Integrated analysis of mRNA and viral miRNAs in the kidney of Carassius auratus gibelio response to cyprinid herpesvirus 2

Abstract

MicroRNAs (miRNAs) are small, non-coding single stranded RNAs that play crucial roles in numerous biological processes. Vertebrate herpesviruses encode multiple viral miRNAs that modulate host and viral genes. However, the roles of viral miRNAs in lower vertebrates have not been fully determined. Here, we used high-throughput sequencing to analyse the miRNA and mRNA expression profiles of Carassius auratus gibelio in response to infection by cyprinid herpesvirus 2 (CyHV-2). RNA sequencing obtained 26,664 assembled transcripts, including 2,912 differentially expressed genes. Based on small RNA sequencing and secondary structure predictions, we identified 17 CyHV-2 encoded miRNAs, among which 14 were validated by stem-loop quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and eight were validated by northern blotting. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNAs-mRNA pairs revealed diverse affected immune signalling pathways, including the RIG-I-like receptor and JAK-STAT pathways. Finally, we presented four genes involved in RIG-I-like pathways, including host gene IRF3, RBMX, PIN1, viral gene ORF4, which are negatively regulated by CyHV-2 encoded miRNA miR-C4. The present study is the first to provide a comprehensive overview of viral miRNA-mRNA co-regulation, which might have a key role in controlling post-transcriptomic regulation during CyHV-2 infection.

Cyprinid Herpesvirus 2 miR-C12 Attenuates Virus-Mediated Apoptosis and Promotes Virus Propagation by Targeting Caspase 8

“…Next, we determined the mechanism of miR-C4 and miR-C12 in CyHV-2-induced cell apoptosis. In our previous research, caspase 8 was predicted as one of the candidate target gene of miR-C12, and we did not find a target gene directly related to apoptosis in miR-C4 (Lu et al, 2017). Therefore, miR-C12 was the main research object of this study.…”

“…Hela cells transfected with pGL3 reporter plasmids and miR-C12 mimics, miR-NC, miR-C12 inhibitor, or inhibitor NC were analyzed post-transfection. The luciferase assays were conducted as previously described (Lu et al, 2017). The Dual-Glo luciferase assay kit (Promega, USA) was used to detect the quantity of firefly and Renilla luciferase by GloMax-Multi Detection System (Promega, USA).…”

“…In Cyprinivirus genus, Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), and Cyprinid herpesvirus 3 (CyHV-3) infect common carp, crucian carp, or goldfish (Davison et al, 2013). Many reports have demonstrated that Cyprinivirus, mainly CyHV-2 and CyHV-3, can lead to latent infection (Reed et al, 2014;Wei et al, 2019), produce virus-encoded MicroRNAs (Donohoe et al, 2015;Lu et al, 2017), and facilitate viral invasion by exploiting various intracellular signaling pathways (Adamek et al, 2014;Zhou et al, 2019). However, the molecular mechanism employed by CyHV-2 is largely unknown.…”

“…A number of herpesviruses, including CyHV-2 and CyHV-3, encode miRNAs in infected cells. The regulatory effects of miRNAs involved in viral infection include apoptosis, oncogenic transformation, and modulation of viral life cycle (Donohoe et al, 2015;Bhela and Rouse, 2017;Lu et al, 2017). Viral miRNAs play an important role in the regulation of cell apoptosis, mostly by decreasing pro-apoptotic or anti-apoptotic genes expression (Pfeffer et al, 2004).…”

“…For example, BCL2associated X (BAX) was demonstrated to be a target of viral miRNA ebv-miR-BART16 (Marquitz et al, 2011); BCL2associated death promoter (BAD) and the pro-apoptotic protein caspase 3 are targets of the ebv-miR-BART20-5p, ebv-miR-BART16 or ebv-miR-BART1-3p, respectively (Vereide et al, 2014). Our previous studies have identified 17 viral miRNAs from CyHV-2-infected crucian carp kidney, and have shown that the host genes PIN1, IRF3, and RBMX are the targets of CyHV-2-encoded miR-C4 (Lu et al, 2017). Recently, it was reported that host miRNAs are involved in CyHV-2 infection and participate in the regulation of apoptosis and immune-related genes (Lu et al, 2018b).…”