Reduction of microRNA-221 in BVDV infection enhances viral replication by targeting the ATG7-mediated autophagy pathway

Abstract

Background: Bovine viral diarrhoea (BVD), a condition triggered by bovine viral diarrhoea virus (BVDV), is recognized globally as a prevalent pathogen among ruminants and markedly affects the economics of animal husbandry. MicroRNAs, a class of small noncoding RNAs, play pivotal roles in regulating a myriad of biological processes.The ATG7-LC3 pathway, a canonical autophagy mechanism, is integral in defending against pathogenic invasion and maintaining cellular homeostasis.

Results: In this study, we observed significant downregulation of bta-miR-221 in cells infected with BVDV. We further established that overexpression of bta-miR-221 markedly attenuated BVDV replication in Madin‒Darby bovine kidney (MDBK) cells. Through bioinformatics prediction analysis, we identified ATG7, an autophagy-related gene, as a direct downstream target of bta-miR-221. However, the intricate relationships among bta-miR-221, the ATG7-LC3 pathway, and BVDV infection remained unclear. Our study revealed that ATG7 expression was significantly elevated in BVDV-infected cells, whereas bta-miR-221 mimics repressed both endogenous and exogenous ATG7 expression. Following BVDV infection, we noted a decrease in LC3I expression, its conversion to LC3II, a significant increase in ATG7 expression, and a notable decrease in SQSTM1/p62 expression. By employing laser confocal microscopy and immunoprecipitation assays, we elucidated the regulation of the ATG7-LC3 pathway by bta-miR-221 in MDBK cells. Our findings recealed that BVDV infection enhanced the ATG7-LC3 interaction, inducing autophagy through the suppression of bta-miR-221 in MDBK cells. Consequently, bta-miR-221 emerged as a potent inhibitor of BVDV, impacting its proliferation and replication within the host.

Conclusions: This research sheds light on novel aspects of virus-host interactions and lays a foundation for the development of antiviral therapeutics.

Keywords: ATG7-LC3; Bovine viral Diarrhoea virus; Cellular autophagy; MicroRNA; Viral replication.

Fig. 1

Classical Degradative Autophagy [18]. The depicted schematic elucidates the pivotal role of the Unc-51-like kinase 1 (ULK1) complex in orchestrating a spectrum of upstream signals, culminating in the initiation of an autophagic cascade. This process involves the synthesis of ATG5-ATG12 and ATG8 (LC3 II), which, under the catalytic influence of ATG7, coalesce to assemble autophagosomes. Concurrently, cellular constituents exert their influence via a dualistic engagement mechanism: a direct liaison with the LC3 protein and an indirect association mediated by the p62 receptor

Fig. 2

bta-miR-221 Expression Dynamics in BVDV-Infected Cells. Cells infected with BVDV at a multiplicity of infection (MOI) of 1 underwent miRNA library construction for detailed expression analysis. A and B, Clustering heatmaps showing the differential expression of miRNAs; a gradient from red to blue indicates expression levels from high to low, respectively. C, Differential expression volcano plot for miRNAs. D, Gene Ontology (GO) analysis results for commonly differentially expressed miRNAs. E, Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of these miRNAs. F, Quantification of the relative expression of bta-miR-221

Fig. 3

Suppression of BVDV Replication by bta-miR-221 Mimics. A, Transfection of MDBK cells with bta-miR-221 mimics and inhibitors; the transfection efficiency was assessed via quantitative PCR (qPCR) at 12 h posttransfection. B, qPCR analysis of BVDV 5’UTR mRNA levels across different treatment groups, indicating the impact of bta-miR-221 modulation on viral mRNA expression. C and D, Western blot analyses evaluating the variations in BVDV E2 protein levels among the treatment groups

Fig. 4

bta-miR-221 Targets and Suppresses ATG7 Expression. A, bioinformatics tools were used to predict the interaction site between bta-miR-221 and the ATG7 3’UTR. B, Effect of bta-miR-221 on ATG7 expression. HEK-293T cells were cotransfected with either wild-type or mutant ATG7-3’UTR-psiCHECK2 plasmids along with bta-miR-221 mimics or inhibitors, followed by luciferase assay measurements 24 h posttransfection to assess regulatory impacts. C, Details of the experiment in MDBK cells, where transfection with bta-miR-221 mimics or inhibitors (along with their respective negative controls) preceded qPCR analysis at 12 and 24 h to quantify ATG7 mRNA levels. D and E, The influence of bta-miR-221 on ATG7 protein levels was tracked, and the intracellular concentration of mCherry-ATG7 protein in HEK-293T cells 24 h after cotransfection with bta-miR-221 mimics or inhibitors was determined

Fig. 5

Autophagy induction in BVDV-infected MDBK cells. A, Autophagic response in MDBK cells at 48 h post infection with BVDV at a multiplicity of infection (MOI) of 1, as visualized via transmission electron microscopy. Autophagic lysosomes (indicated by blue arrows) and autophagosomes (red arrows) were markedly more prevalent in the BVDV-infected (experimental) group than in the MOCK (control) group. B, Temporal analysis of ATG7 mRNA expression levels following BVDV infection, as quantified via qPCR. C and D, Details of the protein expression dynamics of p62, ATG7, and LC3 during the course of BVDV infection, as determined by Western blotting

Fig. 6

Modulation of the ATG7-LC3 Autophagy Pathway by bta-miR-221 Mimics. A, The initial localization of GFP-LC3 and mCherry-ATG7 in 293T cells was observed via confocal microscopy, establishing baseline cellular distribution patterns of these autophagy markers. B, Examination of the impact of bta-miR-221 mimics and inhibitors on the autophagy pathway. The bta-miR-221 mimics or inhibitors (and their respective controls) were cotransfected with GFP-LC3 and mCherry-ATG7 in HEK-293T cells. Cellular localization and interaction patterns were observed through confocal microscopy imaging 24 h posttransfection. C, HEK-293T cells were transfected with combinations of GFP-LC3 + mCherry-ATG7 and GFP-LC3 + vector-mCherry, followed by immunoprecipitation to analyse the association between the GFP and mCherry proteins, as quantified by Western blotting

Fig. 7

Mechanistic Model of the role of bta-miR-221 in Inhibiting BVDV Replication. This model illustrates the dual role of bta-miR-221 in the cellular response to BVDV infection. BVDV infection leads to a decrease in bta-miR-221 expression, consequently triggering the activation of the ATG7-LC3 autophagy pathway. This autophagic response potentially aids in the viral replication process. Conversely, bta-miR-221 inhibits BVDV replication within the cell, revealing a complex interplay among host miRNA regulation, autophagy, and viral replication dynamics. The diagram was created using FigDraw (https://www.figdraw.com/), providing a visual summary of the inhibitory mechanism of bta-miR-221 on BVDV replication through the modulation of cellular autophagy pathways