Analysis of the Genome of a Korean Isolate of the Pieris rapae Granulovirus Enabled by Its Separation from Total Host Genomic DNA by Pulse-Field Electrophoresis
Background Most traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. However, purification of virions may yield insufficient material for traditional sequencing. The electrophoretic method described here provides a strategy whereby the genomic DNA of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) could be recovered in sufficient amounts for sequencing by purifying it directly from total host DNA by pulse-field gel electrophoresis (PFGE). Methodology/Principal Findings The total genomic DNA of infected P. rapae was embedded in agarose plugs, treated with restriction nuclease and methylase, and then PFGE was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the purified viral DNA. The double-stranded circular genome of PiraGV-K was found to encode 120 open reading frames (ORFs), which covered 92% of the sequence. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF 11), involved in the liquefaction of the host, were found in the genome. Conclusions/Significance The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the electrophoretic method. The method appears to be generally applicable to the analysis of genomes of large viruses.
References
[1]
Garcia-Maruniak A, Maruniak JE, Zanatto PMA, Doumbouya AE, Liu JC, et al. (2004) Sequence analysis of the genome of the Neodiprion sertifera Nucleopolyhedrovirus. J Virol 78: 7036–7051.
[2]
Jehle JA, Lange M, Wang H, Zhihong H, Wang Y, et al. (2006) Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology 346: 180–193.
[3]
Lauzon HA, Garcia-Maruniak A, Zanatto PM, Clemente JC, Herniou EA, et al. (2006) Genomic comparison of Neodiprion sertifera and Neodiprion lecontei nucleopolyhedroviruses and identification of potential hymenopteran baculovirus-specific open reading frames. J Gen Virol 87: 1477–1489.
[4]
Hashimoto Y, Hayakawa T, Ueno Y, Fugita T, Sano Y, et al. (2000) Sequence analysis of the Plutella xylostella granulovirus genome. Virology 275: 358–372.
[5]
Hayakawa T, Ko R, Okano K, Seong S, Goto C, et al. (1999) Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology 262: 277–297.
[6]
Hayakawa T, Rohrmann GF, Hashimoto Y (2000) Patterns of genome organization and content in lepidopteran baculoviruses. Virology 278: 1–12.
[7]
Lange M, Jehle JA (2003) The genome of the Cryptophlebia leucotreta granulovirus. Virology 317: 220–236.
[8]
Afonso CL, Tulman ER, Lu Z, Balinsky CA, Moser BA, et al. (2001) Genome sequence of a baculovirus pathogenic for Culex nigripalpus. J Virol 75: 11157–11165.
[9]
Moser BA, Becnel JJ, White SE, Alfonso C, Kutish G, et al. (2001) Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae. J Gen Virol 82: 283–297.
[10]
Winstanley D, Crook NE (1993) Replication of Cydia pomonella granulosis virus in cell cultures. J Gen Virol 74: 1599–1609.
[11]
Wang XF, Zhang BQ, Xu HJ, Cui YJ, Xu YP, et al. (2011) ODV-associated proteins of the Pieris rapae granulovirus. J Prot Res 10: 2817–2827.
[12]
Zhang BQ, Cheng RL, Wang XF, Zhang CX (2012) The genome of Pieris rapae granulovirus (Genome announcement). J Virol 86: 9544.
[13]
Yanisch-Perron CJ, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequencing of the M13mp18 and pUC9 vectors. Gene 33: 103–119.
[14]
Ewing B, Green P (1998) Base-calling of automated sequencer traces using PHRED. II. Error probabilities. Genome Res 8: 186–194.
[15]
Ewing B, Hillier L, Wendl M, Green P (1998) Base-calling of automated sequencer traces using PHRED. I. Accuracy assessment. Genome Res 8: 175–185.
[16]
Borodovsky M, McIninch JD (1993) GeneMark: Parallel gene recognition for both DNA strands. Comp Chem 17: 123–133.
[17]
Delcher AL, Harmon D, Kasif S, White O, Salzberg SL (1999) Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: 4636–4641.
[18]
Bocs S, Cruveiller S, Vallenet D, Nuel G, Medique C (2003) AMIGene: Annotation of microbial genes. Nucleic Acids Res 31: 3723–3726.
[19]
Wormleaton S, Kuzio J, Winstanley D (2003) The complete sequence of the Adoxophyes orana granulovirus genome. Virology 311: 350–365.
[20]
Escasa SR, Lauzon HAM, Mathur AC, Krell PJ, Arif BM (2006) Sequence analysis of the Choristoneura occidentalis granulovirus genome. J Gen Virol 87: 1917–1933.
[21]
Luque T, Finch R, Crook N, O’Reilly, Winstanley D (2001) The complete sequence of the Cydia pomonella granulovirus genome. J Gen Virol 82: 2531–2547.
[22]
Chen XW, Ijkel WFJ, Tarchini R, Sun XL, Sandbrink H, et al. (2001) The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 82: 241–257.
[23]
Wang Y, Choi JY, Roh JY, Liu Q, Tao XY, et al. (2011) Genomic sequence analysis of granulovirus isolated from the tobacco cutworm, Spodoptera litura. PloS ONE 6(11): e28163 Doi:10:1371/journal.pone.0028163.
[24]
Park TH, Park BS, Kim JA, Hong JK, Jin M, et al. (2011) Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project. J Gent Genomics 38: 47–53.
[25]
Huang SW, Lin YY, You EM, Liu TT, Shu HY, et al. (2011) Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon. BMC Genomics 12: 242.
[26]
Ariyadasa R, Stein N (2012) Advances in BAC-based physical mapping and map integration strategies in plants. J Biomed Biotech doi: 10.1155/2012/184854.
[27]
Feuillet C, Leach JE, Rogers J, Schnable PS, Eversole K (2011) Crop genome sequencing: lessons and rationales. Trends Plant Sci 16: 77–88.
[28]
Alkan C, Sajjadian S, Eichler EE (2011) Limitations of next generation genome sequence assembly. Nature Methods 8: 61–65.
[29]
Schatz MC, Delcher AL, Salzberg SL (2010) Assembly of large genomes using second-generation sequencing. Genome Res doi: 10.1101/gr.101360.109.
[30]
Kim CG, Fujiyama A, Saitou N (2003) Construction of a gorilla fosmid library and its PCR screening system. Genomics 82: 571–574.
[31]
Meyer JDF, Deleu W, Garcia-Mas J, Havey MJ (2008) Construction of a fosmid library of cucumber (Cucumis sativus) and comparative analyses of the Eif4E and eIF(iso)4E regions from cucumber and melon (Cucumis melo). Mol Genet Genomics 279: 473–480.
[32]
Hao DC, Ge G, Xiao P, Zhang Y, Yang L (2011) The first insight into the tissue specific Taxus transcriptome via Illumina second generation sequencing. PLoS one 6, doi:10.1371/journal.pone.0021220.
[33]
Coupland P, Chandra T, Quail M, Reik W, Swerdlow H (2012) Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation. BioTechniques 53: 365–372.
[34]
Mizutani T, Endoh D, Okamoto M, Shirato K, Shimizu H, et al. (2007) Rapid genome sequencing of RNA Viruses. Emerging Infectious Diseases 13: 322–324.
[35]
Malboeuf CM, Yang X, Charlebois P, Qu J, Berlin AM, et al. (2013) Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification. Nucleic Acids Res 41(1): e13 Doi:10.1093/nar/gks794.
[36]
Ninomiya M, Ueno Y, Funayama R, Nagashima T, Nishida Y, et al. (2012) Use of illumina deep sequencing technology to differentiate hepatitis C virus variants. J Clin Microbiol 50: 857–866.
[37]
Li L, Donly C, Li Q, Willis LG, Keddie BA, et al. (2002a) Identification and genomic analysis of a second species of nucleopolyhedrovirus isolated from Mamestra configurata. Virology 297: 226–244.
[38]
Li Q, Donly C, Li L, Willis LG, Theilmann DA, et al. (2002b) Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome. Virology 294: 106–121.
[39]
Lange M, Wang H, Zhihong H, Jehle JA (2004) Towards a molecular identification and classification system of lepidopteran-specific baculovirus. Virology 325: 36–47.
[40]
Harrison RL, Popham HJ (2008) Genomic sequence analysis of a granulovirus isolated from the Old World bollworm, Helicoverpa armigera. Virus Genes 36: 565–581.
[41]
Wan H, Wootton JC (2000) A global compositional complexity measure for biological sequences: AT-rich and GC-rich genomes encode less complex proteins. Comput Chem 24: 71–94.
[42]
Jukes TH, Bhushan V (1986) Silent nucleotide substitutions and G+C content of some mitochondrial and bacterial genes. J Mol Evol 24: 39–44.
[43]
D’Onofrio G, Mouchiroud D, Aissani B, Gautier C, Bernardi G (1991) Correlations between the compositional properties of human genes, codon usage, and amino acid composition of proteins. J Mol Evol 32: 504–510.
[44]
Mount DW (2001) Bioinformatics: Sequence and Genome analysis. Cold Spring Harbor, NY, p.308.
[45]
Hilton S, Winstanley D (2008) Genomic sequence and biological characterization of a nucleopolyhedrovirus isolated from the summer fruit tortrix, Adoxophyes orana. J Gen Virol 89: 2898–2908.
[46]
Karlin S (1998) Global dinucleotide signatures and analysis of genomic heterogeneity. Curr Opin Microbiol 1: 598–610.
[47]
Hyink O, Dellow RA, Olsen MJ, Caradoc-Davies KMB, Drake K, et al. 2002. Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus. J Gen Virol 83: 957–971.
[48]
Pang Y, Yu J, Wang L, Hu X, Li W, et al. (2001) Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287: 391–404.
[49]
Leisy DJ, Rasmussen C, Kim HT, Rohrmann GF (1995) The Autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for DNA replication activity and transcriptional enhancer function. Virology 208: 742–752.
[50]
Ko R, Okano K, Maeda S (2000) Structural and functional analysis of the Xestia c-nigrum granulovirus matrix metalloproteinase. J Virol 74: 11240–11246.
[51]
Wolgamot GM, Gross CH, Russell RLQ, Rohrmann GF (1993) Immunocytochemical characterization of p24, a baculovirus capsid-associated protein. J Gen Virol 74: 103–107.
[52]
van Oers MM, Vlak JM (1997) The baculovirus 10-kDa protein. J Invertebr Pathol 70: 1–17.
[53]
Monsma SA, Oomens AG, Blissard GW (1996) The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70: 4607–4616.
[54]
Pearson MN, Groten C, Rohrmann GF (2000) Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J Virol 74: 6126–6131.
[55]
Ijkel WFJ, Westenberg M, Goldbach RW, Blissard GW, Vlak JM, et al. (2000) A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site. Virology 275: 30–41.
[56]
Rapp JC, Wilson JA, Miller LK (1998) Nineteen baculovirus open reading frames, including LEF-12, support late gene expression. J Virol 72: 10197–10206.
[57]
Friesen PD (1997) Regulation of baculovirus early gene expression. In: L.K. Miller (ed). The Baculoviruses, Plenum Press. 141–170.
[58]
Lu A, Miller LK (1997) Regulation of baculovirus late and very late gene expression. In: L.K. Miller (ed). The Baculoviruses, Plenum Press. 193–216.
[59]
Gomi S, Zhou CE, Yih W, Majima K, Maeda S (1997) Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230: 35–47.
[60]
Morris TD, Todd JW, Fisher B, Miller LK (1994) Identification of lef-7: a baculovirus gene affecting late gene expression. Virology 200: 360–369.
[61]
Pearson MN, Rohrmann GF (1998) Characterization of a baculovirus encoded ATP-dependent DNA ligase. J Virol 72: 9142–9149.
[62]
Kuzio J, Pearson MN, Harwood SH, Funk CJ, Evans JT, et al. (1999) Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253: 17–34.
[63]
Ayres MD, Howard SC, Kuzio J, Lopez-Ferber M, Possee RD (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202: 586–605.
[64]
Gomi S, Majima K, Maeda S (1999) Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80: 1323–1337.
[65]
Lu A, Miller LK (1997) Regulation of baculovirus late and very late gene expression. In: L.K. Miller (ed.) The Baculoviruses, Plenum Press. 193–216.
[66]
Oh S, Kim DH, Patnaik BB, Jo YH, Noh MY, et al.. (2013) Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus. Arch Virol DOI 10.1007/s00705-013-1649-z.
[67]
Hawtin RE, Zarkowska T, Arnold K, Thomas CK, Gooday GW, et al. (1997) Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes. Virology 238: 243–253.
[68]
Tomalski MD, Eldridge R, Miller LK (1991) A baculovirus homolog of a Cu/Zn superoxide dismutase gene. Virology 184: 149–161.
[69]
Oh S, Kim DH, Patnaik BB, Jo YH, Noh MY, et al. (2013) Molecular and immunohistochemical characterization of granulin gene encoded in Pieris rapae granulovirus genome. J Invertebr Pathol 113: 7–17.
[70]
Wang RR, Deng F, Hou DH, Zhao Y, Guo L, et al. (2010) Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J Virol 84: 7233–7242.
[71]
Xiang XW, Chen L, Guo AQ, Yu SF, Yang R, et al. (2011) The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor. Virus Res 155: 69–75.
[72]
Song JJ, Wang RR, Deng F, Wang HL, Hu ZH (2008) Functional studies of per os infectivity factors of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. J Gen Virol 89: 2331–2338.
[73]
Wang P, Grandos RR (1998) Observations on the presence of the peritrophic membrane in larval Trichoplusia ni and its role in limiting baculovirus infection. J Invertebr Pathol 72: 57–62.
[74]
Jiang H, Liu S, Zhao P, Pope C (2009) Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae.. Insect Biochem Mol Biol 39: 646–653.
[75]
Dall D, Luque T, O’Reilly D (2001) Insect-virus relationships: sifting by informatics. Bioessays 23: 184–193.
[76]
Birnbaum MJ, Clem RJ, Miller LK (1994) An apoptosis inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. J Virol 68: 2521–2528.
[77]
Vucic D, Kaiser WJ, Harvey AJ, Miller LK (1997) Inhibition of Reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs). Proc Natl Acad Sci USA 94: 10183–10188.
[78]
Crook NE, Clem RJ, Miller LK (1993) An apoptosis-inhibiting baculovirus gene with a zinc-finger motif. J Virol 67: 2168–2174.
