Profiling of alternative polyadenylation and gene expression in PEDV-infected IPEC-J2 cells

Abstract

Since 2010, porcine epidemic diarrhea virus (PEDV) has received global attention with the emergence of variant strains characterized with high pathogenicity. The pathogen-host interaction after PEDV infection is still unclear. To investigate this issue, high-throughput-based sequencing technology is one of the optimal choices. In this study, we used in vitro transcription sequencing alternative polyadenylation sites (IVT-SAPAS) method, which allowed accurate profiling of gene expression and alternative polyadenylation (APA) sites to profile APA switching genes and differentially expressed genes (DEGs) in IPEC-J2 cells during PEDV variant strain infection. We found 804 APA switching genes, including switching in tandem 3' UTRs and switching between coding region and 3' UTR, and 1,677 DEGs in host after PEDV challenge. These genes participated in variety of biological processes such as cellular process, metabolism and immunity reactions. Moreover, 413 genes, most of which are the "focus" genes in interaction networks, were found to be involved in both APA switching genes and DEGs, suggesting these genes were synchronously regulated by different mechanisms. In summary, our results gave a relatively comprehensive insight into dynamic host-pathogen interactions in the regulation of host gene transcripts during PEDV infection.

Keywords: Alternative polyadenylation; Differential expression of host genes; Dynamic host–pathogen interactions; Porcine epidemic diarrhea virus.

Fig. 1

PEDV replication in IPEC-J2 cells. a Light microscopy and immunofluorescence of IPEC-J2 cells infected with PEDV strain GDS01 at 7 and 13 hpi. Mock-infected cells at 13 hpi were used as control. b One-step growth curve of GDS01 in IPEC-J2 cells. Infected cells were collected at 6-h interval. The virus titers were determined by plaque assay. The error bars represent SEM

Fig. 2

IVT-SAPAS reads and poly(A) sites in IPEC-J2 cells. a Genomic location of reads. b Distribution of poly(A) sites. c Genes with different numbers of tandem poly(A) sites. d Boxplot of the median distances between stop codons and poly(A) sites in genes with single poly(A) sites and distances between stop codons and closest poly(A) sites or farthest poly(A) sites in genes with APA in the 3′-end. The elements of box means the upper limit of outliers; 75th percent (Q1); 50th percent (Q2); 25th percent (Q3); the lower limit of outliers. The circles in box plot means outlier. The upper limit of outliers = Q3 + 1.5*(Q3-Q1); the lower limit of outliers = Q1-1.5*(Q3-Q1). (E) PAS usage of poly(A) sites. ucsc: database of University of California, Santa Cruz

Fig. 3

Tandem APA switching genes in IPEC-J2 cells after PEDV infection. a Summary of tandem APA switching genes between samples at different time points. b Venn diagrams of tandem APA sites switching genes. c Functional classification of tandem APA switching genes. d Pathway analysis of tandem APA switching genes

Fig. 4

Differentially expressed genes in IPEC-J2 cells after PEDV-infected. a Summary of differentially expressed genes between samples at different time points. b Venn diagrams of DGEs. c Functional classification of differentially expressed genes. d Pathway analysis of differentially expressed genes

Fig. 5

RT-qPCR analysis of the dynamic expression of selected genes. IPEC-J2 cells infected with PEDV GDS01 at 0.5 MOI were collected at six time points and RT-qPCRs were performed. The x-axis denotes different time points and the y-axis denotes the relative expression in infected samples comparing to uninfected samples. SAPAs represent the relative expression of genes calculated from IVT-SAPAS sequencing database

Fig. 6

Genes with APA switching among coding region and 3′ UTR region. a Venn diagrams of UTR-prefer genes and CDS-prefer genes. b Summary of genes with APA switching between coding region and 3′ UTR region at different time points. c Functional classification of CDS-prefer genes

Fig. 7

Relationship between tandem APA switching genes and differentially expressed genes. a Venn diagram of tandem APA switching genes and differentially expressed genes. b Pathway enrichment of genes subjected to changes of 3′ UTR length and differential expression level

Fig. 8

Ingenuity pathway analysis of genes significantly altered in IPEC-J2 cells during PEDV infection. Tandem APA switching genes are marked green. Differently expressed genes are marked red; Genes exhibiting both APA switching and differential expression are marked yellow