in our laboratory. 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) and sequenced on the Illumina HiSeq 4000 platform by Zhongke Genome Company, Ltd. (Shanghai, China). Raw reads were quality-filtered to remove low-quality sequences. Differentially expressed lncRNAs and mRNAs (q-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. Virus infection MDBK cells were seeded at 5 × 10⁴ cells/dish in 10 cm dishes. When cells reached ~70% confluence, the medium was replaced with serum-free DMEM, and BVDV was inoculated at 1,000 TCID₅₀/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). Western blotting Cells were lysed in RIPA buffer (Roche) containing protease inhibitors. Protein concentration was determined using a Bio‑Rad protein assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes (GE Healthcare), and blocked with 5% skim milk. Membranes were incubated with primary antibodies against dsRNA (J2, Scicons, Hungary), GAPDH (Wuhan Sanying Biotechnology), or Rab3a (Wuhan Sanying Biotechnology), followed by HRP‑conjugated secondary antibodies (Wuhan Sanying Biotechnology). Blots were visualized using BeyoECL Plus (Biosharp Biotechnology). Quantitative real-time PCR (qRT-PCR) Total RNA was extracted using TRNzol reagent (TIANGEN) and reverse transcribed with the ReverTra Ace qPCR RT Kit (Toyobo). qRT-PCR was performed using SYBR Green qPCR Master Mix (GenStar) on a QuantStudio system (Applied Biosystems). GAPDH served as an internal control. Primer sequences are listed in the Supplementary Table. 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 (3,000 × 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). 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. 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. 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). RESULTS The expression of lncSMIM14 is significantly elevated in MDBK cells after BVDV infection. The expression of lncSMIM14 is significantly elevated in MDBK cells following BVDV infection. To investigate the involvement of long non-coding RNAs (lncRNAs) in bovine viral diarrhea virus (BVDV) infection, RNA sequencing (RNA-seq) was conducted to identify differentially expressed lncRNAs in MDBK cells infected with BVDV for 6 or 12 hours (Figure 1A). The top 13 upregulated lncRNAs responsive to BVDV infection were further analyzed using quantitative PCR (qPCR). Our data showed that the expression patterns of these 13 upregulated lncRNAs in MDBK cells were consistent with the RNA-seq results following BVDV infection (Figure 1B). KEGG analysis revealed that lncSMIM14, lncWIPF3, and lncGPC6 were upregulated at 6, 12, and 24 hours post-BVDV infection, respectively (Figure 1C). Furthermore, Gene Ontology (GO) and KEGG pathway enrichment analyses indicated that the differentially expressed lncRNAs were predominantly enriched in the endocytosis pathway (Figure 1D). We used Lnclocator to predict the subcellular localizations of lncSMIM14, lncWIPF3, and lncGPC6, and found that lncSMIM14 and lncGPC6 were localized to the cytoplasm, whereas lncWIPF3 was localized to the cytoplasmic matrix (Table 1). Given that lncSMIM14 expression exhibited a stable positive correlation with the duration of BVDV infection in MDBK cells (Figure 1C), further investigation of lncSMIM14 was warranted LncSMIM14 influences intracellular replication of BVDV. To elucidate the role of lncSMIM14 in intracellular BVDV replication, we performed qRT-PCR to assess lncSMIM14 expression in MDBK cells following BVDV infection. Our results showed that lncSMIM14 expression increased in a time-dependent manner during BVDV infection (Figure 2A). Subsequently, we generated lncSMIM14-overexpressing (lncSMIM14+MDBK) cells (Figures 2B–C) as well as lncSMIM14-knockdown (lncSMIM14−MDBK) cells. LncSMIM14-overexpressing cells exhibited increased BVDV mRNA levels, higher progeny virus titers, more cytopathic effects (CPE), and enhanced fluorescence intensity and distribution of BVDV dsRNA (Figures 2D–G). We further knocked down lncSMIM14 to verify its functional role (Figure 3A). LncSMIM14-knockdown cells exhibited reduced BVDV mRNA levels, lower progeny virus titers, fewer cytopathic effects (CPE), and diminished fluorescence intensity and distribution of BVDV dsRNA (Figures 3B–E). In summary, our findings demonstrate that lncSMIM14 promotes intracellular BVDV replication. LncSMIM14 targets and regulates the endocytosis-related protein Rab3a. To further investigate the mechanism by which lncSMIM14 affects BVDV replication, we predicted its potential target genes using bioinformatics analysis (Figure 4A). Subsequently, RNA–protein interaction prediction (RPISeq) was performed to evaluate the binding probability between lncSMIM14 and candidate proteins using random forest (RF) and support vector machine (SVM) scores. This analysis identified Rab3a, an endocytosis-related protein, as a potential binding target of lncSMIM14 (Figure 4B). To examine whether lncSMIM14 regulates Rab3a expression, MDBK cells were transfected with Myc, Myc–lncSMIM14, shNC, or sh–lncSMIM14. Overexpression of lncSMIM14 increased both Rab3a mRNA and protein levels, whereas lncSMIM14 knockdown reduced Rab3a expression (Figures 4C–F). We next infected the transfected cells with BVDV. After BVDV infection, Rab3a protein levels gradually increased in lncSMIM14-overexpressing cells; however, Rab3a expression declined at 48 h compared with levels at 24 h and 36 h. Conversely, Rab3a protein levels gradually decreased in lncSMIM14-knockdown cells following BVDV infection (Figures 4G–H). RNA immunoprecipitation (RIP) assays further showed that Strep–Rab3a bound more strongly to lncSMIM14 than to controls (Figure 4I). These results suggest a direct interaction between lncSMIM14 and Rab3a. The endocytosis-related protein Rab3a promotes intracellular replication of BVDV. To assess the role of Rab3a in BVDV replication, Rab3a expression was measured in MDBK cells following BVDV infection using qRT-PCR. Rab3a mRNA levels showed a significant increase at 48 h post-infection (P < 0.05) (Figure 5A). Western blot analysis corroborated these findings, revealing a similar trend in Rab3a protein expression (Figure 5B). We subsequently generated Rab3a-overexpressing cells (mCherry–Rab3a) (Figures 5C–G), which exhibited elevated BVDV mRNA levels, increased progeny virus titers, more cytopathic effects (CPE), and enhanced fluorescence intensity and distribution of BVDV dsRNA (Figure 5H). In contrast, Rab3a-knockdown cells (Rab3a–sg1) (Figures 6A–D) displayed markedly reduced BVDV mRNA levels, lower progeny virus titers, fewer CPE, and diminished fluorescence intensity and distribution of BVDV dsRNA (Figures 6E–G). Collectively, these results demonstrate that Rab3a facilitates intracellular replication of BVDV. Rab3a co-localizes with key endocytic regulators Rab5a and Rab7a and promotes endocytic vesicle formation. To investigate the role of Rab3a in BVDV replication, we examined its spatial relationship with the key endocytic regulators Rab5a and Rab7a. Recombinant plasmids pCDH–CMV–MCS–EF1–Blast–mCherry–Rab3a–puro were co-transfected with pCDH–CMV–MCS–EF1–Blast–mCherry–Rab5a–puro or pCDH–CMV–MCS–EF1–Blast–mCherry–Rab7a–puro, respectively. Laser confocal microscopy revealed marked spatial co-localization of Rab3a with Rab5a and Rab7a in HEK-293T cells (Figure 7A), confirming the association of Rab3a with the endocytic pathway. We next investigated whether Rab3a affects endocytic vesicle formation under BVDV infection. Cells transfected with mCherry, mCherry–Rab3a, Myc, or Myc–lncSMIM14 were mock-infected or infected with BVDV, and endocytic vesicles were examined by electron microscopy. BVDV infection alone significantly increased the number of endocytic vesicles compared with the mock control. Furthermore, compared with BVDV infection alone, overexpression of Rab3a or lncSMIM14 further enhanced intracellular endocytic vesicle formation (Figure 7B). Collectively, these findings indicate that lncSMIM14 interacts with Rab3a, which in turn associates with Rab5a and Rab7a to enhance endosome formation, thereby promoting intracellular BVDV replication. DISCUSSION Endocytosis plays a crucial role in numerous intracellular signaling pathways [22] . Pathogens frequently exploit endocytic pathways to facilitate their entry into host cells [23] , leading to severe or persistent infections. In this study, we identified lncSMIM14—induced by BVDV in MDBK cells—as a significant factor in the endocytic pathway that facilitates increased intracellular BVDV replication. Furthermore, lncSMIM14 interacts with the endocytosis-related protein Rab3a and key endocytic regulators Rab5a and Rab7a, thereby promoting endosome formation. This study provides mechanistic insights into the processes by which BVDV enters host cells to facilitate viral replication. Several published studies have implicated lncRNAs in viral replication. For instance, during hepatitis C virus (HCV) infection, lncRNA-CMPK2 promotes viral replication and is regulated by interferon-α (IFN-α) [24] , whereas lncRNA GAS5 inhibits replication by binding to the HCV NS3 protein [25] . Additionally, lncRNA NRAV enhances the replication and virulence of influenza A virus (IAV) [26] . Current research suggests that lncRNAs can modulate BVDV replication through apoptotic pathways [27] . In this study, we found that lncSMIM14 promotes interactions among endocytosis-related proteins Rab3a, Rab5a, and Rab7a, thereby facilitating endocytic vesicle formation and subsequently enhancing BVDV replication. These findings suggest that a critical and potentially universal function of lncSMIM14 is the regulation of viral entry, which is particularly important during the early stages of viral infection. Therefore, we hypothesize that key signaling pathways involved in the formation of cell membrane receptors or endocytic vesicles may be regulated by lncSMIM14, warranting further investigation to elucidate the underlying molecular mechanisms. VDV-induced lncSMIM14 was localized to the cytoplasm, where it specifically interacts with Rab3a. Our results indicate that lncSMIM14 plays a critical role in BVDV infection by promoting Rab3a upregulation. Additionally, we found that Rab3a modulates BVDV replication in MDBK cells. Conversely, BVDV infection enhanced lncSMIM14 expression, suggesting a potentially reciprocal regulatory relationship among BVDV, lncSMIM14, and Rab3a. The specific molecular mechanisms underlying these interactions warrant further investigation. In addition, we confirmed the interactions between Rab3a and both Rab5a and Rab7a. The roles of Rab5a and Rab7a in viral infection are multifaceted. Published studies indicate that Rab5a knockdown inhibits the early expression of the rabies virus nucleoprotein [28] In respiratory epithelial cells infected with respiratory syncytial virus, Rab5a expression increases and suppresses the antiviral immune response by downregulating interferon lambda (IFN-λ) via interferon regulatory factor 1 (IRF1) [29] . Rab7a is involved in host–pathogen interactions [30] , and studies have shown that late endosome-associated Rab7a is essential for HIV-1 transmission [31] . Furthermore, Zeyen et al. demonstrated that Rab7a plays a bidirectional role in the viral life cycle, promoting hepatitis B virus endocytosis in the early stage while restricting the release of exogenous viral particles in the later stage [32] . In our study, we observed Rab3a co-localization with Rab5a and Rab7a, which correlated with increased intracellular BVDV replication. Additionally, overexpression of lncSMIM14 and Rab3a promoted endosome production. Our study demonstrates that the non-coding RNA lncSMIM14 interacts with Rab3a and key endocytic proteins, thereby enhancing endosome generation and promoting BVDV replication. Further studies are required to elucidate the specific interaction sites, molecular mechanisms, and processes involved in endosome production. The relationship between Rab3a co-localization with Rab5a and Rab7a and the regulation of endosome generation remains to be investigated. In summary, we found that lncSMIM14 is induced following BVDV infection and facilitates BVDV replication. We describe a molecular mechanism whereby lncSMIM14 promotes BVDV replication through the Rab3a-mediated endocytic pathway. Table 1 Predictive analysis of lncRNA positions by lnclocator Subcellular locations score Endocytosis related lncRNAs lncSMIM14 lncWIPF3 lncGPC6 Cytoplasm 0.369671600163 0.181414846852 0.747400390359 Nucleus 0.287521215258 0.0335444274812 0.167841284931 Ribosome 0.157724942096 0.0785417971393 0.012327340771 Cytosol 0.118447661906 0.678341753356 0.0339009144726 Exosome 0.0666345805775 0.028157175172 0.0385300694608 Predicted location Cytoplasm Cytosol Cytoplasm Declarations Ethics approval and consent to participate “Not applicable” in this section. Consent for publication “Not applicable” in this section. Availability of data and materials The sequence data supporting this study have been deposited in the NCBI database, with the main accession number being PRJNA1333864. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Autonomous Region's 'Tianshan Talents' Young Top Scientific and Technological Talent Program (Project No.: 2022TSYCCX0049); the Central Guidance Local Science and Technology Development Special Fund Project — 'Integration and Demonstration of Rapid Screening Technologies for Common Pathogens in Calves and Lambs'; and the National Natural Science Foundation Regional Fund Project (Project No.: 32160829). Authors' contributions Zhiran Shao contributed to conceptualization, methodology, investigation, and writing (original draft). Siqi Ma performed investigation, validation, and formal analysis. FengSiyue Gao, Yang Lou, Rulong Chen, and Xinyi Liu were involved in software development, formal analysis, and visualization. Li Yang, Zhanhai Mai, Lixia Wang, and Areayi haiyilati provided resources, writing (review and editing), and supervision. Huijun Shi and Qiang Fu were responsible for conceptualization, writing (review and editing), supervision, project administration, and funding acquisition. Acknowledgments “Not applicable” in this section. References Kadir Yeşilbağ,Gizem Alpay,Paul Becher. Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus[J]. Viruses, 2017, 9(6): 128. Christopher C L Chase,Neelu Thakur,Mahmoud F Darweesh, et al. Immune response to bovine viral diarrhea virus—looking at newly defined targets[J]. Animal Health Research Reviews, 2015, 16(1): 4-14. Yulong Zhou,Jiawen Zhang,Hailiang Wu, et al. First linear B-cell epitope identified on the nucleocapsid protein of bovine coronavirus[J]. Virology, 2025, 610: 110581. Bertone Paul,Stolc Viktor,Royce Thomas-E, et al.. Global identification of human transcribed sequences with genome tiling arrays.[J]. Science (New York, N.Y.), 2004, (5705): 2242-6. Alessandro Fatica,Irene Bozzoni. Long non-coding RNAs: new players in cell differentiation and development[J]. Nature Reviews. Genetics, 2014, 15(1): 7-21. Yang Xiaoping,Yuan Zuohui,Gou Lingzhu, et al. FEZF1-AS1 drives autophagy-mediated progression of colon cancer and reduces chemosensitivity through inhabiting the PI3K/AKT/mTOR signaling pathway[J]. Frontiers in genetics, 2025, 16: 1514205. Anindita Sarma,Parul Suri,Megan Justice, et al. An Emphasis on the Role of Long Non-Coding RNAs in Viral Gene Expression, Pathogenesis, and Innate Immunity in Viral Chicken Diseases[J]. Non-coding Rna, 2025, 11(3): 42. Driéle B Dos Santos,Geysson J Fernandez,Letícia T Silva, et al. lncRNAs as Biomarkers of Hepatocellular Carcinoma Risk and Liver Damage in Advanced Chronic Hepatitis C[J]. Current Issues in Molecular Biology, 2025, 47(5): 348. Wenjun Yan,Xue Fu,Hao Li, et al. The long non-coding RNA lncRNA-DRNR enhances infectious bronchitis virus replication by targeting chicken JMJD6 and modulating interferon-stimulated genes expression via the JAK-STAT signalling pathway[J]. Veterinary Research, 2024, 55(1): 141. Aatish Mahajan,Sivasankar Chandran,Ashok Kumar, et al. LncRNA-536 and RNA-Binding Protein RBM25 Interactions in Pulmonary Artery Smooth Muscle Cells: Implications in Pulmonary Hypertension[J]. Arteriosclerosis, Thrombosis, and Vascular Biology, 2025, 45(8): e374-e391. Chundi Gao,Huayao Li,Jing Zhuang, et al. The construction and analysis of ceRNA networks in invasive breast cancer: a study based on The Cancer Genome Atlas[J]. Cancer Management and Research, 2019, Volume 11: 1-11. Nihal Kaplan,Rosa Ventrella,Han Peng, et al. EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling[J]. Investigative Ophthalmology & Visual Science, 2018, 59(1): 393-406. Marsh Mark,Helenius Ari. Virus entry: open sesame.[J]. Cell, 2006, (4): 729-40. Rahul Sharma,Sujay K Maity,Partha Chakrabarti, et al. PIMT Controls Insulin Synthesis and Secretion through PDX1[J]. International Journal of Molecular Sciences, 2023, 24(9): 8084. Coppola Thierry,Perret-menoud Véronique,Gattesco Sonia, et al. The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.[J]. The Biochemical journal, 2002, (Pt 2): 273-9. Lemahieu Grégoire,Moreno-layseca Paulina,Hub Tobias, et al. RAB5A Promotes Active Fluid Wetting by Reprogramming Breast Cancer Spheroid Mechanics.[J]. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2025: e03569. Shi Mo,Wei Tang,Jun Xie, et al. Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells[J]. Journal of Virology, 2021, 95(8). Wing Hei Wong,Stephanie Z Liu,Annie Shi Ru Li, et al. Evidence for Rab7b and Its Splice Isoforms Having Distinct Biological Functions from Rab7a[J]. International Journal of Molecular Sciences, 2025, 26(6): 2610. Marina Caillet,Katy Janvier,Annegret Pelchen-Matthews, et al. Rab7A Is Required for Efficient Production of Infectious HIV-1[J]. Plos Pathogens, 2011, 7(11): e1002347. Chao Yang,Yan Cheng,Tianxi Zhang, et al. Multi-omics analysis provides new insights into the molecular mechanisms underlying colostral immunoglobulin G absorption in the gut of neonatal goat kids[J]. Animal Nutrition (zhongguo Xu Mu Shou Yi Xue Hui), 2025, 21: 166-178. Murielle P Serres,Ronan Shaughnessy,Sophie Escot, et al. MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis[J]. Developmental Cell, 2023, 58(22): 2477-2494.e8. Lewis P Hancock,Ellen G Allwood,John S Palmer, et al. Agent-based modelling of the early stages of actin polymerisation required to drive endocytosis in Saccharomyces cerevisiae[J]. Scientific Reports, 2025, 15(1): 28951. Andreas Santamaria,Daniel Pereira,Nisha Pawar, et al. Structural molecular details of the endocytic adaptor protein CALM upon binding with phosphatidylinositol 4,5-bisphosphate-containing model membranes[J]. Communications Chemistry, 2025, 8(1): 219. Hiroto Kambara,Farshad Niazi,Lenche Kostadinova, et al. Negative regulation of the interferon response by an interferon-induced long non-coding RNA[J]. Nucleic Acids Research, 2014, 42(16): 10668-80. Guo Yong,Li Chunxue,Zhang Rongrong, et al. Epigenetically-regulated serum GAS5 as a potential biomarker for patients with chronic hepatitis B virus infection[J]. Cancer biomarkers : section A of Disease markers, 2021, 32(2): 137-146. Correction to “Investigation of Long Noncoding RNA‐NRAV and Long Noncoding RNA‐Lethe Expression in Crimean−Congo Hemorrhagic Fever”[J]. Journal of Medical Virology, 2025, 97(2): e70245. Qiman Ma,Liangyuan Li,Yan Tang, et al. Analyses of long non-coding RNAs and mRNA profiling through RNA sequencing of MDBK cells at different stages of bovine viral diarrhea virus infection[J]. Research in Veterinary Science, 2017, 115: 508-516. Feilong Chen,Dingding Zhang,Lifang Cheng, et al. Xiaowugui decoction alleviates experimental rheumatoid arthritis by suppressing Rab5a-mediated TLR4 internalization in macrophages[J]. Phytomedicine : International Journal of Phytotherapy and Phytopharmacology, 2024, 132: 155762. Shi Mo,Wei Tang,Jun Xie, et al. Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells[J]. Journal of Virology, 2021, 95(8). Marina Caillet,Katy Janvier,Annegret Pelchen-Matthews, et al. Rab7A Is Required for Efficient Production of Infectious HIV-1[J]. Plos Pathogens, 2011, 7(11): e1002347. Zeyen Lisa,Prange Reinhild. Host Cell Rab GTPases in Hepatitis B Virus Infection[J]. Frontiers in cell and developmental biology, 2018, 6: 154. Christina Bartusch,Tatjana Döring,Reinhild Prange. Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing[J]. Viruses, 2017, 9(6): 157. Additional Declarations No competing interests reported. Supplementary Files WesternBlot.zip file.zip Figure5G6F.zip Figure2F3D.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7956623","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542859301,"identity":"c39362d8-0655-4658-94bc-4eb4d9362532","order_by":0,"name":"Zhiran Shao","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhiran","middleName":"","lastName":"Shao","suffix":""},{"id":542859302,"identity":"67df2767-f919-4735-97f7-dc40b4a9c8c5","order_by":1,"name":"Siqi Ma","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Ma","suffix":""},{"id":542859303,"identity":"1b307706-9b25-4991-8e95-7dd1948f489b","order_by":2,"name":"FengSiyue Gao","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"FengSiyue","middleName":"","lastName":"Gao","suffix":""},{"id":542859304,"identity":"4cf52aba-afe0-4bdd-85c5-31bb28b2d303","order_by":3,"name":"Yang Lou","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Lou","suffix":""},{"id":542859305,"identity":"8f4591dd-32de-4709-84fa-fd014344fa1b","order_by":4,"name":"Rulong Chen","email":"","orcid":"","institution":"Xinjiang Tianlai Agriculture and Animal Husbandry Group Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Rulong","middleName":"","lastName":"Chen","suffix":""},{"id":542859306,"identity":"06f6d720-ff0e-4bbd-89cb-99ac4c1fbdd4","order_by":5,"name":"Xinyi Liu","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Liu","suffix":""},{"id":542859307,"identity":"d876e94f-fed2-496a-b362-902f0ca2fa09","order_by":6,"name":"Li Yang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Yang","suffix":""},{"id":542859308,"identity":"3de532ee-9d2d-4879-b7d8-e20cd40a7ec8","order_by":7,"name":"Zhanhai Mai","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhanhai","middleName":"","lastName":"Mai","suffix":""},{"id":542859309,"identity":"68cbd873-031b-4a75-a364-440a0c3b9989","order_by":8,"name":"Lixia Wang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lixia","middleName":"","lastName":"Wang","suffix":""},{"id":542859310,"identity":"19d10da0-62ab-494c-88cd-e96d20b60910","order_by":9,"name":"Areayi haiyilati","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Areayi","middleName":"","lastName":"haiyilati","suffix":""},{"id":542859311,"identity":"5258f411-e81c-4032-b959-613b8f0a2e46","order_by":10,"name":"Huijun Shi","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huijun","middleName":"","lastName":"Shi","suffix":""},{"id":542859312,"identity":"50e62373-e405-4555-827f-5127df895ecd","order_by":11,"name":"Qiang Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYBAC9gYGhgMQJpD6wMAGZkrg08JzAEkL4wxitcABMw+UgV+LRPLGAz931DIYHDz8TNq2jU/e4ADzwds8DHZyug24tKQVHOw9c5xBsuGYmXTOGTbDDQfYkq15GJKNzQ5g12IvkWNwgLftGAM/wwGglgo2xg0HeMykeRgOJG7DoYUHqOXgX6AWNobj36QtDNjsNxzg/0ZQy2HethqgLWfMpBkq2BKBtrDh18LzrOCwbNsBoF/OFFv2nGFLnnmYzdhyjgFuv/CwJ2/++LatjsHgxvGNN362HbPtO9788MabCjs5XFqAwACID9c3SIBVHAPGDkwQNwDJ1jEw8DeAODV4lY6CUTAKRsHIBAD6J1xBJTPNtAAAAABJRU5ErkJggg==","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2025-10-27 11:52:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7956623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7956623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95792379,"identity":"6c784943-26bf-4d58-8282-37712524732b","added_by":"auto","created_at":"2025-11-13 07:02:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":311998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated expression of lncSMIM14 in MDBK cells following BVDV infection.\u003c/strong\u003e\u003cbr\u003e\n (A) RNA sequencing of MDBK cells infected with BVDV for 6, 12, and 24 hours.\u003cbr\u003e\n(B) qRT-PCR validation of differentially expressed lncRNAs identified by RNA-seq.\u003cbr\u003e\n(C) qRT-PCR validation of genes upregulated at all three time points (6, 12, and 24 hours post-infection).\u003cbr\u003e\n(D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of genes upregulated at all three time points. Data are presented as the mean ± standard error (**P \u0026lt; 0.01; ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/b6f3cfa146430e0e768a2772.png"},{"id":95802327,"identity":"5f9ed514-4bd9-4880-add6-09897775779d","added_by":"auto","created_at":"2025-11-13 08:27:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of lncSMIM14 promotes intracellular replication of BVDV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of lncSMIM14 expression at different time points following BVDV infection.\u003c/p\u003e\n\u003cp\u003e(B) Representative images of pLVX-AcGFP1-N1 plasmid transfection in HEK-293T cells (Brightfield: brightfield microscopy; GFP: fluorescence channel; Merge: merged image).\u003c/p\u003e\n\u003cp\u003e(C) qRT-PCR analysis of lncSMIM14 transcription levels in transfected MDBK cells.\u003c/p\u003e\n\u003cp\u003e(D) qRT-PCR analysis of BVDV 5′UTR mRNA levels.\u003c/p\u003e\n\u003cp\u003e(E) Progeny virus titers measured by TCID₅₀ assay.\u003c/p\u003e\n\u003cp\u003e(F) Representative cytopathic effects in infected MDBK cells.\u003c/p\u003e\n\u003cp\u003e(G) Immunofluorescence staining of BVDV dsRNA in MDBK cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/dbb8fb3f152b7b808ac2fa69.png"},{"id":95792378,"identity":"35cf6ddc-4b14-40ac-b455-39bf712c9a58","added_by":"auto","created_at":"2025-11-13 07:02:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":481760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing of lncSMIM14 suppresses intracellular replication of BVDV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transfection of pLKO.1–copGFP–PURO–SMIM14 plasmid and qRT-PCR analysis of sh–lncSMIM14 knockdown efficiency in MDBK cells.\u003cbr\u003e\n(B) qRT-PCR analysis of BVDV 5′UTR mRNA levels.\u003cbr\u003e\n(C) Progeny virus titers determined by TCID₅₀ assay.\u003cbr\u003e\n(D) Representative cytopathic effects in infected MDBK cells.\u003cbr\u003e\n(E) Immunofluorescence staining of BVDV dsRNA in MDBK cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/2476134e023239b2415866cf.png"},{"id":95792376,"identity":"c92cbdf1-b879-400e-9cc4-2f863d2b3432","added_by":"auto","created_at":"2025-11-13 07:02:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncSMIM14 targets and regulates the endocytosis-related protein Rab3a.\u003cbr\u003e\n \u003c/strong\u003e(A) Predicted interaction network between lncSMIM14 and mRNAs.\u003cbr\u003e\n(B) RNA–protein interaction (RPISeq) analysis of lncSMIM14 and differentially expressed mRNAs.\u003cbr\u003e\n(C) qRT-PCR analysis of Rab3a mRNA levels after lncSMIM14 overexpression (Myc–lncSMIM14).\u003cbr\u003e\n(D) qRT-PCR analysis of Rab3a mRNA levels after lncSMIM14 knockdown (sh–lncSMIM14).\u003cbr\u003e\n(E) Western blot analysis of Rab3a protein levels following lncSMIM14 overexpression or knockdown.\u003cbr\u003e\n(F) Densitometric quantification of Western blot results.\u003cbr\u003e\n(G) Western blot analysis of Rab3a protein levels at different time points after BVDV infection in lncSMIM14-overexpressing cells.\u003cbr\u003e\n(H) Western blot analysis of Rab3a protein levels at different time points after BVDV infection in lncSMIM14-knockdown cells.\u003cbr\u003e\n(I) RNA immunoprecipitation (RIP) assay showing the interaction between Rab3a and lncSMIM14.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/4f6c6cb353c9330ec61ffed2.png"},{"id":95792381,"identity":"3ff50e04-b5c8-4c57-b9ac-47154d8fd96e","added_by":"auto","created_at":"2025-11-13 07:02:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":662542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of Rab3a enhances intracellular replication of BVDV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of Rab3a mRNA levels.\u003c/p\u003e\n\u003cp\u003e(B) Western blot analysis of Rab3a protein levels with densitometric quantification.\u003c/p\u003e\n\u003cp\u003e(C) Fluorescence microscopy of HEK‑293T cells transfected with mCherry or mCherry–Rab3a plasmids under brightfield, mCherry channel, and merged images, with corresponding qRT-PCR analysis of Rab3a mRNA expression.\u003c/p\u003e\n\u003cp\u003e(D) Western blot analysis and densitometric quantification of Rab3a protein levels after mCherry or mCherry–Rab3a transfection.\u003c/p\u003e\n\u003cp\u003e(E) qRT-PCR analysis of BVDV 5′UTR mRNA levels.\u003c/p\u003e\n\u003cp\u003e(F) Progeny virus titers determined by TCID₅₀ assay.\u003c/p\u003e\n\u003cp\u003e(G) Representative cytopathic effects in cells transfected with mCherry or mCherry–Rab3a plasmids at different time points after BVDV infection.\u003c/p\u003e\n\u003cp\u003e(H) Confocal microscopy of BVDV dsRNA immunofluorescence in mCherry- or mCherry–Rab3a-transfected cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/d3a0c1603a9a06b719197a69.png"},{"id":95802792,"identity":"b8b083f4-89cc-41f7-ac32-43922b1a36a0","added_by":"auto","created_at":"2025-11-13 08:28:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":368381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing of Rab3a suppresses intracellular replication of BVDV.\u003cbr\u003e\n \u003c/strong\u003e(A, B) Western blot analysis of Rab3a protein levels with densitometric quantification in Rab3a‑sg1‑transfected cells.\u003cbr\u003e\n(C) qRT-PCR analysis of Rab3a mRNA levels.\u003cbr\u003e\n(D) qRT-PCR analysis of BVDV 5′UTR mRNA levels.\u003cbr\u003e\n(E) Progeny virus titers determined by TCID₅₀ assay.\u003cbr\u003e\n(F) Representative cytopathic effects in Rab3a‑sg1‑transfected cells at different time points after BVDV infection.\u003cbr\u003e\n(G) Confocal microscopy of BVDV dsRNA immunofluorescence in Rab3a‑sg1‑transfected cells.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/86be048602489a5d8496c517.png"},{"id":95792382,"identity":"1c94bb58-fcd1-412b-90c0-6e8946628607","added_by":"auto","created_at":"2025-11-13 07:02:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":791098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRab3a co-localizes with endocytic regulators Rab5a and Rab7a and promotes endocytic vesicle formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Confocal microscopy showing spatial co-localization of mCherry–Rab3a with mCherry–Rab5a or mCherry–Rab7a in HEK‑293T cells.\u003c/p\u003e\n\u003cp\u003e(B) Transmission electron microscopy (TEM) analysis of endocytic vesicle formation in cells overexpressing Rab3a or lncSMIM14, with or without BVDV infection.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/e1d809caceb95c55d4de9eb9.png"},{"id":98435481,"identity":"bde2dd5f-1048-4b49-b132-4de41ed997a7","added_by":"auto","created_at":"2025-12-17 16:53:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4160577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/0ecabc8f-ea21-4010-b0d8-b3d68dd2d5a5.pdf"},{"id":95792383,"identity":"8a78cad6-ac15-4ca4-a4a0-8b39aa529233","added_by":"auto","created_at":"2025-11-13 07:02:51","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10385320,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlot.zip","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/f7f5e858c5e5d96ad2ba4cdb.zip"},{"id":95792386,"identity":"e304fcc8-fa1a-471f-953f-dfa6738bd217","added_by":"auto","created_at":"2025-11-13 07:03:15","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":551135063,"visible":true,"origin":"","legend":"","description":"","filename":"file.zip","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/9705fd464a83892ce277baa4.zip"},{"id":95792385,"identity":"2b7641dc-f378-4ef3-850d-3b8f464b97e3","added_by":"auto","created_at":"2025-11-13 07:02:55","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":101655819,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5G6F.zip","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/f85253608f6b8ae66702cae3.zip"},{"id":95792402,"identity":"cd823290-8c01-4a76-bdfa-174c02f52426","added_by":"auto","created_at":"2025-11-13 07:03:55","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1470683175,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2F3D.zip","url":"https://assets-eu.researchsquare.com/files/rs-7956623/v1/17f6d3d6b7228354001b27fc.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"LncSMIM14 Hijacks Rab3a-Mediated Endocytosis to Promote Bovine Viral Diarrhea Virus Replication","fulltext":[{"header":"Novelty statement","content":"\u003cp\u003eWe identified lncSMIM14 as a previously unknown host factor that significantly promotes BVDV replication. lncSMIM14 enhances viral propagation by upregulating Rab3a, an endocytosis-related GTPase essential for efficient infection. The lncSMIM14\u0026ndash;Rab3a axis facilitates endocytic vesicle formation, revealing a new mechanism exploited by BVDV and potential antiviral targets.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eBovine viral diarrhea virus (BVDV) is the main etiological agent causing viral diarrhea and mucosal disease in cattle, collectively known as bovine viral diarrhea\u0026ndash;mucosal disease (BVD/MD or BVD-MD\u003csup\u003e[1]\u003c/sup\u003e. It belongs to the genus Pestivirus within the family Flaviviridae, and its major clinical manifestations include inflammatory diarrhea, enteritis, and mucosal necrosis\u003csup\u003e[2]\u003c/sup\u003e. BVDV poses a global threat to cattle populations and imposes a significant economic burden, resulting in substantial losses to the cattle industry worldwide\u003csup\u003e[3]\u003c/sup\u003e. To date, BVDV has evolved multiple strategies to establish infection in its host; however, the underlying pathogenic mechanisms remain incompletely understood.\u003c/p\u003e\n\u003cp\u003eLong non-coding RNAs (lncRNAs), once dismissed as \u0026apos;transcriptional noise\u0026apos;, are defined as transcripts longer than 200 nucleotides that lack protein-coding capabilities\u003csup\u003e[4]\u003c/sup\u003e. In recent years, lncRNAs have attracted significant attention as emerging and potentially crucial regulators of biological processes, including cell proliferation, differentiation, and development\u003csup\u003e[5-6]\u003c/sup\u003e . Accumulating evidence underscores the vital role of lncRNAs in the initiation, modulation, and progression of various viral infections, including influenza\u003csup\u003e[7]\u003c/sup\u003e, hepatitis\u003csup\u003e[8]\u003c/sup\u003e, coronaviruses\u003csup\u003e[9]\u003c/sup\u003e, and human immunodeficiency virus\u003csup\u003e[10]\u003c/sup\u003e. Therefore, identifying key lncRNAs will enable targeted analysis of their functional roles, thereby elucidating their infection-related targets and regulatory mechanisms\u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEndocytosis is the process through which cells internalize membrane lipids, membrane proteins, and extracellular material, thereby playing a vital role in regulating numerous intracellular signaling cascades \u003csup\u003e[11-12]\u003c/sup\u003e. Pathogens often exploit the endocytic pathway to facilitate their internalization within host cells \u003csup\u003e[13]\u003c/sup\u003e. Rab proteins, a family of small GTP-binding proteins involved in vesicular transport, are crucial for intracellular membrane vesicle trafficking \u003csup\u003e[14]\u003c/sup\u003e. he mammalian genome contains over 60 Rab genes \u003csup\u003e[15]\u003c/sup\u003e among which Rab3a acts as a molecular switch by binding GTP and GDP, thus participating in the regulation of synaptic vesicle exocytosis and hormone secretion \u003csup\u003e[15]\u003c/sup\u003e. Rab5a, another member of the Rab family, contributes to signal transduction, receptor downregulation, and the phagocytosis of pathogens\u003csup\u003e[16]\u003c/sup\u003e. Studies indicate that Rab5a upregulation can inhibit the antiviral immune response in epithelial cells\u003csup\u003e[17]\u003c/sup\u003e. Similarly, Rab7a, another small GTP-binding protein, plays a significant role in the intracellular transport of viruses\u003csup\u003e[18]\u003c/sup\u003e. Located primarily in late endosomes, Rab7a regulates viral spread \u003csup\u003e[19]\u003c/sup\u003e and serves a bidirectional regulatory function throughout the viral replication cycle, participating in host-pathogen interactions to promote viral infection \u003csup\u003e[20-21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDespite limited research on the relationship between lncRNA and BVDV replication, our study provides significant insights. We conducted lncRNA sequencing (accession number: [to be provided]) and discovered that lncSMIM14 was significantly upregulated. We identified lncSMIM14 as an lncRNA that is significantly induced by BVDV and plays a crucial role in viral replication. LncSMIM14 enhances the expression of Rab3a, thereby promoting BVDV replication. Moreover, Rab3a co-localizes with key endocytic regulatory factors Rab5a and Rab7a, facilitating the formation of endocytic vesicles, particularly following BVDV infection. Our findings reveal a novel mechanism through which BVDV exploits the host lncRNA lncSMIM14 to hijack Rab3a-mediated endocytosis, thereby enhancing viral replication. This study identifies the lncSMIM14-Rab3a axis as a critical host pathway subverted by BVDV, providing a molecular mechanism that influences BVDV intracellular replication and offering new potential targets for antiviral intervention.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCell culture and virus\u003c/p\u003e\n\u003cp\u003eMadin–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) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. Transfections were performed using a DNA transfection reagent (Roche) according to the manufacturer’s protocol. The bovine viral diarrhea virus (BVDV) TC strain was preserved in our laboratory.\u003c/p\u003e\n\u003cp\u003ePlasmids\u003c/p\u003e\n\u003cp\u003eThe 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. Competent Escherichia coli Stbl3 cells were prepared and stored in our laboratory.\u003c/p\u003e\n\u003cp\u003eLncRNA sequencing analysis\u003c/p\u003e\n\u003cp\u003eTotal 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) and sequenced on the Illumina HiSeq 4000 platform by Zhongke Genome Company, Ltd. (Shanghai, China). Raw reads were quality-filtered to remove low-quality sequences. Differentially expressed lncRNAs and mRNAs (q-value \u0026lt; 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.\u003c/p\u003e\n\u003cp\u003eVirus infection\u003c/p\u003e\n\u003cp\u003eMDBK cells were seeded at 5 × 10⁴ cells/dish in 10 cm dishes. When cells reached ~70% confluence, the medium was replaced with serum-free DMEM, and BVDV was inoculated at 1,000 TCID₅₀/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).\u003c/p\u003e\n\u003cp\u003eWestern blotting\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer (Roche) containing protease inhibitors. Protein concentration was determined using a Bio‑Rad protein assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes (GE Healthcare), and blocked with 5% skim milk. Membranes were incubated with primary antibodies against dsRNA (J2, Scicons, Hungary), GAPDH (Wuhan Sanying Biotechnology), or Rab3a (Wuhan Sanying Biotechnology), followed by HRP‑conjugated secondary antibodies (Wuhan Sanying Biotechnology). Blots were visualized using BeyoECL Plus (Biosharp Biotechnology).\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR (qRT-PCR)\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRNzol reagent (TIANGEN) and reverse transcribed with the ReverTra Ace qPCR RT Kit (Toyobo). qRT-PCR was performed using SYBR Green qPCR Master Mix (GenStar) on a QuantStudio system (Applied Biosystems). GAPDH served as an internal control. Primer sequences are listed in the Supplementary Table.\u003c/p\u003e\n\u003cp\u003eLentiviral packaging\u003c/p\u003e\n\u003cp\u003eHEK‑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 (3,000 × 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).\u003c/p\u003e\n\u003cp\u003eImmunofluorescence microscopy\u003c/p\u003e\n\u003cp\u003eMDBK 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.\u003c/p\u003e\n\u003cp\u003eRNA immunoprecipitation (RIP)\u003c/p\u003e\n\u003cp\u003eCells 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.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eAll 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 \u0026lt; 0.05 (P \u0026lt; 0.05; *P \u0026lt; 0.01; ns, not significant).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eThe expression of lncSMIM14 is significantly elevated in MDBK cells after BVDV infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of lncSMIM14 is significantly elevated in MDBK cells following BVDV infection. To investigate the involvement of long non-coding RNAs (lncRNAs) in bovine viral diarrhea virus (BVDV) infection, RNA sequencing (RNA-seq) was conducted to identify differentially expressed lncRNAs in MDBK cells infected with BVDV for 6 or 12 hours (Figure 1A). The top 13 upregulated lncRNAs responsive to BVDV infection were further analyzed using quantitative PCR (qPCR). Our data showed that the expression patterns of these 13 upregulated lncRNAs in MDBK cells were consistent with the RNA-seq results following BVDV infection (Figure 1B). KEGG analysis revealed that lncSMIM14, lncWIPF3, and lncGPC6 were upregulated at 6, 12, and 24 hours post-BVDV infection, respectively (Figure 1C). Furthermore, Gene Ontology (GO) and KEGG pathway enrichment analyses indicated that the differentially expressed lncRNAs were predominantly enriched in the endocytosis pathway (Figure 1D). We used Lnclocator to predict the subcellular localizations of lncSMIM14, lncWIPF3, and lncGPC6, and found that lncSMIM14 and lncGPC6 were localized to the cytoplasm, whereas lncWIPF3 was localized to the cytoplasmic matrix (Table 1). Given that lncSMIM14 expression exhibited a stable positive correlation with the duration of BVDV infection in MDBK cells (Figure 1C), further investigation of lncSMIM14 was warranted\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLncSMIM14 influences intracellular replication of BVDV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the role of lncSMIM14 in intracellular BVDV replication, we performed qRT-PCR to assess lncSMIM14 expression in MDBK cells following BVDV infection. Our results showed that lncSMIM14 expression increased in a time-dependent manner during BVDV infection (Figure 2A). Subsequently, we generated lncSMIM14-overexpressing (lncSMIM14+MDBK) cells (Figures 2B\u0026ndash;C) as well as lncSMIM14-knockdown (lncSMIM14\u0026minus;MDBK) cells. LncSMIM14-overexpressing cells exhibited increased BVDV mRNA levels, higher progeny virus titers, more cytopathic effects (CPE), and enhanced fluorescence intensity and distribution of BVDV dsRNA (Figures 2D\u0026ndash;G). We further knocked down lncSMIM14 to verify its functional role (Figure 3A). LncSMIM14-knockdown cells exhibited reduced BVDV mRNA levels, lower progeny virus titers, fewer cytopathic effects (CPE), and diminished fluorescence intensity and distribution of BVDV dsRNA (Figures 3B\u0026ndash;E). In summary, our findings demonstrate that lncSMIM14 promotes intracellular BVDV replication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLncSMIM14 targets and regulates the endocytosis-related protein Rab3a.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the mechanism by which lncSMIM14 affects BVDV replication, we predicted its potential target genes using bioinformatics analysis (Figure 4A). Subsequently, RNA\u0026ndash;protein interaction prediction (RPISeq) was performed to evaluate the binding probability between lncSMIM14 and candidate proteins using random forest (RF) and support vector machine (SVM) scores. This analysis identified Rab3a, an endocytosis-related protein, as a potential binding target of lncSMIM14 (Figure 4B). To examine whether lncSMIM14 regulates Rab3a expression, MDBK cells were transfected with Myc, Myc\u0026ndash;lncSMIM14, shNC, or sh\u0026ndash;lncSMIM14. Overexpression of lncSMIM14 increased both Rab3a mRNA and protein levels, whereas lncSMIM14 knockdown reduced Rab3a expression (Figures 4C\u0026ndash;F). We next infected the transfected cells with BVDV. After BVDV infection, Rab3a protein levels gradually increased in lncSMIM14-overexpressing cells; however, Rab3a expression declined at 48 h compared with levels at 24 h and 36 h. Conversely, Rab3a protein levels gradually decreased in lncSMIM14-knockdown cells following BVDV infection (Figures 4G\u0026ndash;H). RNA immunoprecipitation (RIP) assays further showed that Strep\u0026ndash;Rab3a bound more strongly to lncSMIM14 than to controls (Figure 4I). These results suggest a direct interaction between lncSMIM14 and Rab3a.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe endocytosis-related protein Rab3a promotes intracellular replication of BVDV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the role of Rab3a in BVDV replication, Rab3a expression was measured in MDBK cells following BVDV infection using qRT-PCR. Rab3a mRNA levels showed a significant increase at 48 h post-infection (P \u0026lt; 0.05) (Figure 5A). Western blot analysis corroborated these findings, revealing a similar trend in Rab3a protein expression (Figure 5B). We subsequently generated Rab3a-overexpressing cells (mCherry\u0026ndash;Rab3a) (Figures 5C\u0026ndash;G), which exhibited elevated BVDV mRNA levels, increased progeny virus titers, more cytopathic effects (CPE), and enhanced fluorescence intensity and distribution of BVDV dsRNA (Figure 5H). In contrast, Rab3a-knockdown cells (Rab3a\u0026ndash;sg1) (Figures 6A\u0026ndash;D) displayed markedly reduced BVDV mRNA levels, lower progeny virus titers, fewer CPE, and diminished fluorescence intensity and distribution of BVDV dsRNA (Figures 6E\u0026ndash;G). Collectively, these results demonstrate that Rab3a facilitates intracellular replication of BVDV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRab3a co-localizes with key endocytic regulators Rab5a and Rab7a and promotes endocytic vesicle formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of Rab3a in BVDV replication, we examined its spatial relationship with the key endocytic regulators Rab5a and Rab7a. Recombinant plasmids pCDH\u0026ndash;CMV\u0026ndash;MCS\u0026ndash;EF1\u0026ndash;Blast\u0026ndash;mCherry\u0026ndash;Rab3a\u0026ndash;puro were co-transfected with pCDH\u0026ndash;CMV\u0026ndash;MCS\u0026ndash;EF1\u0026ndash;Blast\u0026ndash;mCherry\u0026ndash;Rab5a\u0026ndash;puro or pCDH\u0026ndash;CMV\u0026ndash;MCS\u0026ndash;EF1\u0026ndash;Blast\u0026ndash;mCherry\u0026ndash;Rab7a\u0026ndash;puro, respectively. Laser confocal microscopy revealed marked spatial co-localization of Rab3a with Rab5a and Rab7a in HEK-293T cells (Figure 7A), confirming the association of Rab3a with the endocytic pathway.\u003c/p\u003e\n\u003cp\u003eWe next investigated whether Rab3a affects endocytic vesicle formation under BVDV infection. Cells transfected with mCherry, mCherry\u0026ndash;Rab3a, Myc, or Myc\u0026ndash;lncSMIM14 were mock-infected or infected with BVDV, and endocytic vesicles were examined by electron microscopy. BVDV infection alone significantly increased the number of endocytic vesicles compared with the mock control. Furthermore, compared with BVDV infection alone, overexpression of Rab3a or lncSMIM14 further enhanced intracellular endocytic vesicle formation (Figure 7B).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that lncSMIM14 interacts with Rab3a, which in turn associates with Rab5a and Rab7a to enhance endosome formation, thereby promoting intracellular BVDV replication.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eEndocytosis plays a crucial role in numerous intracellular signaling pathways\u003csup\u003e[22]\u003c/sup\u003e. Pathogens frequently exploit endocytic pathways to facilitate their entry into host cells\u003csup\u003e[23]\u003c/sup\u003e, leading to severe or persistent infections. In this study, we identified lncSMIM14\u0026mdash;induced by BVDV in MDBK cells\u0026mdash;as a significant factor in the endocytic pathway that facilitates increased intracellular BVDV replication. Furthermore, lncSMIM14 interacts with the endocytosis-related protein Rab3a and key endocytic regulators Rab5a and Rab7a, thereby promoting endosome formation. This study provides mechanistic insights into the processes by which BVDV enters host cells to facilitate viral replication. Several published studies have implicated lncRNAs in viral replication. For instance, during hepatitis C virus (HCV) infection, lncRNA-CMPK2 promotes viral replication and is regulated by interferon-\u0026alpha; (IFN-\u0026alpha;)\u003csup\u003e[24]\u003c/sup\u003e, whereas lncRNA GAS5 inhibits replication by binding to the HCV NS3 protein\u003csup\u003e[25]\u003c/sup\u003e\u003csup\u003e.\u003c/sup\u003e Additionally, lncRNA NRAV enhances the replication and virulence of influenza A virus (IAV)\u003csup\u003e[26]\u003c/sup\u003e. Current research suggests that lncRNAs can modulate BVDV replication through apoptotic pathways\u003csup\u003e[27]\u003c/sup\u003e. In this study, we found that lncSMIM14 promotes interactions among endocytosis-related proteins Rab3a, Rab5a, and Rab7a, thereby facilitating endocytic vesicle formation and subsequently enhancing BVDV replication. These findings suggest that a critical and potentially universal function of lncSMIM14 is the regulation of viral entry, which is particularly important during the early stages of viral infection. Therefore, we hypothesize that key signaling pathways involved in the formation of cell membrane receptors or endocytic vesicles may be regulated by lncSMIM14, warranting further investigation to elucidate the underlying molecular mechanisms.\u003c/p\u003e\n\u003cp\u003eVDV-induced lncSMIM14 was localized to the cytoplasm, where it specifically interacts with Rab3a. Our results indicate that lncSMIM14 plays a critical role in BVDV infection by promoting Rab3a upregulation. Additionally, we found that Rab3a modulates BVDV replication in MDBK cells. Conversely, BVDV infection enhanced lncSMIM14 expression, suggesting a potentially reciprocal regulatory relationship among BVDV, lncSMIM14, and Rab3a. The specific molecular mechanisms underlying these interactions warrant further investigation.\u003c/p\u003e\n\u003cp\u003eIn addition, we confirmed the interactions between Rab3a and both Rab5a and Rab7a. The roles of Rab5a and Rab7a in viral infection are multifaceted. Published studies indicate that Rab5a knockdown inhibits the early expression of the rabies virus nucleoprotein\u003csup\u003e[28]\u003c/sup\u003e In respiratory epithelial cells infected with respiratory syncytial virus, Rab5a expression increases and suppresses the antiviral immune response by downregulating interferon lambda (IFN-\u0026lambda;) via interferon regulatory factor 1 (IRF1)\u003csup\u003e[29]\u003c/sup\u003e. Rab7a is involved in host\u0026ndash;pathogen interactions\u003csup\u003e[30]\u003c/sup\u003e, and studies have shown that late endosome-associated Rab7a is essential for HIV-1 transmission\u003csup\u003e[31]\u003c/sup\u003e. Furthermore, Zeyen et al. demonstrated that Rab7a plays a bidirectional role in the viral life cycle, promoting hepatitis B virus endocytosis in the early stage while restricting the release of exogenous viral particles in the later stage\u003csup\u003e[32]\u003c/sup\u003e. In our study, we observed Rab3a co-localization with Rab5a and Rab7a, which correlated with increased intracellular BVDV replication. Additionally, overexpression of lncSMIM14 and Rab3a promoted endosome production. Our study demonstrates that the non-coding RNA lncSMIM14 interacts with Rab3a and key endocytic proteins, thereby enhancing endosome generation and promoting BVDV replication. Further studies are required to elucidate the specific interaction sites, molecular mechanisms, and processes involved in endosome production. The relationship between Rab3a co-localization with Rab5a and Rab7a and the regulation of endosome generation remains to be investigated. In summary, we found that lncSMIM14 is induced following BVDV infection and facilitates BVDV replication. We describe a molecular mechanism whereby lncSMIM14 promotes BVDV replication through the Rab3a-mediated endocytic pathway.\u003c/p\u003e\n\u003cp\u003eTable 1 Predictive analysis of lncRNA positions by lnclocator\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003eSubcellular locations score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 429px;\"\u003e\n \u003cp\u003eEndocytosis related lncRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003elncSMIM14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003elncWIPF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003elncGPC6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eCytoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e0.369671600163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.181414846852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.747400390359\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e0.287521215258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.0335444274812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.167841284931\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eRibosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e0.157724942096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.0785417971393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.012327340771\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eCytosol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e0.118447661906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.678341753356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.0339009144726\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eExosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e0.0666345805775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.028157175172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.0385300694608\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003ePredicted location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eCytoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eCytosol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eCytoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Not applicable\u0026rdquo;\u0026nbsp;in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Not applicable\u0026rdquo;\u0026nbsp;in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence data supporting this study have been deposited in the NCBI database, with the main accession number being PRJNA1333864.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Autonomous Region\u0026apos;s \u0026apos;Tianshan Talents\u0026apos; Young Top Scientific and Technological Talent Program (Project No.: 2022TSYCCX0049); the Central Guidance Local Science and Technology Development Special Fund Project\u0026nbsp;\u0026mdash;\u0026nbsp;\u0026apos;Integration and Demonstration of Rapid Screening Technologies for Common Pathogens in Calves and Lambs\u0026apos;; and the National Natural Science Foundation Regional Fund Project (Project No.: 32160829).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhiran Shao contributed to conceptualization, methodology, investigation, and writing (original draft). Siqi Ma performed investigation, validation, and formal analysis. FengSiyue Gao, Yang Lou, Rulong Chen, and Xinyi Liu were involved in software development, formal analysis, and visualization. Li Yang, Zhanhai Mai, Lixia Wang, and Areayi haiyilati provided resources, writing (review and editing), and supervision. Huijun Shi and Qiang Fu were responsible for conceptualization, writing (review and editing), supervision, project administration, and funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Not applicable\u0026rdquo; in this section.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKadir Yeşilbağ,Gizem Alpay,Paul Becher. Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus[J]. Viruses, 2017, 9(6): 128.\u003c/li\u003e\n\u003cli\u003eChristopher C L Chase,Neelu Thakur,Mahmoud F Darweesh, et al. Immune response to bovine viral diarrhea virus\u0026mdash;looking at newly defined targets[J]. Animal Health Research Reviews, 2015, 16(1): 4-14.\u003c/li\u003e\n\u003cli\u003eYulong Zhou,Jiawen Zhang,Hailiang Wu, et al. First linear B-cell epitope identified on the nucleocapsid protein of bovine coronavirus[J]. Virology, 2025, 610: 110581.\u003c/li\u003e\n\u003cli\u003eBertone Paul,Stolc Viktor,Royce Thomas-E, et al.. Global identification of human transcribed sequences with genome tiling arrays.[J]. Science (New York, N.Y.), 2004, (5705): 2242-6.\u003c/li\u003e\n\u003cli\u003eAlessandro Fatica,Irene Bozzoni. Long non-coding RNAs: new players in cell differentiation and development[J]. Nature Reviews. Genetics, 2014, 15(1): 7-21.\u003c/li\u003e\n\u003cli\u003eYang Xiaoping,Yuan Zuohui,Gou Lingzhu, et al. FEZF1-AS1 drives autophagy-mediated progression of colon cancer and reduces chemosensitivity through inhabiting the PI3K/AKT/mTOR signaling pathway[J]. Frontiers in genetics, 2025, 16: 1514205.\u003c/li\u003e\n\u003cli\u003eAnindita Sarma,Parul Suri,Megan Justice, et al. An Emphasis on the Role of Long Non-Coding RNAs in Viral Gene Expression, Pathogenesis, and Innate Immunity in Viral Chicken Diseases[J]. Non-coding Rna, 2025, 11(3): 42.\u003c/li\u003e\n\u003cli\u003eDri\u0026eacute;le B Dos Santos,Geysson J Fernandez,Let\u0026iacute;cia T Silva, et al. lncRNAs as Biomarkers of Hepatocellular Carcinoma Risk and Liver Damage in Advanced Chronic Hepatitis C[J]. Current Issues in Molecular Biology, 2025, 47(5): 348.\u003c/li\u003e\n\u003cli\u003eWenjun Yan,Xue Fu,Hao Li, et al. The long non-coding RNA lncRNA-DRNR enhances infectious bronchitis virus replication by targeting chicken JMJD6 and modulating interferon-stimulated genes expression via the JAK-STAT signalling pathway[J]. Veterinary Research, 2024, 55(1): 141.\u003c/li\u003e\n\u003cli\u003eAatish Mahajan,Sivasankar Chandran,Ashok Kumar, et al. LncRNA-536 and RNA-Binding Protein RBM25 Interactions in Pulmonary Artery Smooth Muscle Cells: Implications in Pulmonary Hypertension[J]. Arteriosclerosis, Thrombosis, and Vascular Biology, 2025, 45(8): e374-e391.\u003c/li\u003e\n\u003cli\u003eChundi Gao,Huayao Li,Jing Zhuang, et al. The construction and analysis of ceRNA networks in invasive breast cancer: a study based on The Cancer Genome Atlas[J]. Cancer Management and Research, 2019, Volume 11: 1-11.\u003c/li\u003e\n\u003cli\u003eNihal Kaplan,Rosa Ventrella,Han Peng, et al. EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling[J]. Investigative Ophthalmology \u0026amp;amp; Visual Science, 2018, 59(1): 393-406.\u003c/li\u003e\n\u003cli\u003eMarsh Mark,Helenius Ari. Virus entry: open sesame.[J]. Cell, 2006, (4): 729-40.\u003c/li\u003e\n\u003cli\u003eRahul Sharma,Sujay K Maity,Partha Chakrabarti, et al. PIMT Controls Insulin Synthesis and Secretion through PDX1[J]. International Journal of Molecular Sciences, 2023, 24(9): 8084.\u003c/li\u003e\n\u003cli\u003eCoppola Thierry,Perret-menoud V\u0026eacute;ronique,Gattesco Sonia, et al. The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.[J]. The Biochemical journal, 2002, (Pt 2): 273-9.\u003c/li\u003e\n\u003cli\u003eLemahieu Gr\u0026eacute;goire,Moreno-layseca Paulina,Hub Tobias, et al. RAB5A Promotes Active Fluid Wetting by Reprogramming Breast Cancer Spheroid Mechanics.[J]. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2025: e03569.\u003c/li\u003e\n\u003cli\u003eShi Mo,Wei Tang,Jun Xie, et al. Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells[J]. Journal of Virology, 2021, 95(8).\u003c/li\u003e\n\u003cli\u003eWing Hei Wong,Stephanie Z Liu,Annie Shi Ru Li, et al. Evidence for Rab7b and Its Splice Isoforms Having Distinct Biological Functions from Rab7a[J]. International Journal of Molecular Sciences, 2025, 26(6): 2610.\u003c/li\u003e\n\u003cli\u003eMarina Caillet,Katy Janvier,Annegret Pelchen-Matthews, et al. Rab7A Is Required for Efficient Production of Infectious HIV-1[J]. Plos Pathogens, 2011, 7(11): e1002347.\u003c/li\u003e\n\u003cli\u003eChao Yang,Yan Cheng,Tianxi Zhang, et al. Multi-omics analysis provides new insights into the molecular mechanisms underlying colostral immunoglobulin G absorption in the gut of neonatal goat kids[J]. Animal Nutrition (zhongguo Xu Mu Shou Yi Xue Hui), 2025, 21: 166-178.\u003c/li\u003e\n\u003cli\u003eMurielle P Serres,Ronan Shaughnessy,Sophie Escot, et al. MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis[J]. Developmental Cell, 2023, 58(22): 2477-2494.e8.\u003c/li\u003e\n\u003cli\u003eLewis P Hancock,Ellen G Allwood,John S Palmer, et al. Agent-based modelling of the early stages of actin polymerisation required to drive endocytosis in Saccharomyces cerevisiae[J]. Scientific Reports, 2025, 15(1): 28951.\u003c/li\u003e\n\u003cli\u003eAndreas Santamaria,Daniel Pereira,Nisha Pawar, et al. Structural molecular details of the endocytic adaptor protein CALM upon binding with phosphatidylinositol 4,5-bisphosphate-containing model membranes[J]. Communications Chemistry, 2025, 8(1): 219.\u003c/li\u003e\n\u003cli\u003eHiroto Kambara,Farshad Niazi,Lenche Kostadinova, et al. Negative regulation of the interferon response by an interferon-induced long non-coding RNA[J]. Nucleic Acids Research, 2014, 42(16): 10668-80.\u003c/li\u003e\n\u003cli\u003eGuo Yong,Li Chunxue,Zhang Rongrong, et al. Epigenetically-regulated serum GAS5 as a potential biomarker for patients with chronic hepatitis B virus infection[J]. Cancer biomarkers : section A of Disease markers, 2021, 32(2): 137-146.\u003c/li\u003e\n\u003cli\u003eCorrection to \u0026ldquo;Investigation of Long Noncoding RNA‐NRAV and Long Noncoding RNA‐Lethe Expression in Crimean\u0026minus;Congo Hemorrhagic Fever\u0026rdquo;[J]. Journal of Medical Virology, 2025, 97(2): e70245.\u003c/li\u003e\n\u003cli\u003eQiman Ma,Liangyuan Li,Yan Tang, et al. Analyses of long non-coding RNAs and mRNA profiling through RNA sequencing of MDBK cells at different stages of bovine viral diarrhea virus infection[J]. Research in Veterinary Science, 2017, 115: 508-516.\u003c/li\u003e\n\u003cli\u003eFeilong Chen,Dingding Zhang,Lifang Cheng, et al. Xiaowugui decoction alleviates experimental rheumatoid arthritis by suppressing Rab5a-mediated TLR4 internalization in macrophages[J]. Phytomedicine : International Journal of Phytotherapy and Phytopharmacology, 2024, 132: 155762.\u003c/li\u003e\n\u003cli\u003eShi Mo,Wei Tang,Jun Xie, et al. Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells[J]. Journal of Virology, 2021, 95(8).\u003c/li\u003e\n\u003cli\u003eMarina Caillet,Katy Janvier,Annegret Pelchen-Matthews, et al. Rab7A Is Required for Efficient Production of Infectious HIV-1[J]. Plos Pathogens, 2011, 7(11): e1002347.\u003c/li\u003e\n\u003cli\u003eZeyen Lisa,Prange Reinhild. Host Cell Rab GTPases in Hepatitis B Virus Infection[J]. Frontiers in cell and developmental biology, 2018, 6: 154.\u003c/li\u003e\n\u003cli\u003eChristina Bartusch,Tatjana D\u0026ouml;ring,Reinhild Prange. Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing[J]. Viruses, 2017, 9(6): 157.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bovine Viral Diarrhea Virus, Long non-coding RNA, lncSMIM14, Rab3a, Viral eplication","lastPublishedDoi":"10.21203/rs.3.rs-7956623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7956623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"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.","manuscriptTitle":"LncSMIM14 Hijacks Rab3a-Mediated Endocytosis to Promote Bovine Viral Diarrhea Virus Replication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 07:02:46","doi":"10.21203/rs.3.rs-7956623/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5d77c914-4720-461b-8558-94ae0dbfb634","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T05:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 07:02:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7956623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7956623","identity":"rs-7956623","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}