ABSTRACT
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5' untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.
Subject(s)
Bombyx/virology , Inhibitor of Apoptosis Proteins/metabolism , MicroRNAs/metabolism , RNA, Viral/metabolism , Reoviridae , Animals , Bombyx/metabolism , Gene Expression Profiling , HEK293 Cells , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , Insect Proteins/metabolism , Larva/metabolism , Larva/virology , Reoviridae/genetics , Reoviridae/metabolism , Sequence Analysis, RNAABSTRACT
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. SIGNIFICANCE: The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes the silkworm to death, which negatively affects the sericulture industry. Studies on insect antiviral immunity and on interactive mechanisms between host cells and BmCPV are in their infancy and remain insufficient. In order to obtain an overall view of silkworm response to BmCPV infection, we performed a proteomic analysis of the midgut of silkworm responses to BmCPV infection by iTRAQ. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection.
