Caspase-1 Interacts with Bovine Viral Diarrhea Virus NS2-3 protein to modulate the viral titer in MDBK

Caspase-1 Interacts with Bovine Viral Diarrhea Virus NS2-3 protein to modulate the viral titer in MDBK | 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 Caspase-1 Interacts with Bovine Viral Diarrhea Virus NS2-3 protein to modulate the viral titer in MDBK Héctor Daniel Najera-Rivera, Isabela Ruelas-Mesa, Meztli Miroslava Cantera-Bravo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9359986/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Bovine Viral Diarrhea Virus (BVDV) belongs to the Flaviviridae viral family and is widely distributed worldwide, affecting millions of animals and causing significant clinical manifestations and production losses. Its genome encodes a single polyprotein that is processed into 10–12 viral proteins. Polyprotein processing is essential for viral replication, and is mediated by viral autoproteases such as Npro and NS2-3, which activate the replication machinery. Inflammasomes are innate immune system receptors/sensors that regulate the activation of caspase-1 and promote inflammation in response to viral infections. Previous studies have shown that NLRP3 and IFI16 inflammasomes are activated in bovine macrophages infected with BVDV and that this activation is related to viral titer. In this study, we hypothesized that caspase-1 interacts with BVDV proteins to cleave them during viral replication, contributing to polyprotein processing and promoting the start of the viral replication process, which has an impact on viral progeny. In silico analysis identified four putative caspase-1 cleavage sites within the Erns, NS2-3, and NS5A proteins. These sites are structurally and phylogenetically conserved across the Pestivirus genus, suggesting a common evolutionary mechanism. We observed colocalization of NS2-3 with caspase-1 in BVDV-infected MDBK. Additionally, modulation of caspases-1 activation resulted in significant changes in viral titers. Furthermore, pharmacological modulation of caspase-1 activity significantly altered viral replication levels, indicating that caspase-1 plays a key role in efficient BVDV replication. Our findings suggest that BVDV may exploit caspase-1-mediated proteolytic processing of its polyprotein to enhance replication. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bovine Viral Diarrhea Virus (BVDV) is a member of the Flaviviridae family within the Pestivirus genus and shares structural similarities with Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV) ( 1 ). Although BVDV primarily infects cattle, it can also infect other ungulate species. Its global distribution and high prevalence in cattle represent a major economic burden to the livestock industry, causing infertility, abortions, and reduced milk production ( 2 , 3 ). The BVDV genome consists of a single-stranded, positive-sense RNA that encodes a single polyprotein. This polyprotein is processed by both cellular and viral proteases in order to generate structural proteins (C, Erns, E1, and E2) and nonstructural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) ( 4 , 5 ). Nonstructural proteins (NSPs) are responsible for viral replication ( 6 ). Among these, NS2 and NS3 play a critical role in the polyprotein processing ( 7 ). Formation of the viral replication complex depends on coordinated interactions among NS3-NS4A-NS4B-NS5A-NS5B proteins, with NS3 and NS5B being particularly important. NS5B functions as an RNA-dependent RNA polymerase (RdRp), which is indispensable for viral genome replication ( 8 ). The C-terminal region of the NS2 protein exhibits cysteine protease activity responsible for the autoproteolytic cleavage of between NS2 and NS3. This cleavage is important for early stages of RNA replication ( 9 ) and is regulated by the activation of the DNAJC14 chaperone, also known as Jiv domain. NS3 is a multifunctional protein with protease, helicase, and NTPase activities and is critical for viral replication. It has been observed that free NS3 is essential for proper formation of the viral replication complex ( 10 , 9 , 5 ). The processing of these two proteins is associated with the classification of BVDV into two biotypes: cytopathic (cp) and non-cytopathic (ncp) based on their effects in cell culture. In the cytopathic biotype, the presence and accumulation of free NS3 in late stages of infection can induce apoptosis. In contrast, in the non-cytopathic biotype, the cleavage of NS2 and NS3 occurs only in early stages of infection; thus, in late stages of infection, NS2-3 remains uncleaved and the infection continues without cytopathic effects ( 9 , 11 ). During viral infection, host cells activate innate immune response mechanisms by recognizing Damage-Associated Molecular Patterns (DAMPs) or Pathogen-Associated Molecular Patterns (PAMPs) through pattern recognition receptors (PRRs), such as NOD-like receptors (NLRs) or Toll-like receptors (TLRs). Activation of these receptors triggers signaling cascades that promote the transcription of proinflammatory cytokines such as type 1 and 3 interferons (IFN), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) ( 12 , 4 , 6 ). Cytokines of the IL-1 family (IL-1β, IL-18 and IL-33) require post-translational processing for secretion. This processing is mediated by a multiprotein complex known as the inflammasome ( 12 , 13 ). This inflammasome consists of an NLR family receptor, the adaptor molecule ASC, and caspase-1. The classical activation of this complex occurs through ligand-receptor interaction, followed by coupling to ASC through the pyrin domain, which in turn promotes autocatalytic activation of procaspase-1. Activated caspase-1 then cleaves proIL-1β into its mature form, enabling its secretion and amplification of the inflammatory response ( 12 ). The most common associated inflammasome with viral infections are NLRP3, AIM2, and IFI16, which can recognize DNA, RNA, and viral proteins ( 13 , 14 ). Although inflammasome activation primarily promotes cytokine release, several viruses have evolved mechanisms to modulate this pathway, by either suppressing or exploiting it to enhance replication ( 15 ). For example, Influenza, Measles, and Nipah viruses can inhibit inflammasome assembly ( 16 ), while Yellow Fever, Dengue, or Zika viruses interfere with cytokine production signaling pathways through their NS5 protein ( 4 ). Among the DNA viruses reported to activate inflammasomes complex some examples are the varicella-zoster virus, adenovirus type 5, and bovine herpesvirus type 1 ( 14 , 17 ). Regarding RNA viruses, BVD, influenza, Zika, dengue, chikungunya, hepatitis C, and west Nile encephalitis virus (WNV) have been described as capable of producing an immune response through the inflammasome ( 14 , 18 ). In silico predictions provide insight into potential interactions between cellular and viral molecules, identifying signaling pathways and potential therapeutic targets ( 19 ). In BVDV infection, the activation of the NLRP3 ( 20 ) and IFI16 ( 21 ) inflammasomes has been identified. Moreover, inhibition of caspase-1 and the NLRP3 receptor has been shown to reduce BVDV viral titer, suggesting that caspase-1 that may play a role in promoting viral replication, potentially through interaction with viral proteins ( 21 ). However, the mechanisms underlying this proviral effect remain unclear. Therefore, the present study aims to identify the effect of inflammasome activation role on the modulation of viral titer through its effector molecule, caspase-1. Materials and methods Bioinformatic analysis Protein sequences reported for the BVDV NADL strain and viruses belonging to the Flaviviridae family were retrieved from the National Center for Biotechnology Information (NCBI) sequence database. Sequence alignment was performed using the protein Basic Local Alignment Search Tool (BLASTp) ( 22 ). Separately, each of these sequences was submitted to the ExPASy Swiss-Model program, where protein homology modeling was conducted using crystallized proteins from viruses of the same viral family using templates within the Swiss-Model database ( 23 ). De novo structural models were also created using the AlphaFold 3 program ( 24 ). Accession numbers for all sequences and templates are provided in Supplementary Material 1. Models exhibiting the highest sequence identity and the lowest QMEANDisCo scores were selected for downstream analyses. Structural comparisons were conducted using the MatchMaker tool within Chimera. Sequence identity percentages and Root Mean Square Deviation (RMSD) were obtained with an iteration cutoff of 2 Å. Visualization and analysis of these proteins and their domains were performed using the Chimera program 1.19. ( 25 ). Prediction of Caspase-1 Cleavages A total of 89 sequences of viral proteins from different members of the Flaviviridae family were used. These sequences showed a similarity in BLASTp analyses, this was evaluated for potential caspase-1 cleavage sites. Using ExPASy PeptideCutter tool (Swiss-Model variant) cleavage predictions were performed. Cleavage motifs consistent with known caspase-1 recognition sequences to analyze comparatively across taxa were identified. Phylogenetic Analysis Phylogenetic trees were constructed using RAxML under a Maximum Likelihood evolutionary model ( 26 ). Protein sequence alignments were generated by comparing BVDV-1a strain NADL against homologous sequences of proteins from viruses within the Flaviviridae family obtained from the NCBI sequence database (38 viruses and 144,948 sequences) using BLASTp ( 22 ). Accession numbers for the sequences are listed in Supplementary Material 1. Sequence alignment was performed using MAFFT ( 27 ), and alignments were visualized in JalView ( 28 ). Incomplete or redundant sequences were removed. Phylogenetic trees were generated with 10,000 bootstraps for each tree, and the bipartition tree was visualized using the FigTree program. To root the trees, the most taxonomically distant viral sequences from the Flaviviridae family were selected as outgroups. Viral propagation For propagation and acquisition of the viral stock of cp-BVDV (NADL-1) we used Madin-Darby Bovine Kidney Cells (MDBK) (ATCC® CCL-22, USA.). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA) incubated at 37°C with 5% CO 2 . For the infection assays, the medium was changed to DMEM supplemented with 2% FBS, agitated for 2 hours, and then incubated for 72 hours for cp-BVDV (NADL-1). Cells were freezed in order to lysate them to obtain the viral stock needed for the experiments. For the viral titration of cp-BVDV (NADL-1), MDBK cells were harvested on 96-well plates, performing serial tenfold dilutions of the viral inoculum, and the titer was determined by the Reed-Müench Method. Cell infection and inflammasome modulation The MDBK cells were cultured on 24-well plates at a density of 8×10 4 cells per well to perform the infection, activation or inhibition assays. Multiplicity of infection (MOI) of 2:1 was used for cp-BVDV with 500µL of DMEM medium supplemented with 2% FBS (Gibco, New York, USA). Negative control was MDBK cells cultured with DMEM and 2% FBS, positive control of inflammasome activation was treated 24 h postinfection with 300ng/mL of LPS from E. coli O26:B26, strain is ATCC 12795 (Merck, Boston, USA). For the inhibition treatments, the MDBK cells were incubated with CRID3 24 h post infection (50µM) (Merck, Boston, USA). The supernatants recovered from 48 hours post-infection. Confocal Microscopy 8×10 3 MDBK cells were harvested in a chamber slide and infected with BVDV NADL at MOI 2:1 during 24h, followed the cells were fixed with 4% PFA and washed three times with PBS 1x, after the cells were blocked during one hour (PBS 1x 5% equine sera and 0.3% de triton X-100) at room temperature, after they were washed three times with PBS and incubated 4°C overnight with primary antibodies 1/500, anti-NS2/3 (Santa Cruz, California US sc-101592) and anti-caspase-1 p10 (Santa Cruz, California US sc-515). Incubation was followed by five washes with PBS 1x and after, were incubated with secondary antibodies for one hour at room temperature (Texas Red and Alexa Fluor 488), subsequently was washed three times with PBS 1x and the Vectashield with DAPI was added (Vector laboratories). The images were captured using a confocal microscope (Nikon A1R + STORM, Tokyo, Japan). Results BVDV Erns, NS2-3, and NS5A proteins contain putative caspase-1 cleavage sites To identify potential in silico interactions between caspase-1 and the Pestivirus polyprotein, cleavage site prediction analysis were performed. Seven potential cleavage sites were identified within the Erns, E2, NS2-3, and NS5A proteins. These sites were, specifically located at aspartic acid residues as follows: position 48 in Erns; position 36 in E2; positions 60, 74 and 157 in NS2-3; and position 48 in NS5A (Fig. 1 a- 1 f and Table 1 ). The predicted cleavage sites for each protein are highlighted in the protein models (Fig. 1 g- 1 l). Table 1 Predicted cleavage sites by caspase-1 in pestivirus polyprotein Virus Protein Likely cleavage by caspase-1 (AA position) BVDV Erns NS2-3 NS5A D48 D74 and D157 D48 CSFV E2 NS2-3 D36 D60 BDV NS2-3 D157 Supplemental 1. Accession numbers for all sequences and templates used NS2-3 protein exhibits structural and phylogenetic similarities across the Flaviviridae family Following the identification of potential In silico interactions, it was observed that the predicted sites in the NS2-3 protein were conserved across genus. Phylogenetic analyses were conducted to determine evolutionary relationships and assess cleavage site conservation within the Flaviviridae family. NS2-3 protein phylogeny was constructed with four sequences of the Pestivirus genus. The phylogenetic tree for BVDV NS2-3 protein revealed that this protein forms an exclusive monophyletic group together with Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV) (Fig. 2 a), consisting with their classification within the Pestivirus genus. Structural comparison analysis demonstrated high homology, with sequence identity percentages exceeding 88% and RMSD values of 0.928 Å for CSFV and 0.896 Å for BDV (Fig. 2 b). Among the proteins with predicted caspase-1 cleavage sites, NS2-3 plays a central role in viral replication. Notably, the predicted caspase-1 cleavage sites were located proximal to the natural cleavage site by other viral proteases such as NS3/4A, which cut the complete protein in NS2 and NS3 proteins. NS3 protein phylogeny was constructed with 37 sequences from the viruses of the Flaviviridae family. This protein is a highly conserved protein throughout the Flaviviridae family (Fig. 2 c) and also shows a high homology in terms of sequence identity percentages, exceeding 89% and structural similarities between the pestiviruses analyzed with RMSD values of less than 0.8 Å (Fig. 2 d). Since the predicted cleavage In silico in NS2-3, would favor the release of NS3, it was determined whether the domains of this protein were conserved after caspase-1 cleavage. It was identified that none of the domains were cut by the cleavage site predicted by caspase-1 (Fig. 2 e) and most likely conserved its biological activity, therefore, it was analyzed for its potential interaction with caspase-1 In vitro . Caspase-1 Colocalizes with BVDV NS2-3 Protein in Infected MDBK Cells To experimentally validate the interaction suggested by the In silico predictions, MDBK cells were infected with the BVDV NADL strain. Cytopathic effects were observed to be characterized by vacuolization and monolayer lysis, which were most evident at 48 hours post-infection (hpi) (Fig. 3 a). Infection was confirmed by RT-PCR (Fig. 3 c). As expected, non-cytopathic effects were observed in uninfected control cells. Additionally, viral titers were calculated using the Reed-Muench method, showing a time-dependent increase (Fig. 3 b). To assess potential In vitro interactions between caspase-1 and the BVDV NS2-3 protein, confocal microscopy was performed. Infected cells showed an increased presence of caspase-1 compared to the negative control (Fig. 3 d). Merged images of caspase-1 and the viral NS2-3 protein revealed yellow spots corresponding to a possible colocalization. This suggests a close physical association between caspase-1 and the viral NS2-3 protein expressed only during the viral replicative cycle. Besides, it was identified as the presence of caspase-1 active at 24 hpi by Western Blot (Fig. 3 e) Modulation of Caspase-1 Activity Directly Impacts BVDV Viral Replication in MDBK Cells To explore the functional consequences of the caspase-1/NS2-3 interaction, caspase-1 activity was modulated using LPS and CRID3 in BVDV cp-NADL-infected MDBK cells (MOI 2:1). Stimulating infected cells with LPS resulted in an increase in viral titer (Fig. 4 b). Conversely, inhibition of the NLRP3 inflammasome pathway drastically reduced the viral load (Fig. 4 c). Representative images for both assays are shown in Fig. 4 a. Discussion Our results demonstrate that the modulation of caspase-1 activity has a significant impact on the viral titer of BVDV in MDBK cells. We observed that stimulation with LPS increases viral titers, whereas inhibition with CRID3 drastically decreases them. These findings are consistent with previous reports; for instance, luteolin has been shown to attenuate p65 phosphorylation, reducing the expression of inflammasome-related genes and, causes the recruitment of pro-caspase-1, which ultimately limits caspase-1 activation and viral replication ( 29 ). Likewise, Gallegos-Rodarte et al. (2023) reported that the use of CRID3 in bovine macrophages infected with the NADL strain decreased viral titers. Overall, these findings suggest that inflammasome components, in both the upstream and downstream of the inflammasome pathway, exert a direct effect on the viral replication cycle. However, the precise molecular mechanism has yet to be fully elucidated. In this context, the NS2-3 protein is a key candidate. Since NS2-3 is essential for Pestivirus replication, its interaction with caspase-1 (suggested by our In silico and In vitro analyses) could explain the observed biological changes. The proteolytic processing of viral proteins by host caspases is not an isolated phenomenon; it has been documented in coronaviruses, such as Transmissible Gastroenteritis Virus (TGEV) and Porcine Epidemic Diarrhea Virus (PEDV), where the nucleocapsid protein is susceptible to cleavage by caspases 6 and 7 ( 30 , 31 ). Similarly, in Feline Calicivirus, caspase-2 can process capsid proteins, and in sarcoma-associated herpesviruses, similar interactions that modulate pathogenesis have been detected ( 32 , 33 ). These precedents support the possibility that BVDV may exploit host proteases to optimize replication. The colocalization between caspase-1 and the NS2-3 protein provides physical evidence linking innate immune response to the viral replication machinery. However, biochemical validation experiments such as In vitro cleavage assays or targeted Western Blotting are necessary to confirm the caspase-1 mediated proteolytic processing. It is particularly relevant to determine whether this mechanism differs between the cytopathic (cp) and non-cytopathic (ncp) biotypes, considering their distinct replication kinetics and that the ncp biotype specifically activates the IFI16 inflammasome. Since both biotypes induce caspase-1-mediated IL-1β release ( 34 , 21 ), the interaction observed with NS2-3 could represent a point of convergence in BVDV pathogenesis, regardless of the pattern recognition receptor (PRR) initially activated. The strong conservation observed among BVDV-1, CSFV and BDV, together with the stability of their predicted protein structures, suggest evolutionary constraints that may preserve functional interactions across pestiviruses.. When comparing NS2-3 BVDV-1 protein to CSFV and BDV, we observed a low RMSD value 0.928 Å and 0.896 Å, respectively; besides, the identity percentage is high with both viruses (BDV: 90.76% and CSFV: 88.9%). The pattern is similar when comparing the NS3 protein among these viruses; high identity percentage (CSFV: 89.9% and BDV: 91.65%) and low RMSD which is reflected in the phylogenetic tree. This parameter is lower for BDV (0.246 Å) than for CSFV (0.782 Å) which is consistent with keeping the predicted cut site by caspase-1 at the same position for BDV. Although there are subtle differences, that suggest certain amino acid changes in BDV resulted in structural modifications not observed in CSFV, which may have had a decisive evolutionary weight in the divergence of these species within the Pestivirus genus. In this study, we focused solely on the cp biotype (NADL reference strain). It has been reported that the inflammasome acts as a regulator of viral load not only through caspase-1 interaction but also through various inflammasome components ( 16 , 14 ). This has been observed in viruses such as Hepatitis and Classical Swine Fever, p7 viroporin induces IL-1β secretion through caspase-1 activation as a viral regulation mechanism ( 12 ). Therefore, future studies should expand the analysis to other viral proteins, other inflammasome components, and the ncp biotype in the future. However, since Gallegos-Rodarte et al. (2023) demonstrated that caspase-1 activation in the ncp biotype is specifically linked to the IFI16 inflammasome, the stimuli used in this work (targeted at the canonical pathway) might not be suitable for that model, since the innate immune response and the heterogeneity of molecules involved in the replication of both biotypes. In silico predictions are fundamental for inferring biological functions, especially for proteins that have not yet been crystallized. In addition to NS2-3, we identified potential caspase-1 cleavage sites in the Erns and NS5A proteins, which could enhance the observed biological effect. Nonetheless, molecular docking studies are required to precisely characterize these interactions. These tools have previously enabled the demonstration of genomic proximity between Ebola and Marburg viruses ( 35 ), the evolutionary tracking of SARS-CoV-2 ( 36 ), and the homology-based susceptibility analysis of the ACE2 protein in different hosts ( 37 ). In our case, the integration of computational models optimizes In vitro and In vivo resources by directing experimentation toward molecular targets with a higher probability of functional relevance. Conclusion Therefore, the present study identified the effect of inflammasome activation role on the modulation of viral titer through its effector molecule, caspase-1. The cytopathic NADL strain of BVDV colocalizes with caspase-1 in infected MDBK cells, and the stimulation or inhibition of this pathway directly affects the viral titer. This results in an increase or decrease of viral load, respectively. These findings suggest that caspase-1 plays a functional role in the BVDV replication cycle and may represent a novel target for antiviral strategies. Declarations Funding This work was financed by the DGAPA-PAPIIT IN214724. Competing interests All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Author Contribution Data curation: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Cervantes-Torres J., Benítez-Guzmán A., Formal análisis: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Gutiérrez-Guerrero YT., Benítez-Guzmán A., Investigation: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Benítez-Guzmán A., Methodology: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Gutiérrez-Guerrero YT., Supervision: Nájera-Rivera H.D., Benítez-Guzmán A., Validation: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Writing - original draft: Nájera-Rivera H.D., Gutiérrez-Guerrero YT., Benítez-Guzmán A., Writing - Review and Editing: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Gutiérrez-Guerrero YT., Cervantes-Torres J., Benítez-Guzmán A., Conceptualization: Nájera-Rivera H.D., Ruelas-Mesa I., Gutiérrez-Guerrero YT., Benítez-Guzmán A., Resources: Benítez-Guzmán A., Visualization: Nájera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Gutiérrez-Guerrero YT., Funding acquisition: Benítez-Guzmán A., Project administration: Nájera-Rivera H.D., Benítez-Guzmán A. Acknowledgement Sincere thanks to Dr. Miguel Tapia (Unidad de Microscopía Confocal, Institututo de Investigaciones Biomédicas) and Dr René Segura from the Unidad de Investigación-FMVZ for their technical assistance to carrying out this project. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. No ethical approval was required, as no animals or human volunteers were involved in this study. References Simmonds P, Becher P, Bukh J, Gould EA, Meyers G, Monath T et al (2017) ICTV Virus Taxonomy Profile: Flaviviridae. 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Nat 13 de junio de 630(8016):493–500. 10.1038/s41586-024-07487-w Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC et al (2004) UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem octubre de 25(13):1605–1612. 10.1002/jcc.20084 Stamatakis A (2014) Bioinf 1 de mayo de 30(9):1312–1313. 10.1093/bioinformatics/btu033 . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies Rozewicki J, Li S, Amada KM, Standley DM, Katoh K MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 7 de mayo de 2019;gkz342. 10.1093/nar/gkz342 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinf 1 de mayo de 25(9):1189–1191. 10.1093/bioinformatics/btp033 Cai D, Liu Q, Shen Z, Tian B, Gao J, Lin Y et al (2026) Luteolin Inhibits Bovine Viral Diarrhea Virus Replication by Disrupting Viral Internalization and Replication and Interfering with the NF-κB/STAT3-NLRP3 Inflammasome Pathway. Vet Sci 7 de enero de 13(1):57. 10.3390/vetsci13010057 Eléouët JF, Slee EA, Saurini F, Castagné N, Poncet D, Garrido C et al (2000) The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and – 7 during TGEV-Induced Apoptosis. J Virol mayo de 74(9):3975–3983. 10.1128/JVI.74.9.3975-3983.2000 Oh C, Kim Y, Chang KO (2020) Caspase-mediated cleavage of nucleocapsid protein of a protease-independent porcine epidemic diarrhea virus strain. Virus Res agosto de 285:198026. 10.1016/j.virusres.2020.198026 Al-Molawi N, Beardmore VA, Carter MJ, Kass GEN, Roberts LO (2003) Caspase-mediated cleavage of the feline calicivirus capsid protein. J Gen Virol 1 de mayo de 84(5):1237–1244. 10.1099/vir.0.18840-0 Davis DA, Astter Y, Treco EN, Shrestha P, Stream A, Haque M et al (2025) Caspase cleavage of Kaposi sarcoma-associated herpesvirus proteins: a role for K5 in preventing caspase-mediated cell death during lytic replication. Jung JU, editor. J Virol 23 de septiembre de 99(9):e00622–e00625. 10.1128/jvi.00622-25 La Polla R, Testard MC, Goumaidi A, Chapot E, Legras-Lachuer C, De Saint-Vis B (2022) Identification of differentially expressed gene pathways between cytopathogenic and non-cytopathogenic BVDV-1 strains by analysis of the transcriptome of infected primary bovine cells. Virol febrero de 567:34–46. 10.1016/j.virol.2021.12.005 Suzuki Y, Gojobori T (1997) The origin and evolution of Ebola and Marburg viruses. Mol Biol Evol 1 de agosto de 14(8):800–806. 10.1093/oxfordjournals.molbev.a025820 Benvenuto D, Giovanetti M, Ciccozzi A, Spoto S, Angeletti S, Ciccozzi M (2020) The 2019-new coronavirus epidemic: Evidence for virus evolution. J Med Virol abril de 92(4):455–459. 10.1002/jmv.25688 Piplani S, Singh PK, Winkler DA, Petrovsky N (2021) In silico comparison of SARS-CoV-2 spike protein-ACE2 binding affinities across species and implications for virus origin. Sci Rep 24 de junio de 11(1):13063. 10.1038/s41598-021-92388-5 Additional Declarations No competing interests reported. Supplementary Files supplementarymaterial1.xlsx Supplemental 1. Accession numbers for all sequences and templates used Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 May, 2026 Reviews received at journal 30 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 09 Apr, 2026 Submission checks completed at journal 09 Apr, 2026 First submitted to journal 08 Apr, 2026 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. 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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-9359986","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629664558,"identity":"173788e2-409b-4fb7-b034-a3e458f56cb3","order_by":0,"name":"Héctor Daniel Najera-Rivera","email":"","orcid":"","institution":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Héctor","middleName":"Daniel","lastName":"Najera-Rivera","suffix":""},{"id":629664562,"identity":"eafcb1c9-9c44-427d-9141-ecebc24e29bf","order_by":1,"name":"Isabela Ruelas-Mesa","email":"","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":false,"prefix":"","firstName":"Isabela","middleName":"","lastName":"Ruelas-Mesa","suffix":""},{"id":629664563,"identity":"89fff21f-6b34-457f-88c4-445609a2f4ca","order_by":2,"name":"Meztli Miroslava Cantera-Bravo","email":"","orcid":"","institution":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México.","correspondingAuthor":false,"prefix":"","firstName":"Meztli","middleName":"Miroslava","lastName":"Cantera-Bravo","suffix":""},{"id":629664566,"identity":"8a241630-52a9-4c57-8a02-cf2572c2345f","order_by":3,"name":"Claudia Gallegos-Rodarte","email":"","orcid":"","institution":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México.","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Gallegos-Rodarte","suffix":""},{"id":629664570,"identity":"22b8042f-e73d-4172-a003-d8085df92d82","order_by":4,"name":"Yocelyn Teresa Gutiérrez-Guerrero","email":"","orcid":"","institution":"University of California, Berkeley","correspondingAuthor":false,"prefix":"","firstName":"Yocelyn","middleName":"Teresa","lastName":"Gutiérrez-Guerrero","suffix":""},{"id":629664571,"identity":"17cb3d72-4602-44ac-9adb-b76223fd6a00","order_by":5,"name":"Jacquelynne Cervantes-Torres","email":"","orcid":"","institution":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México.","correspondingAuthor":false,"prefix":"","firstName":"Jacquelynne","middleName":"","lastName":"Cervantes-Torres","suffix":""},{"id":629664572,"identity":"d97d9d33-6c88-44d9-997f-e62866d80593","order_by":6,"name":"Alejandro Benítez-Guzmán","email":"data:image/png;base64,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","orcid":"","institution":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México.","correspondingAuthor":true,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Benítez-Guzmán","suffix":""}],"badges":[],"createdAt":"2026-04-08 17:39:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9359986/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9359986/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108006678,"identity":"8c0e27d1-afd4-4af6-9182-7a203a7ed2ec","added_by":"auto","created_at":"2026-04-28 12:56:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":481412,"visible":true,"origin":"","legend":"\u003cp\u003eStructural models of BVDV, CSFV, and BDV proteins and predicted caspase-1 cleavage sites. a) Cleavage site for BVDV Erns protein. b) Cleavage sites for BVDV NS2-3 protein. c) Cleavage site for BVDV NS5A protein. d) Cleavage site for CSFV E2 protein. e) Cleavage site for CSFV NS2-3 protein. f) Cleavage site for BDV NS2-3 protein. g) BVDV Erns protein model. h) BVDV NS2-3 protein model. i) BVDV NS5A protein model. j) CSFV E2 protein model. k) CSFV NS2-3 protein model. l) BDV NS2-3 protein model. Green circles indicate the predicted cleavage sites within the protein structures.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/b6a0f204ea3bc8d4e3946632.png"},{"id":108006390,"identity":"f13deaaa-cea7-4813-9db5-a9947371f23c","added_by":"auto","created_at":"2026-04-28 12:55:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1128524,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic, structural, and functional domain analysis of NS2-3 and NS3 proteins. a) Phylogenetic tree of the NS2-3 protein. b) Structural superposition of NS2-3 protein models: BVDV-1 (gold) and CSFV (blue) or BDV (blue). c) Phylogenetic tree of the NS3 protein. d) Structural comparison of NS3 protein models: BVDV-1 (gold) and CSFV (blue) or BDV (blue). e) Functional domains of the BVDV NS2-3 protein: Peptidase C74 (Yellow), Jiv90 (Pink), Peptidase S31 (Orange), DEXH-box helicase domain of NS3 protease-helicase (Cyan), C-terminal helicase domain of the RNA helicase A (RHA) family helicases (Blue), and predicted cleavage sites (Green).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/b1cfde60ea05265c5542e493.png"},{"id":108006715,"identity":"c40d5cfb-023b-436d-a548-c55f9b110d11","added_by":"auto","created_at":"2026-04-28 12:56:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1443586,"visible":true,"origin":"","legend":"\u003cp\u003eBVDV NS2-3 protein colocalizes with caspase-1 in MDBK cells. a) Cytopathic effect of BVDV at 24 and 48 h post-infection. b) TCID50% of BVDV infection in MDBK cells at 24 and 48 hpi. c) Identification of the BVDV 5’ UTR in the infected culture. d) Colocalization of BVDV NS2-3 viral protein with caspase-1 in MDBK cells. e) Identification of active caspase-1 in MDBK cells by Western Blot (WB).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/ee7d89fc60fcda9e05cd572b.png"},{"id":107905864,"identity":"27af4511-a513-4f4e-a115-835445cd0c2c","added_by":"auto","created_at":"2026-04-27 12:36:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":948826,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of caspase-1 modulation on BVDV replication in MDBK cells. a) Cytopathic effect of BVDV at 48 h post-infection in MDBK cells with LPS or CRID3. b) TCID50% in BVDV-infected MDBK cells at 48 hpi with LPS stimulation. c) TCID50% in BVDV-infected MDBK cells at 48 with CRID3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/8b8d17ad7e24ce8e09405aed.png"},{"id":108490985,"identity":"15814b58-1dc4-4761-8493-1b3de1cc1c8f","added_by":"auto","created_at":"2026-05-05 09:50:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4240027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/72becb08-0c3c-44a4-b610-9fea2fc394e4.pdf"},{"id":107905863,"identity":"59c8fa17-eb0c-4dfe-9ca0-070c0323ce6e","added_by":"auto","created_at":"2026-04-27 12:36:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25728,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental 1. Accession numbers for all sequences and templates used\u003c/p\u003e","description":"","filename":"supplementarymaterial1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9359986/v1/66248cbce2c88e879bd45ad5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Caspase-1 Interacts with Bovine Viral Diarrhea Virus NS2-3 protein to modulate the viral titer in MDBK","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine Viral Diarrhea Virus (BVDV) is a member of the \u003cem\u003eFlaviviridae\u003c/em\u003e family within the \u003cem\u003ePestivirus\u003c/em\u003e genus and shares structural similarities with Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although BVDV primarily infects cattle, it can also infect other ungulate species. Its global distribution and high prevalence in cattle represent a major economic burden to the livestock industry, causing infertility, abortions, and reduced milk production (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe BVDV genome consists of a single-stranded, positive-sense RNA that encodes a single polyprotein. This polyprotein is processed by both cellular and viral proteases in order to generate structural proteins (C, Erns, E1, and E2) and nonstructural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Nonstructural proteins (NSPs) are responsible for viral replication (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Among these, NS2 and NS3 play a critical role in the polyprotein processing (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Formation of the viral replication complex depends on coordinated interactions among NS3-NS4A-NS4B-NS5A-NS5B proteins, with NS3 and NS5B being particularly important. NS5B functions as an RNA-dependent RNA polymerase (RdRp), which is indispensable for viral genome replication (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe C-terminal region of the NS2 protein exhibits cysteine protease activity responsible for the autoproteolytic cleavage of between NS2 and NS3. This cleavage is important for early stages of RNA replication (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and is regulated by the activation of the DNAJC14 chaperone, also known as Jiv domain. NS3 is a multifunctional protein with protease, helicase, and NTPase activities and is critical for viral replication. It has been observed that free NS3 is essential for proper formation of the viral replication complex (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The processing of these two proteins is associated with the classification of BVDV into two biotypes: cytopathic (cp) and non-cytopathic (ncp) based on their effects in cell culture. In the cytopathic biotype, the presence and accumulation of free NS3 in late stages of infection can induce apoptosis. In contrast, in the non-cytopathic biotype, the cleavage of NS2 and NS3 occurs only in early stages of infection; thus, in late stages of infection, NS2-3 remains uncleaved and the infection continues without cytopathic effects (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring viral infection, host cells activate innate immune response mechanisms by recognizing Damage-Associated Molecular Patterns (DAMPs) or Pathogen-Associated Molecular Patterns (PAMPs) through pattern recognition receptors (PRRs), such as NOD-like receptors (NLRs) or Toll-like receptors (TLRs). Activation of these receptors triggers signaling cascades that promote the transcription of proinflammatory cytokines such as type 1 and 3 interferons (IFN), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCytokines of the IL-1 family (IL-1β, IL-18 and IL-33) require post-translational processing for secretion. This processing is mediated by a multiprotein complex known as the inflammasome (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This inflammasome consists of an NLR family receptor, the adaptor molecule ASC, and caspase-1. The classical activation of this complex occurs through ligand-receptor interaction, followed by coupling to ASC through the pyrin domain, which in turn promotes autocatalytic activation of procaspase-1. Activated caspase-1 then cleaves proIL-1β into its mature form, enabling its secretion and amplification of the inflammatory response (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most common associated inflammasome with viral infections are NLRP3, AIM2, and IFI16, which can recognize DNA, RNA, and viral proteins (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although inflammasome activation primarily promotes cytokine release, several viruses have evolved mechanisms to modulate this pathway, by either suppressing or exploiting it to enhance replication (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). For example, Influenza, Measles, and Nipah viruses can inhibit inflammasome assembly (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), while Yellow Fever, Dengue, or Zika viruses interfere with cytokine production signaling pathways through their NS5 protein (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Among the DNA viruses reported to activate inflammasomes complex some examples are the varicella-zoster virus, adenovirus type 5, and bovine herpesvirus type 1 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Regarding RNA viruses, BVD, influenza, Zika, dengue, chikungunya, hepatitis C, and west Nile encephalitis virus (WNV) have been described as capable of producing an immune response through the inflammasome (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e predictions provide insight into potential interactions between cellular and viral molecules, identifying signaling pathways and potential therapeutic targets (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn BVDV infection, the activation of the NLRP3 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) and IFI16 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) inflammasomes has been identified. Moreover, inhibition of caspase-1 and the NLRP3 receptor has been shown to reduce BVDV viral titer, suggesting that caspase-1 that may play a role in promoting viral replication, potentially through interaction with viral proteins (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, the mechanisms underlying this proviral effect remain unclear. Therefore, the present study aims to identify the effect of inflammasome activation role on the modulation of viral titer through its effector molecule, caspase-1.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eBioinformatic analysis\u003c/p\u003e \u003cp\u003eProtein sequences reported for the BVDV NADL strain and viruses belonging to the \u003cem\u003eFlaviviridae\u003c/em\u003e family were retrieved from the National Center for Biotechnology Information (NCBI) sequence database. Sequence alignment was performed using the protein Basic Local Alignment Search Tool (BLASTp) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Separately, each of these sequences was submitted to the ExPASy Swiss-Model program, where protein homology modeling was conducted using crystallized proteins from viruses of the same viral family using templates within the Swiss-Model database (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). De novo structural models were also created using the AlphaFold 3 program (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Accession numbers for all sequences and templates are provided in Supplementary Material 1. Models exhibiting the highest sequence identity and the lowest QMEANDisCo scores were selected for downstream analyses. Structural comparisons were conducted using the MatchMaker tool within Chimera. Sequence identity percentages and Root Mean Square Deviation (RMSD) were obtained with an iteration cutoff of 2 \u0026Aring;. Visualization and analysis of these proteins and their domains were performed using the Chimera program 1.19. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrediction of Caspase-1 Cleavages\u003c/p\u003e \u003cp\u003eA total of 89 sequences of viral proteins from different members of the Flaviviridae family were used. These sequences showed a similarity in BLASTp analyses, this was evaluated for potential caspase-1 cleavage sites. Using ExPASy PeptideCutter tool (Swiss-Model variant) cleavage predictions were performed. Cleavage motifs consistent with known caspase-1 recognition sequences to analyze comparatively across taxa were identified.\u003c/p\u003e \u003cp\u003ePhylogenetic Analysis\u003c/p\u003e \u003cp\u003ePhylogenetic trees were constructed using RAxML under a Maximum Likelihood evolutionary model (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Protein sequence alignments were generated by comparing BVDV-1a strain NADL against homologous sequences of proteins from viruses within the \u003cem\u003eFlaviviridae\u003c/em\u003e family obtained from the NCBI sequence database (38 viruses and 144,948 sequences) using BLASTp (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Accession numbers for the sequences are listed in Supplementary Material 1. Sequence alignment was performed using MAFFT (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and alignments were visualized in JalView (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Incomplete or redundant sequences were removed. Phylogenetic trees were generated with 10,000 bootstraps for each tree, and the bipartition tree was visualized using the FigTree program. To root the trees, the most taxonomically distant viral sequences from the \u003cem\u003eFlaviviridae\u003c/em\u003e family were selected as outgroups.\u003c/p\u003e \u003cp\u003eViral propagation\u003c/p\u003e \u003cp\u003eFor propagation and acquisition of the viral stock of cp-BVDV (NADL-1) we used Madin-Darby Bovine Kidney Cells (MDBK) (ATCC\u0026reg; CCL-22, USA.). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA) incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. For the infection assays, the medium was changed to DMEM supplemented with 2% FBS, agitated for 2 hours, and then incubated for 72 hours for cp-BVDV (NADL-1). Cells were freezed in order to lysate them to obtain the viral stock needed for the experiments. For the viral titration of cp-BVDV (NADL-1), MDBK cells were harvested on 96-well plates, performing serial tenfold dilutions of the viral inoculum, and the titer was determined by the Reed-M\u0026uuml;ench Method.\u003c/p\u003e \u003cp\u003eCell infection and inflammasome modulation\u003c/p\u003e \u003cp\u003eThe MDBK cells were cultured on 24-well plates at a density of 8\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well to perform the infection, activation or inhibition assays. Multiplicity of infection (MOI) of 2:1 was used for cp-BVDV with 500\u0026micro;L of DMEM medium supplemented with 2% FBS (Gibco, New York, USA). Negative control was MDBK cells cultured with DMEM and 2% FBS, positive control of inflammasome activation was treated 24 h postinfection with 300ng/mL of LPS from \u003cem\u003eE. coli\u003c/em\u003e O26:B26, strain is ATCC 12795 (Merck, Boston, USA). For the inhibition treatments, the MDBK cells were incubated with CRID3 24 h post infection (50\u0026micro;M) (Merck, Boston, USA). The supernatants recovered from 48 hours post-infection.\u003c/p\u003e \u003cp\u003eConfocal Microscopy\u003c/p\u003e \u003cp\u003e8\u0026times;10\u003csup\u003e3\u003c/sup\u003e MDBK cells were harvested in a chamber slide and infected with BVDV NADL at MOI 2:1 during 24h, followed the cells were fixed with 4% PFA and washed three times with PBS 1x, after the cells were blocked during one hour (PBS 1x 5% equine sera and 0.3% de triton X-100) at room temperature, after they were washed three times with PBS and incubated 4\u0026deg;C overnight with primary antibodies 1/500, anti-NS2/3 (Santa Cruz, California US sc-101592) and anti-caspase-1 p10 (Santa Cruz, California US sc-515). Incubation was followed by five washes with PBS 1x and after, were incubated with secondary antibodies for one hour at room temperature (Texas Red and Alexa Fluor 488), subsequently was washed three times with PBS 1x and the Vectashield with DAPI was added (Vector laboratories). The images were captured using a confocal microscope (Nikon A1R\u0026thinsp;+\u0026thinsp;STORM, Tokyo, Japan).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eBVDV Erns, NS2-3, and NS5A proteins contain putative caspase-1 cleavage sites\u003c/p\u003e \u003cp\u003eTo identify potential \u003cem\u003ein silico\u003c/em\u003e interactions between caspase-1 and the \u003cem\u003ePestivirus\u003c/em\u003e polyprotein, cleavage site prediction analysis were performed. Seven potential cleavage sites were identified within the Erns, E2, NS2-3, and NS5A proteins. These sites were, specifically located at aspartic acid residues as follows: position 48 in Erns; position 36 in E2; positions 60, 74 and 157 in NS2-3; and position 48 in NS5A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The predicted cleavage sites for each protein are highlighted in the protein models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePredicted cleavage sites by caspase-1 in pestivirus polyprotein\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLikely cleavage by caspase-1 (AA position)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBVDV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eErns\u003c/p\u003e \u003cp\u003eNS2-3\u003c/p\u003e \u003cp\u003eNS5A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD48\u003c/p\u003e \u003cp\u003eD74 and D157\u003c/p\u003e \u003cp\u003eD48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSFV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE2\u003c/p\u003e \u003cp\u003eNS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD36\u003c/p\u003e \u003cp\u003eD60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBDV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD157\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSupplemental 1. Accession numbers for all sequences and templates used\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNS2-3 protein exhibits structural and phylogenetic similarities across the \u003cem\u003eFlaviviridae\u003c/em\u003e family\u003c/p\u003e \u003cp\u003eFollowing the identification of potential \u003cem\u003eIn silico\u003c/em\u003e interactions, it was observed that the predicted sites in the NS2-3 protein were conserved across genus. Phylogenetic analyses were conducted to determine evolutionary relationships and assess cleavage site conservation within the \u003cem\u003eFlaviviridae\u003c/em\u003e family. NS2-3 protein phylogeny was constructed with four sequences of the \u003cem\u003ePestivirus\u003c/em\u003e genus. The phylogenetic tree for BVDV NS2-3 protein revealed that this protein forms an exclusive monophyletic group together with Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), consisting with their classification within the \u003cem\u003ePestivirus\u003c/em\u003e genus. Structural comparison analysis demonstrated high homology, with sequence identity percentages exceeding 88% and RMSD values of 0.928 \u0026Aring; for CSFV and 0.896 \u0026Aring; for BDV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Among the proteins with predicted caspase-1 cleavage sites, NS2-3 plays a central role in viral replication. Notably, the predicted caspase-1 cleavage sites were located proximal to the natural cleavage site by other viral proteases such as NS3/4A, which cut the complete protein in NS2 and NS3 proteins. NS3 protein phylogeny was constructed with 37 sequences from the viruses of the \u003cem\u003eFlaviviridae\u003c/em\u003e family. This protein is a highly conserved protein throughout the \u003cem\u003eFlaviviridae\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and also shows a high homology in terms of sequence identity percentages, exceeding 89% and structural similarities between the pestiviruses analyzed with RMSD values of less than 0.8 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Since the predicted cleavage \u003cem\u003eIn silico\u003c/em\u003e in NS2-3, would favor the release of NS3, it was determined whether the domains of this protein were conserved after caspase-1 cleavage. It was identified that none of the domains were cut by the cleavage site predicted by caspase-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and most likely conserved its biological activity, therefore, it was analyzed for its potential interaction with caspase-1 \u003cem\u003eIn vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCaspase-1 Colocalizes with BVDV NS2-3 Protein in Infected MDBK Cells\u003c/p\u003e \u003cp\u003eTo experimentally validate the interaction suggested by the \u003cem\u003eIn silico\u003c/em\u003e predictions, MDBK cells were infected with the BVDV NADL strain. Cytopathic effects were observed to be characterized by vacuolization and monolayer lysis, which were most evident at 48 hours post-infection (hpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Infection was confirmed by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). As expected, non-cytopathic effects were observed in uninfected control cells. Additionally, viral titers were calculated using the Reed-Muench method, showing a time-dependent increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess potential \u003cem\u003eIn vitro\u003c/em\u003e interactions between caspase-1 and the BVDV NS2-3 protein, confocal microscopy was performed. Infected cells showed an increased presence of caspase-1 compared to the negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Merged images of caspase-1 and the viral NS2-3 protein revealed yellow spots corresponding to a possible colocalization. This suggests a close physical association between caspase-1 and the viral NS2-3 protein expressed only during the viral replicative cycle. Besides, it was identified as the presence of caspase-1 active at 24 hpi by Western Blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee)\u003c/p\u003e \u003cp\u003eModulation of Caspase-1 Activity Directly Impacts BVDV Viral Replication in MDBK Cells\u003c/p\u003e \u003cp\u003eTo explore the functional consequences of the caspase-1/NS2-3 interaction, caspase-1 activity was modulated using LPS and CRID3 in BVDV cp-NADL-infected MDBK cells (MOI 2:1). Stimulating infected cells with LPS resulted in an increase in viral titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Conversely, inhibition of the NLRP3 inflammasome pathway drastically reduced the viral load (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Representative images for both assays are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results demonstrate that the modulation of caspase-1 activity has a significant impact on the viral titer of BVDV in MDBK cells. We observed that stimulation with LPS increases viral titers, whereas inhibition with CRID3 drastically decreases them. These findings are consistent with previous reports; for instance, luteolin has been shown to attenuate p65 phosphorylation, reducing the expression of inflammasome-related genes and, causes the recruitment of pro-caspase-1, which ultimately limits caspase-1 activation and viral replication (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Likewise, Gallegos-Rodarte et al. (2023) reported that the use of CRID3 in bovine macrophages infected with the NADL strain decreased viral titers. Overall, these findings suggest that inflammasome components, in both the upstream and downstream of the inflammasome pathway, exert a direct effect on the viral replication cycle. However, the precise molecular mechanism has yet to be fully elucidated.\u003c/p\u003e \u003cp\u003eIn this context, the NS2-3 protein is a key candidate. Since NS2-3 is essential for \u003cem\u003ePestivirus\u003c/em\u003e replication, its interaction with caspase-1 (suggested by our \u003cem\u003eIn silico\u003c/em\u003e and \u003cem\u003eIn vitro\u003c/em\u003e analyses) could explain the observed biological changes. The proteolytic processing of viral proteins by host caspases is not an isolated phenomenon; it has been documented in coronaviruses, such as Transmissible Gastroenteritis Virus (TGEV) and Porcine Epidemic Diarrhea Virus (PEDV), where the nucleocapsid protein is susceptible to cleavage by caspases 6 and 7 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Similarly, in Feline Calicivirus, caspase-2 can process capsid proteins, and in sarcoma-associated herpesviruses, similar interactions that modulate pathogenesis have been detected (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). These precedents support the possibility that BVDV may exploit host proteases to optimize replication.\u003c/p\u003e \u003cp\u003eThe colocalization between caspase-1 and the NS2-3 protein provides physical evidence linking innate immune response to the viral replication machinery. However, biochemical validation experiments such as \u003cem\u003eIn vitro\u003c/em\u003e cleavage assays or targeted Western Blotting are necessary to confirm the caspase-1 mediated proteolytic processing. It is particularly relevant to determine whether this mechanism differs between the cytopathic (cp) and non-cytopathic (ncp) biotypes, considering their distinct replication kinetics and that the ncp biotype specifically activates the IFI16 inflammasome. Since both biotypes induce caspase-1-mediated IL-1β release (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), the interaction observed with NS2-3 could represent a point of convergence in BVDV pathogenesis, regardless of the pattern recognition receptor (PRR) initially activated.\u003c/p\u003e \u003cp\u003eThe strong conservation observed among BVDV-1, CSFV and BDV, together with the stability of their predicted protein structures, suggest evolutionary constraints that may preserve functional interactions across pestiviruses.. When comparing NS2-3 BVDV-1 protein to CSFV and BDV, we observed a low RMSD value 0.928 \u0026Aring; and 0.896 \u0026Aring;, respectively; besides, the identity percentage is high with both viruses (BDV: 90.76% and CSFV: 88.9%). The pattern is similar when comparing the NS3 protein among these viruses; high identity percentage (CSFV: 89.9% and BDV: 91.65%) and low RMSD which is reflected in the phylogenetic tree. This parameter is lower for BDV (0.246 \u0026Aring;) than for CSFV (0.782 \u0026Aring;) which is consistent with keeping the predicted cut site by caspase-1 at the same position for BDV. Although there are subtle differences, that suggest certain amino acid changes in BDV resulted in structural modifications not observed in CSFV, which may have had a decisive evolutionary weight in the divergence of these species within the \u003cem\u003ePestivirus\u003c/em\u003e genus.\u003c/p\u003e \u003cp\u003eIn this study, we focused solely on the cp biotype (NADL reference strain). It has been reported that the inflammasome acts as a regulator of viral load not only through caspase-1 interaction but also through various inflammasome components (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This has been observed in viruses such as Hepatitis and Classical Swine Fever, p7 viroporin induces IL-1β secretion through caspase-1 activation as a viral regulation mechanism (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Therefore, future studies should expand the analysis to other viral proteins, other inflammasome components, and the ncp biotype in the future. However, since Gallegos-Rodarte et al. (2023) demonstrated that caspase-1 activation in the ncp biotype is specifically linked to the IFI16 inflammasome, the stimuli used in this work (targeted at the canonical pathway) might not be suitable for that model, since the innate immune response and the heterogeneity of molecules involved in the replication of both biotypes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e predictions are fundamental for inferring biological functions, especially for proteins that have not yet been crystallized. In addition to NS2-3, we identified potential caspase-1 cleavage sites in the Erns and NS5A proteins, which could enhance the observed biological effect. Nonetheless, molecular docking studies are required to precisely characterize these interactions. These tools have previously enabled the demonstration of genomic proximity between Ebola and Marburg viruses (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), the evolutionary tracking of SARS-CoV-2 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and the homology-based susceptibility analysis of the ACE2 protein in different hosts (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In our case, the integration of computational models optimizes \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003eIn vivo\u003c/em\u003e resources by directing experimentation toward molecular targets with a higher probability of functional relevance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTherefore, the present study identified the effect of inflammasome activation role on the modulation of viral titer through its effector molecule, caspase-1. The cytopathic NADL strain of BVDV colocalizes with caspase-1 in infected MDBK cells, and the stimulation or inhibition of this pathway directly affects the viral titer. This results in an increase or decrease of viral load, respectively. These findings suggest that caspase-1 plays a functional role in the BVDV replication cycle and may represent a novel target for antiviral strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financed by the DGAPA-PAPIIT IN214724.\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eData curation: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Cervantes-Torres J., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Formal an\u0026aacute;lisis: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Guti\u0026eacute;rrez-Guerrero YT., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Investigation: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Methodology: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Guti\u0026eacute;rrez-Guerrero YT., Supervision: N\u0026aacute;jera-Rivera H.D., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Validation: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Writing - original draft: N\u0026aacute;jera-Rivera H.D., Guti\u0026eacute;rrez-Guerrero YT., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Writing - Review and Editing: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Guti\u0026eacute;rrez-Guerrero YT., Cervantes-Torres J., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Conceptualization: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Guti\u0026eacute;rrez-Guerrero YT., Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Resources: Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Visualization: N\u0026aacute;jera-Rivera H.D., Ruelas-Mesa I., Cantera-Bravo MM, Gallegos-Rodarte C., Guti\u0026eacute;rrez-Guerrero YT., Funding acquisition: Ben\u0026iacute;tez-Guzm\u0026aacute;n A., Project administration: N\u0026aacute;jera-Rivera H.D., Ben\u0026iacute;tez-Guzm\u0026aacute;n A.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSincere thanks to Dr. Miguel Tapia (Unidad de Microscop\u0026iacute;a Confocal, Institututo de Investigaciones Biom\u0026eacute;dicas) and Dr Ren\u0026eacute; Segura from the Unidad de Investigaci\u0026oacute;n-FMVZ for their technical assistance to carrying out this project.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003cp\u003eEthics approval\u003c/p\u003e \u003cp\u003eThe authors confirm that the ethical policies of the journal, as noted on the journal\u0026rsquo;s author guidelines page, have been adhered to. No ethical approval was required, as no animals or human volunteers were involved in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimmonds P, Becher P, Bukh J, Gould EA, Meyers G, Monath T et al (2017) ICTV Virus Taxonomy Profile: Flaviviridae. J Gen Virol 1 de enero de 98(1):2\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/jgv.0.000672\u003c/span\u003e\u003cspan address=\"10.1099/jgv.0.000672\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinior B, Firth CL, Richter V, Lebl K, Trauffler M, Dzieciol M et al (2017) A systematic review of financial and economic assessments of bovine viral diarrhea virus (BVDV) prevention and mitigation activities worldwide. 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Sci Rep 24 de junio de 11(1):13063. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-021-92388-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-92388-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9359986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9359986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine Viral Diarrhea Virus (BVDV) belongs to the Flaviviridae viral family and is widely distributed worldwide, affecting millions of animals and causing significant clinical manifestations and production losses. Its genome encodes a single polyprotein that is processed into 10\u0026ndash;12 viral proteins. Polyprotein processing is essential for viral replication, and is mediated by viral autoproteases such as Npro and NS2-3, which activate the replication machinery. Inflammasomes are innate immune system receptors/sensors that regulate the activation of caspase-1 and promote inflammation in response to viral infections. Previous studies have shown that NLRP3 and IFI16 inflammasomes are activated in bovine macrophages infected with BVDV and that this activation is related to viral titer. In this study, we hypothesized that caspase-1 interacts with BVDV proteins to cleave them during viral replication, contributing to polyprotein processing and promoting the start of the viral replication process, which has an impact on viral progeny. In silico analysis identified four putative caspase-1 cleavage sites within the Erns, NS2-3, and NS5A proteins. These sites are structurally and phylogenetically conserved across the Pestivirus genus, suggesting a common evolutionary mechanism. We observed colocalization of NS2-3 with caspase-1 in BVDV-infected MDBK. Additionally, modulation of caspases-1 activation resulted in significant changes in viral titers. Furthermore, pharmacological modulation of caspase-1 activity significantly altered viral replication levels, indicating that caspase-1 plays a key role in efficient BVDV replication. Our findings suggest that BVDV may exploit caspase-1-mediated proteolytic processing of its polyprotein to enhance replication.\u003c/p\u003e","manuscriptTitle":"Caspase-1 Interacts with Bovine Viral Diarrhea Virus NS2-3 protein to modulate the viral titer in MDBK","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 12:36:03","doi":"10.21203/rs.3.rs-9359986/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"29418415344721429314762606832675842113","date":"2026-05-04T14:36:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T13:42:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56587339558607150073030732708120923328","date":"2026-04-22T08:15:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-19T07:39:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-09T04:47:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-09T04:47:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2026-04-08T17:21:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8171b0af-5d61-41fb-b587-618273867c3e","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"29418415344721429314762606832675842113","date":"2026-05-04T14:36:01+00:00","index":19,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T13:42:19+00:00","index":18,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T12:36:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 12:36:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9359986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9359986","identity":"rs-9359986","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}