Pseudorabies Virus Infected Porcine Epithelial Cell Line Generates a Diverse Set of Host MicroRNAs and a Special Cluster of Viral MicroRNAs

Yi-Quan Wu,Hua-Bin He,Ling-Ling Chen,Zheng-Fei Liu,Huan-Chun Chen,Dong-Sheng Chen,Di-Jun Chen

doi:10.1371/journal.pone.0030988

Abstract

Pseudorabies virus (PRV) belongs to Alphaherpesvirinae subfamily that causes huge economic loss in pig industry worldwide. It has been recently demonstrated that many herpesviruses encode microRNAs (miRNAs), which play crucial roles in viral life cycle. However, the knowledge about PRV-encoded miRNAs is still limited. Here, we report a comprehensive analysis of both viral and host miRNA expression profiles in PRV-infected porcine epithelial cell line (PK-15). Deep sequencing data showed that the ∼4.6 kb intron of the large latency transcript (LLT) functions as a primary microRNA precursor (pri-miRNA) that encodes a cluster of 11 distinct miRNAs in the PRV genome, and 209 known and 39 novel porcine miRNAs were detected. Viral miRNAs were further confirmed by stem-loop RT-PCR and northern blot analysis. Intriguingly, all of these viral miRNAs exhibited terminal heterogeneity both at the 5′ and 3′ ends. Seven miRNA genes produced mature miRNAs from both arms and two of the viral miRNA genes showed partially overlapped in their precursor regions. Unexpectedly, a terminal loop-derived small RNA with high abundance and one special miRNA offset RNA (moRNA) were processed from a same viral miRNA precursor. The polymorphisms of viral miRNAs shed light on the complexity of host miRNA-processing machinery and viral miRNA-regulatory mechanism. The swine genes and PRV genes were collected for target prediction of the viral miRNAs, revealing a complex network formed by both host and viral genes. GO enrichment analysis of host target genes suggests that PRV miRNAs are involved in complex cellular pathways including cell death, immune system process, metabolic pathway, indicating that these miRNAs play significant roles in virus-cells interaction of PRV and its hosts. Collectively, these data suggest that PRV infected epithelial cell line generates a diverse set of host miRNAs and a special cluster of viral miRNAs, which might facilitate PRV replication in cells.

Highlights

Pseudorabies virus (PRV), the etiologic pathogen of Aujeszky’s disease causing huge economic loss in pig industry, belongs to the Alphaherpesvirinae subfamily with a broad host range and pantropic property, and has been shown to be a useful model organism for the studies of herpesvirus biology [1,2,3]
To investigate the miRNA profiles in infected epithelial cell line, PK-15 cells were infected with PRV Ea strain as described in Materials and Methods, and small RNAs were isolated and analyzed by deep sequencing (Solexa/Illumina technology)
Based on the differential expression of miR-8 and moR-8 between in epithelial cells and in dendritic cells (DC), we proposed a biogenesis model of processing mechanism of pri-miR-8 (Figure 3B and 3C). i) In both DC and PK cells, pre-prv-miR-8 is cleaved by Drosha, ii) in DC cells, the moR8-miR8 miRNA is cleaved once by Dicer to generate moR-8, while iii) After that, in PK cells, the moR8-miR8 precursor is cleaved by Dicer to generate moR-8 or by Drosha to generate miR-8 hairpin which is further cleaved by Dicer to generate the mature mature miR-8

Summary

Introduction

Pseudorabies virus (PRV), the etiologic pathogen of Aujeszky’s disease causing huge economic loss in pig industry, belongs to the Alphaherpesvirinae subfamily with a broad host range and pantropic property, and has been shown to be a useful model organism for the studies of herpesvirus biology [1,2,3]. PRV genome is about 142 kb long and encodes 70 proteins and a large latency transcript (LLT). PRV LLT, an 8.4 kb polyadenylated RNA, is the only abundant transcript during the latent stage and is split into different sizes including a ,4.6 kb stable intron as in the case of HSV-1 [4,5,6]. Latency associated transcript (LAT) expression is involved in the latency-reactivation cycle by inhibiting apoptosis of latently infected neurons, viral gene expression, and productive infection [7]. MicroRNAs (miRNAs) are endogenous non-coding RNAs that can direct the inhibition of target mRNAs expression via complementary sites in the 39 untranslated region (39 UTR) either by transcriptional destabilization or translational repression [8]. One strand of this duplex is degraded, whereas the other strand is incorporated into the RNA-induced silencing complex (RISC), where it acts as a guide RNA to direct RISC to target mRNAs [8]

Methods

Results

Conclusion