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Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

ABSTRACT: Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in FB but not in MG. BGI001299, BGI014434, BGI012068, and BGI009201 were MG-specific genes with transmembrane transport function, the expression of which were induced by BmCPV. BGI001299, BGI014434, and BGI012068 expressed in entire MG and may be involved in BmCPV invasion. BGI009201 expressed only in PM and may be necessary for BmCPV proliferation. BmPGRP-S2 and BGI012452 (a putative serine protease) were induced by BmCPV and may be involved in immune defense against BmCPV. The expression level of BmCPV S1, S2, S3, S6, and S7 was high and there was no expression of S9 in MG 72 h, implying that the expression time of structural protein coding genes is earlier. These results provide insights into the mechanism of BmCPV infection and host defense.

SUBMITTER: Jiang L

PROVIDER: S-EPMC4847007 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

Jiang Liang L Peng Zhengwen Z Guo Youbing Y Cheng Tingcai T Guo Huizhen H Sun Qiang Q Huang Chunlin C Zhao Ping P Xia Qingyou Q

Scientific reports 20160427

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in F ...[more]

PMID: 27118345

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Project description:Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host's transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic/metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host's RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following pathogenic infection with CPV, while they also highlight the relative importance of RNAi as an antiviral mechanism.

Project description:The pathogenesis of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is unclear, although accumulating evidence indicates that circular RNAs (circRNAs), which act as competing endogenous RNAs or positive regulators, play important roles in regulating gene expression in eukaryotes and, thus, may play a role in BmCPV infections. To explore the expression and biological functions of circRNAs in the silkworm midgut following BmCPV infection, silkworm circRNA expression profiles of normal midgut tissue (control) and BmCPV-infected midgut tissue (test) were determined using high-through sequencing. A total of 9,753 and 7,475circRNAs were detected from the control and test samples, respectively. The two samples shared 6,085 circRNAs, while 646 and 737 circRNAs were expressed specifically in the control and test samples, respectively. A total of 3,638 circRNAs were shown to be differentially expressed, and 400 circRNAs were substantially differentially expressed with a fold-change ≥ 2.0 (p< 0.05 and a false discover rate < 0.05), of which 294 were up-regulated and 106 were down-regulated following infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to determine the principal functions of the substantially differentially regulated genes. circRNA-miRNA interaction networks were constructed based on a correlation analysis between the differentially expressed circRNAs and the nature of their microRNA (miRNA) binding sites. The network inferred that 13 miRNAs interacting with 193 circRNAs were among the 300 most abundant relationships. bmo-miR-3389-5p, bmo-miR-745-3p, and bmo-miR-3262 were related to 30, 34, and 34 circRNAs, respectively. circRNA_8115, circRNA_9444, circRNA_4553, circRNA_0827, and circRNA_6649 contained six, five, four, four, and four miRNA binding sites, respectively. We further found that alternative circularization of circRNAs is a common feature in silkworms and that the junction sites of many silkworm circRNAs are flanked by canonical GT/AG splicing signals. Our study is the first to show the circRNA response to virus infection. Thus, it provides a novel perspective on circRNA-miRNA interactions during BmCPV pathogenesis, and it lays the foundation for future research of the potential roles of circRNAs in BmCPV pathogenesis.

Project description:BackgroundAntheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized.ResultsIn this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed K(m) of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol).ConclusionViral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.

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Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

Publications

Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

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