A Polydnaviral Genome of Microplitis Bicoloratus Bracovirus and Molecular Interactions Between the Host and Virus Involved in NF-Jb Signaling
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Arch Virol (2016) 161:3095–3124 DOI 10.1007/s00705-016-2988-3 ORIGINAL ARTICLE A polydnaviral genome of Microplitis bicoloratus bracovirus and molecular interactions between the host and virus involved in NF-jB signaling 1 1 1 2 1 Dong-Shuai Yu • Ya-Bin Chen • Ming Li • Ming-Jun Yang • Yang Yang • 1 1 Jian-Sheng Hu • Kai-Jun Luo Received: 15 February 2016 / Accepted: 15 July 2016 / Published online: 13 August 2016 Ó Springer-Verlag Wien 2016 Abstract Polydnaviruses (PDVs) play a critical role in and viral NF-jB/IjB genes were persistent and also varied altering host gene expression to induce immunosuppres- in Spli221 cells undergoing virus-induced pre-apoptosis sion. However, it remains largely unclear how PDV genes cell from 1 to 5 hours postinfection. Both were then affect host genes. Here, the complete genome sequence of expressed in a time-dependent expression in virus-induced Microplitis bicoloratus bracovirus (MbBV), which is apoptotic cells. These data show that viral IjB-like tran- known to be an apoptosis inducer, was determined. The scription does not inhibit host NF-jB/IjB expression, MbBV genome consisted of 17 putative double-stranded suggesting that transcription of these genes might be reg- DNA circles and 179 fragments with a total size of 336,336 ulated by different mechanisms. bp and contained 116 open reading frames (ORFs). Based on conserved domains, nine gene families were identified, of which the IjB-like viral ankyrin (vank) family included Abbreviations 28 members and was one of the largest families. Among MbBV Microplitis bicoloratus bracovirus the 116 ORFs, 13 MbBV genes were expressed in hemo- MdBV Microplitis demolitor bracovirus cytes undergoing MbBV-induced apoptosis and further CcBV Cotesia congregata bracovirus analyzed. Three vank genes (vank86, vank92, vank101) CsBV Cotesia sesamiae bracovirus were expressed in hemocytes collected from Spodoptera CvBV Cotesia vestalis bracovirus litura larvae parasitized by M. bicoloratus, in which host CiBV Chelonus inanitus bracovirus NF-jB/IjBs, including relish, dorsal, and cactus, were PDV polydnavirus also persistently expressed. When Spli221 cells were Vank viral ankyrin infected with MbBV viral particles, mRNA levels of host Introduction D.-S. Yu, Y.-B. Chen and M. Li are equal contributors. Electronic supplementary material The online version of this Delivery of viral particles and eggs to the host hemocoel article (doi:10.1007/s00705-016-2988-3) contains supplementary involves the development of immature parasitoid offspring material, which is available to authorized users. through the stages of eggs, embryos, first-instar, second- instar, and third-instar larvae, which must occur in the & Kai-Jun Luo [email protected] absence of the ability of the larva to protect itself against the host cellular immune response. Braconid and ichneu- 1 Key Laboratory for Animal Genetic Diversity and Evolution monid wasps that carry polydnaviruses (PDVs) [37] require of High Education in Yunnan Province, School of Life the immunosuppressive function of a virus to avoid host Sciences, Yunnan University, Kunming 650091, People’s Republic of China non-self recognition. The viral family Polydnaviridae is divided into two genera, Bracovirus and Ichnovirus. Bra- 2 Shanghai-MOST Key Laboratory of Health and Disease Genomic, Chinese National Human Genome Center at coviruses (BVs) and ichnoviruses (IVs) have independent Shanghai, Shanghai 201203, People’s Republic of China origins, but share a similar life style, probably through a 123 3096 D.-S. Yu et al. convergent evolutionary process [40]. The proviral form of signaling pathways are known to regulate cell survival and the viral genome is transmitted vertically in wasps; viral apoptosis. Thus, the transcription patterns of NF-jB/IjBin DNA replication only occurs at the pupal-adult stage in the apoptotic cells can be used to identify vankyrins that ovarian calyx cells in the female wasp [1]. Mature virus interfere with host NF-jB/IjB gene expression. particles are stored in the lumen of the calyx and oviduct The Microplitis bicoloratus and Spodoptera litura and are injected along with the egg into the lepidopteran model is an excellent system for studying immunosup- host hemocoel. The viral genome is then integrated into the pression occurring via the induction of apoptosis [25–27]. wasp’s host chromosome(s) to transcribe the viral genes to In our previously study, we showed, using gel elec- inhibit host immune responses to ensuring proper devel- trophoresis, that Microplitis bicoloratus bracovirus opment of the wasp larva [3, 6, 12, 35–37]. These (MbBV) contains at least 11 circular dsDNAs from 8 to immunosuppressive processes are controlled mainly by 50 kbp [26]. MbBV-induced apoptosis against host gran- viral genome transcription and its effect on the expression ulocytes causes immunosuppression [25] via viral gene of host genes. expression in host hemocytes [20]. Transcriptomes of non- Several BV genomes have been sequenced. The viral parasitized and parasitized S. litura hemocytes contains genome sizes range from 200 kilobase pairs (kbp) in Mi- sequences of host NF-jB/IjB genes, relish, dorsal, and croplitis demolitor bracovirus (MdBV) to 600 kbp in cactus, and also the transcribed viral genes [20]. Impor- Cotesia vestalis bracovirus (CvBV) in separate circles: 15 tantly, the endogenous Spli221 cell line, which undergoes circles in MdBV and 35 circles in CvBV. The number of low-level apoptosis under normal cell culture conditions predicted open reading frames (ORFs) varies from 61 in [21], allows us to generate pre-apoptotic Spli221 cells MdBV to 157 in CvBV [8, 40]. Over 20 protein families infected by MbBV particles to perform in vitro assays. To have been identified from BVs, Cotesia congregata bra- screen for genes expressed in apoptotic hemocytes, we covirus (CcBV) (total 80 genes in 16 protein families), performed whole-genome sequencing of MbBV and iden- CvBV (total 91 genes in 17 protein families), Glyptapan- tified 116 genes. By comparing those sequences with the teles indiensis bracovirus (GiBV) (total 87 genes in 16 transcriptome of S. litura hemocytes parasitized by the protein families), G. flavicoxis bracovirus (GfBV) (total 79 wasp M. bicoloratus, we identified three IjB-like genes, genes in 15 protein families), and MdBV (total 37 genes in vank86, vank92, and vank101, in 13 screened expressed 7 protein families) [10, 11, 14]. However, it is not known genes. Then, we used a bioinformatics approach to identify exactly which functional genes are expressed in virus-in- NF-jB/IjB-related factors from both the host S. litura and fected host hemocytes. BVs. Using in vivo and in vitro transcriptional analyses of Of the few protein families common to both BV and IV, these NF-jB/IjB factors, we identified potential correla- viral ankyrins (vanks), occur in members of both genera tions in their expression patterns. We found that viral IjB- [40]. Various reports have shown that vanks function as like transcription could not inhibit host NF-jB/IjB IjB mimics, but they lack a regulatory domain for phos- expression at the mRNA level, suggesting that they might phorylation and ubiquitination, which could result in irre- use different mechanisms to regulate transcription. versible binding to NF-jB to inhibit an immune response [4, 7, 38, 39]. IjB-like proteins, containing ankyrin-repeat domains (Anks) that are highly similar to Drosophila IjB, Materials and methods and Cactus, have also been found in BVs, such as CcBV (8 vanks), CvBV (8 vanks), GiBV (9 vanks), GfBV (8 vanks), Insect rearing and experimental animals and MdBV (12 vanks). A recent study demonstrated that two IjB mimics, Ank-H4, and N5, from MdBV bind to The S. litura colony was reared on an artificial diet for- Drosophila NF-jB factors Dorsal and Relish, whereupon mulated as described previously [19]at27± 1 °C, RH the expression of several antimicrobial peptide genes is 60-80 %, with a 12:12 h photoperiod regime. The para- reduced [7]. sitoid M. bicoloratus colony was maintained on S. litura NF-jB, Relish, and Dorsal share an REL homology larvae reared in the laboratory. Adults were also provided domain (RHD), which is essential for dimerization and with honey as a dietary supplement. The parasitoid colony DNA binding [7, 16, 37]. Human NF-jB/IjB proteins was passaged according to established methods [27]. contain two-antiparallel a-helices and a b-hairpin, sharing G-TPLH and LL–GA consensus repeats [28]. Although Cell culture vankyrins are known to inhibit host NF-jB, their effect on host NF-jB gene expression during viral immunosuppres- Spli221 (TUAT-Spli221) adherent cells were derived from sion remains largely unclear. Induction of apoptosis is an S. litura [29]. Cells were cultured in TNM-FH insect cul- effective immunosuppression strategy, and NF-jB/IjB ture medium containing 10 % fetal bovine serum (FBS, 123 Microplitis bicoloratus bracovirus genome sequence 3097 Hyclone). Cells were maintained at 27 °C and passaged in significant differences in gene expression based on a pre- 25-cm2 tissue culture flasks (Corning). viously described method [20]. Isolation and purification of viral particles Isolation of total RNA, cDNA synthesis, and infection of Spli221 cells and qRT-PCR Purified viral particles were prepared based on a previ- Whole tiny larvae collected 1–3 days post-parasitization ously published protocol [25, 26] with minor modifica- (p.p.), hemocytes from 4-7 days p.p. collected from para- tions. Briefly, fresh wasps were frozen at -20 °C for sitized S. litura larvae, and Spli221 cells collected 1-5 h 10 min and then put on ice. Reproductive tracts of postinfection (p.i.) were used for RNA isolation. Isolation female wasps were excised under a binocular stereo of total RNA, cDNA synthesis, and qRT-PCR was per- dissecting microscope, and separated ovaries were col- formed as per previously published protocols [20]. The 2- lected into a 2-ml tube on ice until further use.