Archives of Virology

, Volume 158, Issue 8, pp 1701–1718 | Cite as

Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus

  • Seunghan Oh
  • Dong Hyun Kim
  • Bharat Bhusan Patnaik
  • Yong Hun Jo
  • Mi Young Noh
  • Hyo Jeong Lee
  • Kwang Ho Lee
  • Kwang Ho Yoon
  • Wan-Jong Kim
  • Ju Young Noh
  • Heon Cheon Jeong
  • Yong Seok Lee
  • Chuan-Xi Zhang
  • Yong-Su Song
  • Woo-Jin Jung
  • Kisung Ko
  • Yeon Soo Han
Original Article


The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.


Chitinase Chitinase Gene Immunogold Label Infected Larva Signal Peptide Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the Mid-Career Researcher Program through NRF grant funded by the MEST (No. 2010-0027694).


  1. 1.
    Ahrens CH, Russell RL, Funk CJ, Evans JT, Harwood SH, Rohrmann GF (1997) The sequence of the Orgyia pseudosugata multinucleocapsid nuclear Polyhedrosis virus genome. Virology 229:381–399CrossRefPubMedGoogle Scholar
  2. 2.
    Ayres MD, Howard SC, Kuzio J, Lopez-Ferber M, Possee RD (1994) The complete DNA sequence of Autographa californica nuclear Polyhedrosis virus. Virology 202:586–605CrossRefPubMedGoogle Scholar
  3. 3.
    Bradford MM (1976) A dye binding assay for protein. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. 4.
    Brurberg MB, Synstad B, Klemsdal SS, van Aalten DMF, Sundheim L, Eijsink VGH (2001) Chitinases from Serratia marcescens. Recent Res Dev Microbiol 5:187–204Google Scholar
  5. 5.
    Chen X, Ijkel WFJ, Tarchini R, Sun X, Sandbrink H, Wang H, Peters S, Zuiderma D, Lankhorst RK, Vlak JM, Hu Z (2001) The sequence of the Helicoverpa armigera single nucleocapsids nucleopolyhedrovirus genome. J Gen Virol 82:241–257PubMedGoogle Scholar
  6. 6.
    Combet C, Blanchet C, Geourjon C, Deleage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150CrossRefPubMedGoogle Scholar
  7. 7.
    Corrado G, Arciello S, Fanti P, Fiandra L, Garonna A, Digilio MA, Lorito M, Giordana B, Pennacchio F, Rao R (2008) The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco. Transgenic Res 17:557–571CrossRefPubMedGoogle Scholar
  8. 8.
    Daimon T, Katsuma S, Kang W, Shimada T (2006) Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its ortholog, BmChi-h. Biochem Biophys Res Commun 345:825–833CrossRefPubMedGoogle Scholar
  9. 9.
    Daimon T, Katsuma S, Shimada T (2007) Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus. Virus Res 124:168–175CrossRefPubMedGoogle Scholar
  10. 10.
    Deleage G, Blanchet C, Geourjon C (1997) Protein secondary structure prediction. Implications for the biologist. Biochimie 79:681–686CrossRefPubMedGoogle Scholar
  11. 11.
    Dezianian A, Sajap AS, Lau WH, Omar D, Kadir HA, Mohamed R, Yusoh MRM (2010) Morphological Characteristics of P. xylostella granulovirus and effects on its larval host Diamondback moth Plutella xylostella L. (Lepidoptera: Plutellidae). Amer J Agric Biol Sci 5:43–49CrossRefGoogle Scholar
  12. 12.
    Frigerio L, Pastres A, Prada A, Vitale A (2001) Influence of KDEL on the fate of trimeric or assembly-defective phaseolin: Selective use of an alternative route to vacuoles. Plant Cell 13:1109–1126PubMedGoogle Scholar
  13. 13.
    Girard C, Jouanin L (1999) Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae. Insect Biochem Mol Biol 29:1129–1142CrossRefPubMedGoogle Scholar
  14. 14.
    Gomi S, Majima K, Maeda S (1999) Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80:1323–1337PubMedGoogle Scholar
  15. 15.
    Hawtin RE, Arnold K, Ayres MD, Zanatto PM, Howard SC, Gooday GW, Chappell LH, Kitts PA, King LA, Possee RD (1995) Identification and preliminary characterization of a chitinase gene in the Autographa californica Nuclear Polyhedrosis Virus Genome. Virology 212:673–685CrossRefPubMedGoogle Scholar
  16. 16.
    Hawtin RE, Zarkowska T, Arnold K, Thomas CJ, Gooday GW, King LA, Kuzio JA, Possee RD (1997) Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes. Virology 238:243–253CrossRefPubMedGoogle Scholar
  17. 17.
    Hayakawa T, Ko R, Okano K, Seong SI, Goto C, Maeda S (1999) Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology 262:277–297CrossRefPubMedGoogle Scholar
  18. 18.
    Henrissat B (1999) Classification of chitinase modules. In. Chitin and Chitinases. Muzzarelli RAA (ed) Birkhauser-Verlag, Basel, Switzerland, pp137-159Google Scholar
  19. 19.
    Herniou EA, Olszewski JA, Cory JS, O’Reilly DR (2003) The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48:211–234CrossRefPubMedGoogle Scholar
  20. 20.
    Hodgson JJ, Arif BM, Krell PJ (2011) Interaction of Autographa californica multiple nucleopolyhedrovirus cathepsin protease progenitor (V-CATH) with insect baculovirus chitinase as a mechanism for proV-CATH cellular retention. J Virol 85:3918–3929CrossRefPubMedGoogle Scholar
  21. 21.
    Hom LG, Volkman LE (2000) Autographa californica M nucleopolyhedrovirus chiA is required for processing of V-CATH. Virology 277:178–183CrossRefPubMedGoogle Scholar
  22. 22.
    Hughes AL, Friedman R (2003) Genome-wide survey for genes horizontally transferred from cellular organisms to Baculoviruses. Mol Biol Evol 20:979–987CrossRefPubMedGoogle Scholar
  23. 23.
    Hyink O, Dellow RA, Olsen MJ, Caradoc-Davies KMB, Drake K, Herniou EA, Cory JS, O’Reilly DR, Ward VK (2002) Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus. J Gen Virol 83:957–971PubMedGoogle Scholar
  24. 24.
    Ijkel WFJ, van Strien EA, Heldens JGM, Broer R, Zuidema D, Goldbach RW, Vlak JM (1999) Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. J Gen Virol 80:3289–3304PubMedGoogle Scholar
  25. 25.
    Kang W, Tristem M, Maeda S, Crook NE, O’ Reilly DR (1998) Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes. J Gen Virol 79:2283–2292PubMedGoogle Scholar
  26. 26.
    Kasprzewska A (2003) Plant chitinases-regulation and function. Cell Mol Biol Lett 8:809–824PubMedGoogle Scholar
  27. 27.
    Kim MG, Shin SW, Bae KS, Kim SC, Park HY (1998) Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea. Insect Biochem Mol Biol 28:163–171CrossRefPubMedGoogle Scholar
  28. 28.
    Krishnan A, Nair PN, Jones D (1994) Isolation, cloning and characterization of new chitinase stored in active form in chitin-lined venom reservoir. J Biol Chem 269:20971–20976PubMedGoogle Scholar
  29. 29.
    Kuzio J, Pearson MN, Harwood SH, Funk CJ, Evans JT, Slavicek JM, Rohrmann GF (1999) Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253:17–34CrossRefPubMedGoogle Scholar
  30. 30.
    Lange M, Jehle JA (2003) The genome of the Cryptophlebia leucotreta granulovirus. Virology 317:220–236CrossRefPubMedGoogle Scholar
  31. 31.
    Li Q, Donly C, Li L, Willis LG, Theilmann DA, Erlandson M (2002) Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome. Virology 294:106–121CrossRefPubMedGoogle Scholar
  32. 32.
    Maeda S (1989) Gene transfer vectors of a baculovirus, Bombyx mori nucleopolyhedrovirus, and their use for expression of foreign genes in insect cells. In: Mitsuhasi J (ed) Invertebrate cell system application, vol 1. CRC Press, Boca Raton, pp 167–181Google Scholar
  33. 33.
    McCreath KJ, Gooday GW (1992) A rapid and sensitive micro-assay for determination of chitinolytic activity. J Microbiol Methods 14:229–237CrossRefGoogle Scholar
  34. 34.
    McLysaght A, Baldi PF, Gaut BS (2003) Extensive gene gain associated with adaptive evolution of poxviruses. PNAS 100:15655–15660CrossRefPubMedGoogle Scholar
  35. 35.
    Merzendorfer H, Zimoch L (2003) Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol 206:4393–4412CrossRefPubMedGoogle Scholar
  36. 36.
    Pang Y, Yu J, Wang L, Hu X, Bao W, Li G, Chen C, Han H, Hu S, Yang H (2001) Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287:391–404CrossRefPubMedGoogle Scholar
  37. 37.
    Rashidan KK, Nassoury N, Giannopoulos PN, Mauffette Y, Guertin C (2005) Identification, characterization and phylogenetic analysis of conserved genes within the odvp-6e/odv-e56 gene region of Choristoneura fumiferana Granulovirus. J Biochem Mol Biol 37:206–212CrossRefGoogle Scholar
  38. 38.
    Rao R, Fiandra L, Giordana B, Eguileor M, Congiu T, Burlini N, Arciello S, Corrado G, Pennacchio F (2004) AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae. Insect Biochem Mol Biol 34:1205–1213CrossRefPubMedGoogle Scholar
  39. 39.
    Sahai AS, Manocha MS (1993) Chitinases of fungi and plants: their involvement in morphogenesis and host parasite interaction. FEMS Microbiol Rev 11:317–338CrossRefGoogle Scholar
  40. 40.
    Saville GP, Thomas CJ, Possee RD, King LA (2002) Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif. J Gen Virol 83:685–694PubMedGoogle Scholar
  41. 41.
    Shen Z, Jacobs-Lorena M (1997) Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J Biol Chem 272:28895–28900CrossRefPubMedGoogle Scholar
  42. 42.
    Shinoda T, Kobayashi J, Matsui M, Chinzei Y (2001) Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virus-encoded chitinase gene. Insect Biochem Mol Biol 31:521–532CrossRefPubMedGoogle Scholar
  43. 43.
    Su J, Fu CS, You CB (1990) Characterization of inclusion body protein of granulosis virus from Pieris rapae. Acta Agriculturae Nucleatae Sinica 4:151–156Google Scholar
  44. 44.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  45. 45.
    Thomas CJ, Brown HL, Hawes CR, Lee BY, Min MK, King LA, Possee RD (1998) Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum. J Virol 72:10207–10212PubMedGoogle Scholar
  46. 46.
    Thomas CJ, Gooday GW, King LA, Possee RD (2000) Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene. J Gen Virol 81:1403–1411PubMedGoogle Scholar
  47. 47.
    Watanabe T, Kono M (1997) Isolation of a cDNA encoding a chitinase family protein from cuticular tissues of the Kuruma prawn Penaeus japonicas. Zool Sci 14:65–68CrossRefPubMedGoogle Scholar
  48. 48.
    Watanabe T, Kono M, Aida K, Nagasawa H (1996) Isolation of cDNA encoding a putative chitinase precursor in the Kuruma prawn Penaeus japonicas. MolMar Biol Biotechnol 5:299–303Google Scholar
  49. 49.
    Watanabe T, Kono M, Aida K, Nagasawa H (1998) Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicas. Biochim Biophys Acta 1382:181–185CrossRefPubMedGoogle Scholar
  50. 50.
    Williams SJ, Mark BL, Vocadlo DJ, James MNG, Withers SG (2002) Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate assisted catalysis by orienting 2-acetamido group and stabilizing the transition state. J Biol Chem 277:40055–40065CrossRefPubMedGoogle Scholar
  51. 51.
    Young VL, Simpson RM, Ward VK (2005) Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae). J Gen Virol 86:3253–3261CrossRefPubMedGoogle Scholar
  52. 52.
    Zhang B-Q, Cheng R-L, Wang X-F, Zhang C-X (2012) The genome of Pieris rapae granulovirus (Genome announcement). J Virol 86:9544CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Seunghan Oh
    • 1
  • Dong Hyun Kim
    • 1
  • Bharat Bhusan Patnaik
    • 1
  • Yong Hun Jo
    • 1
  • Mi Young Noh
    • 1
  • Hyo Jeong Lee
    • 1
  • Kwang Ho Lee
    • 2
  • Kwang Ho Yoon
    • 3
  • Wan-Jong Kim
    • 3
  • Ju Young Noh
    • 4
  • Heon Cheon Jeong
    • 4
  • Yong Seok Lee
    • 5
  • Chuan-Xi Zhang
    • 6
  • Yong-Su Song
    • 7
  • Woo-Jin Jung
    • 7
  • Kisung Ko
    • 8
  • Yeon Soo Han
    • 1
  1. 1.Division of Plant BiotechnologyCollege of Agriculture and Life Science, Chonnam National UniversityGwangjuKorea
  2. 2.Department of Wood Science and Landscape ArchitectureChonnam National UniversityGwangjuKorea
  3. 3.Department of BiologySoonchunhyang UniversityAsanKorea
  4. 4.Hampyeong County Insect Institute, Hampyeong County Agricultural Technology CenterHampyeongKorea
  5. 5.Department of Life Science and BiotechnologyCollege of Natural Sciences, Soonchunhyang University AsanKorea
  6. 6.Institute of Insect Science, Zhejiang-California International Nanosystem InstituteZhejiang UniversityHangzhouChina
  7. 7.Division of Applied Bioscience and Biotechnology, Institute of Environmentally Friendly Agriculture (IEFA)College of Agriculture and Life Sciences, Chonnam National UniversityGwangjuKorea
  8. 8.Department of Medicine, Medical Research InstituteCollege of Medicine Chung-Ang UniversitySeoulKorea

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