Classical swine fever virus Shimen infection increases p53 signaling to promote cell cycle arrest in porcine alveolar macrophages

Abstract

Classical swine fever virus (CSFV) replicates in macrophages and causes persistent infection. Despite its role in disastrous economic losses in swine industries, the molecular mechanisms underlying its pathogenesis are poorly understood. The virus evades the neutralizing immune response, subverting the immune system to ensure its own survival and persistence. Our genome-wide analysis of porcine alveolar macrophage transcriptional responses to CSFV Shimen infection using the Solexa/Illumina digital gene expression system revealed that p53 pathway components and cell cycle molecules were differentially regulated during infection compared to controls. Further, we investigated the molecular changes in macrophages infected with CSFV Shimen, focusing on the genes involved in the p53 pathway. CSFV Shimen infection led to phosphorylation and accumulation of p53 in a time-dependent manner. Furthermore, CSFV Shimen infection upregulated cyclin-dependent kinase inhibitor 1A (p21) mRNA and protein. In addition, CSFV Shimen infection induced cell cycle arrest at the G1 phase, as well as downregulation of cyclin E1 and cyclin-dependent kinase 2 (CDK2). The expression of genes in the p53 pathway did not change significantly after p53 knockdown by pifithrin-α during CSFV Shimen infection. Our data suggest that CSFV Shimen infection increases expression of host p53 and p21, and inhibits expression of cyclin E1 and CDK2, leading to cell cycle arrest at the G1 phase. CSFV may utilize this strategy to subvert the innate immune response and proliferate in host cells.

AIF1 was identified as an up-regulated gene contributing to CSFV Shimen infection in porcine alveolar macrophage 3D4/21 cells

“…However, little is known about its role in the pathogenesis of CSF. In previous study, significantly up-regulated and down-regulated genes were obtained from CSFV Shimen infected group which were compared to the C strain and MOCK infected group (Ning et al, 2017). In this work, we paid attention to AIF1 from the obtained differentially expressed genes to focus on its biological significance in macrophages to CSFV infection.…”

“…DGE analysis was performed to screen for differentially expressed genes in CSFV-infected PAMs based on previously reported high-throughput sequencing data (Ning et al, 2017), which identified AIF1 as a key gene, and further testing was conducted to confirm its role during infection by CSFV Shimen. The CSFV Shimen strain was obtained from the Control Institute of Veterinary Bioproducts and Pharmaceuticals.…”

“…The PAM 3D4/21 cell line (American Type Culture Collection: CRL-2843) were seeded at a density of 2 × 10 7 cells per flask. When the macrophages reached 70-80% confluence, CSFV Shimen was added to the cultures at a multiplicity of infection of 5 TCID50 (Ning et al, 2017). After incubation with the virus at 37 • C and 5% CO 2 for 1 h, the virus was removed, and the cells were cultured for 0, 12, 24, and 48 h, and RNA or protein extracts from the cultured cells were collected for subsequent detection (Ning et al, 2016).…”

“…Despite the seriousness of this disease, the inflammatory process that occur in the pathogenesis of CSF are not well understood. As shown by the acute organic damage it causes, classical swine fever virus (CSFV) Shimen strain can proliferate efficiently in macrophages, but does not cause the typical cytopathic effects (Ning et al, 2017). The response to CSFV infection remains focused on macrophages recently.…”

Classical swine fever virus-Shimen infection upregulates SH3GLB1 expression in porcine alveolar macrophages

“…Porcine alveolar macrophages (PAMs) were cultured in RPMI 1640 medium (GE, USA) containing 10% fetal bovine serum (FBS; Gibco Life Technologies, USA) at 37 C with 5% CO 2 . The CSFV Shimen strain is from the Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China) [17]. The PAM cell line 3D4/21 (CRL-2843) was obtained from the American Type Culture Collection (Manassas, VA, USA) [18].…”