Genome And ODV Proteome From Pieris Rapae Granulovirus

The family Baculoviridae is a diverse group of rod-shaped, enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 to 180 kb and its gene content is about 859. Baculoviruses could infect and finally kill insect pests of Lepidoptera, Hymenoptera and Mosquito of Diptera. Baculoviruses have been extensively used for insecticides and some are widely used as vectors in gene expression systems, some for gene therapy in medical study. Presently,58 baculovirus genomes have been sequenced, including 45 NPVs and 13 GVs; however, only 5 baculovirus proteins composition were identified from only NPVs, including 4 proteomes from ODV and only one proteome from BV. This study explored Pieris rapae Granulovirus (PrGV) genome structure and organization, ODV protein composition etc., and the results were as follows1. PrGV genome analysisPrGV genome size was 108,592 with 120 ORFs and coding region accounted for 91% of the whole genome. Of 120 putative ORFs,65 ORFs featured the same transcription direction with granulin and other 55 ORFs did the opposite direction; The longest ORF in PrGV genome had 3402bp (ORF75, helicase-1) and coding protein had 1133 amino acid residues; The genome has two repeative genes (ODV-E66a/b); PrGV genome featured non-transcription region, overlapping genes and 8 homologous regions were dispersed different positions in genome.31 core genes in all sequenced baculoviruses genomes and 31 conservative genes in Lepidoptera baculovirus genomes were detected in PrGV genome.Gene colinearity analysis disclosed that the PrGV ORFs featured high colinearity with the parallel parts ofβ-baculovirus. The highest colinearity occurred between PrGV and ChocGV, ClanGV, AdorGV genomes and the lower colinearity with XecnGV while the lowest with AcNPV.Blast P analysis represented the ORFs in PrGV were classified as follows:1) 8-GV unique genes, which only existed in GV-genomes. They were orf4, orf12, orf28, orf34, orf55, orf95(LEF-3), orf103, orf112.2) 33 ORFs existed in GV genome and other species but not in NPV genome, there were orf2, orf5, orf18, orf19, orf20(PEP1), orf24, orf25, orf27, orf31, orf35, orf36, orf37 (Metalloproteinase), orf41, orf42, orf48(39K), orf54, orf62(FGF-1), orf63, orf67, orf83, orf84, orf86, orf89, orf94, orf97, orf102, orf104, orf105, orf106, orf109, orf111, orf114, orf115; 3) 4 ORFs only existed in PrGV genome and other species but not in NPV genome, they were orf09, orf32, orf53, orf117.4) 75 ORFs shared with homolog with NPV genome.Genome evolution analysis showed that PrGV genome featured extremely high identity with ChocGV genome in whatever LCB (Locally Collinear Blocks) number, direction, arrangement order or size, disclosing that both viruses shared similar evolution traces. The lower identity occurred between PrGV genome and PsunGV, XeniGV, HearGV genomes for comparably big differences in LCB size, which implied that 3 viruses (PsunGV, XeniGV, HearGV) could expand genes to add new functions in order to expand their host limit during the long course of selection pressure. The big difference in LCBs occurred between PrGV and AcNPV, representing that both viruses featured earlier diverge in their individual evolution. The lowest identity in LCBs showed more earlier diverge occurred between PrGV and CuniNPV genome, however, both viruses inherited some ancestral genes.Phylogeny analysis disclosed that the closest genetical relationship occurred between PrGV and ChocGV. As expectedly,13β-baculoviruses cluster into one clade including 6 viruses (HearGV, XeniGV, PsunGV, SpliGV, AgseGV, PlxyGV) in one subclade and other 7 viruses (AdorGV, PhopGV, CrleGV, CypoGV, ClanGV, ChocGV, PrGV) in another subclade.2. PrGV-ODV proteome analysisForty seven proteins were identified by 3 MS approaches from PrGV-ODV. Of 47 proteins,16 proteins were identified by MALDI-TOF/TOF MS,37 proteins were done by LC-LTQ Velos MS while 31 proteins by LTQ-Orbitrap MS. Notably,11 proteins were identified by all 3 MS approaches,15 proteins were done by at least 2 MS approaches while 21 proteins by only one MS method.Of 47 proteins,14 proteins (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were regarded as newly-identified ones, including 7 GV-unique proteins (Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114). Immunoblotting analysis, using antisera prepared from 11 randomly selected recombinant, further confirmed that 11 proteins existed in PrGV-ODV, which disclosed the feasibility of MS approach for protein identification.3. Genes analysis of Pr42 and Pr114Bio-information analysis showed that Pr42 gene codes 803aa, putative MW(molecular weigh) is 93.5kDa; the gene product features 52 phosphorylation sites and 3 O-Glycosylated sites; Pr42 is hydrophilic protein with no signal peptide and phylogeny showed that Pr42 shared highest identity with ClanGV gp022; Pr114 gene codes 323aa, putative MW is 36.9kDa; the gene product features 21 phosphorylation sites and no O-Glycosylated sites; Pr114 is also hydrophilic protein with no signal peptide and phylogeny showed that Pr114 shared highest identity with ChocGV-ORF110.RT-PCR analysis represented that both Pr42 and Pr114 genes began to transcribe at 48h per os infection and did till to 96h. Realtime PCR analysis showed Pr42 and Prll4 transcripts could be detected at 6-96h per os infection, respectively. The relative transcripts level featured big differences during different infection phases.Combined bioinformatics, RT-PCR with real-time PCR analysis, Pr42/Prll4 maybe late expression genes and their genes products could function as structure proteins.