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Thep10 gene ofBombyx mori nucleopolyhedrosis virus encodes a 7.5-kDa protein and is hypertranscribed from a TAAG motif

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Abstract

In baculovirus-based high-level expression of cloned foreign genes, the viral very late gene promoters ofpolyhedrin (polh) andp10 are extensively exploited. Here we report the cloning and characterization of thep10 gene from a local isolate ofBombyx mori nucleopolyhedrosis virus (BmNPV). The gene harbours a 213-bp open reading frame encoding a protein of 70 amino acids with a predicted molecular mass of 7.5 kDa. The BmNPVp10 showed deletion of a single A at +210 nucleotide compared to the prototype baculovirus,Autographa californica multinucleocapsid nucleopolyhedrosis virus (AcMNPV),p10 gene, resulting in a translational frameshift to generate a termination codon and consequently a truncated polypeptide instead of the 10-kDa protein. This protein P7.5 from BmNPV has a putative leucine zipper dimerization motif towards the N-terminal end and the central nuclear disintegration domain but the carboxy-terminal domain implicated in protein association for fibrillar structure formation is absent. Phylogenetic analysis revealed thatp10 is highly conserved among baculoviruses and the BmNPV strains are more closely related to AcMNPV than other baculoviruses. The transcription ofp10 is regulated in a temporal manner, reaching maximal levels by 72 h post-infection. RNAase protection and primer extension analysis mapped the transcription start sites at -70 and -71 nt with respect to the ATG, within the conserved baculovirus late gene motif TAAG. The upstream region showed complete homology to the strong promoter of the AcMNPVp10, suggesting that this promoter from BmNPV could also be exploited for high-level expression of cloned foreign genes in silkworm cells or larvae.

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References

  • Ahrens C. H., Russel R., Funk C. J., Evans J. T., Harwood S. H. and Rohrmann G. F. 1997 The sequence ofOrygia pseudotsugata multinucleocapsid nucleopolyhedrosis virus genome.Virology 229, 381–399.

    Article  PubMed  CAS  Google Scholar 

  • Ayres M. D., Howard S. C., Kazia J., Lofiz-Ferber M. and Possee R. D. 1994 The complete DNA sequence ofAutographa californica nuclear polyhedrosis virus.Virology 202, 586–605.

    Article  PubMed  CAS  Google Scholar 

  • Blissard G. S. and Rohrmann G. E. 1990 Baculovirus diversity and molecular biology.Annu. Rev. Entomol. 53, 127–155.

    Article  Google Scholar 

  • Cheley S., Kosik K. S., Paskevich P., Bakalis S. and Baylev H. 1992 Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in SF9 cells.J. Cell Sci. 102, 739–752.

    PubMed  CAS  Google Scholar 

  • Chomczynski P. and Sacchi N. 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

  • Gomi S., Majima K. and Maeda S. 1999 Sequence analysis of the genome ofBombyx mori nucleopolyhedrosis virus.J. Gen. Virol. 80, 1323–1337.

    PubMed  CAS  Google Scholar 

  • Guarino L. A., Xu B., Jin J and Dong W. 1998 A virus encoded RNA polymerase purified from baculovirus-infected cells.J. Virol. 72, 7985–7991.

    PubMed  CAS  Google Scholar 

  • Harrap K. A. 1970 Cell infection by a nuclear polyhedrosis virus.Virology 42, 311–318.

    Article  PubMed  CAS  Google Scholar 

  • Hu N. T., Lu Y. F., Hashimoto Y., Maeda S. and Hou R. F. 1994 The p10 gene of natural isolates ofBombyx mori polyhedrosis virus encodes a truncated protein with an Mr of 7700.J. Gen. Virol. 75, 2085–2088.

    PubMed  CAS  Google Scholar 

  • Huh N. E. and Weaver R. F. 1990 Identifying the RNA polymerases that synthesize specific transcripts of theAutographa californica nuclear polyhedrosis virus.J. Gen. Virol. 71, 195–202.

    PubMed  CAS  Google Scholar 

  • Inoue H. and Mitsuhashi J. 1984 ABombyx mori cell line susceptible to a nuclear polyhedrosis virus.J. Sericulture Sci. Jpn. 53, 108–113.

    Google Scholar 

  • Kuzio J., Rohel D. Z., Curry C. J., Krebs A, Carstens E. B. and Faulkner P. 1984 Nucleotide sequence of the p10 polypeptide gene ofAutographa californica nuclear polyhedrosis virus.Virology 139, 414–418.

    Article  CAS  PubMed  Google Scholar 

  • Kuzio J., Pearson M. N., Harwood S. H., Funk C. J., Evans J. T., Slavicek J. M. and Rohrmann G. F. 1999 Sequence and analysis of the genome of a baculovirus pathogenic forLymantria dispar.Virology 253, 17–34.

    Article  PubMed  CAS  Google Scholar 

  • Lassar A. B., Martin P. L. and Roeder R. G. 1983 Transcription of class III genes: formation of preinitiation complexes.Science 222, 740–748.

    Article  PubMed  CAS  Google Scholar 

  • Leisy D. J., Rohrmann G. F., Nesson M. H. and Beaudreau G. 1986 Nucleotide sequencing and transcriptional mapping of theOrygia pseudotsugata multiple nuclear polyhedrosis virus p10 gene.Virology 153, 157–167.

    Article  PubMed  CAS  Google Scholar 

  • Maeda S. 1989 Gene transfer vectors of a baculovirus,Bombyx mori nuclear polyhedrosis virus, and their use for expression of foreign genes in insect cells. InInvertebrate cell systems and applications (ed. J. Mitsuhashi), vol. I, pp. 167–182. CRC Press, Boca Raton.

    Google Scholar 

  • Maeda S. and Majima K. 1990 Molecular cloning and physical mapping of the genome ofBombyx mori nuclear polyhedrosis virus.J. Gen. Virol. 71, 1851–1855.

    PubMed  CAS  Google Scholar 

  • Mingchou J., Lo C. F., Huang C. J. and Wang C. H. 1992 Isolation and nucleotide sequence ofPenna nuda multiple nucleocapsid nuclear polyhedrosis virus (PnMNPV) two late genes: polyhedrin and p10. In Proceedings of the XIX International Congress of Entomology, Beijing, China, Abstract No. 21, p. 280.

  • Palhan V. B. and Gopinathan K. P. 1996 Characterization of the local isolate ofBombyx mori nuclear polyhedrosis virus.Curr. Sci. 70, 147–153.

    Google Scholar 

  • Palhan V. B., Sumathy S. and Gopinathan K. P. 1995 Baculovirus mediated high level expression of luciferase in silkworm cells and larvae.BioTechniques 199, 97–104.

    Google Scholar 

  • Roelvink P. W., van Meer M. M. M., de Kort C. A. D., Possee R. D., Hammock B. D. and Vlak J. M. 1992 Dissimilar expression ofAutographa californica multicapsid nuclear polyhedrosis virus polyhedrin and p10 genes.J. Gen. Virol. 73, 1481–1489.

    PubMed  CAS  Google Scholar 

  • Rohrmann G. F. 1986 Polyhedrin structure.J. Gen. Virol. 67, 1499–1513.

    PubMed  CAS  Google Scholar 

  • Sambrook J., Fritsch E. F. and Maniatis T. 1989Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

    Google Scholar 

  • Sehgal D. and Gopinathan K. P. 1998 RecombinantBombyx mon nucleopolyhedrosis virus harboring green fluorescent protein.BioTechniques 25, 997–1006.

    PubMed  CAS  Google Scholar 

  • Smith G. E., Vlak J. M. and Summers M. D. 1982In vitro translation ofAutographa californica nuclear polyhedrosis virus early and late mRNAs.J. Virol. 44, 199–208.

    PubMed  CAS  Google Scholar 

  • Sriram S., Palhan V. B. and Gopinathan K. P. 1997 Heterologous promoter recognition leading to high-level expression of cloned foreign genes inBombyx mori cell lines and larvae.Gene 190, 181–189.

    Article  PubMed  CAS  Google Scholar 

  • Van Der Wilk F., Van Lent J. W. M. and Vlak J. M. 1987 Immunogold detection of polyhedrin, p10 and virion antigens inAutographa californica nuclear polyhedrosis virusinfectedSpodoptera frugiperda cells.J. Gen. Virol. 68, 2615–2624.

    Google Scholar 

  • Van Oers M. M., Elipsen J. T. M., Reusken C. B. E. M., Sliwinsky E. L., Goldbach R. W. and Vlak J. M. 1993 Functional domains of the p10 protein ofAutographa californica nuclear polyhedrosis virus.J. Gen. Virol. 74, 563–574.

    PubMed  Google Scholar 

  • Vlak J. M., Klinkenberg F. A., Zaal K. J. M., Usmany M., Klinge-Roode E. C., Geervliet J. B. F., Roosien J. and Van Lent J. W. M. 1988 Functional studies on the p10 gene ofAutographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-Β-galactosidase fusion gene.J. Gen. Virol. 69, 765–777.

    PubMed  CAS  Google Scholar 

  • Volkman L. E. and Zaal K. J. M. 1990Autographa californica M nuclear polyhedrosis virus: Microtubules and replication.Virology 175, 292–302.

    Article  PubMed  CAS  Google Scholar 

  • Williams G. V., Rohel D. Z., Kuzio J. and Faulkner P. 1989 A cytopathological investigation ofAutographa californica nuclear polyhedrosis virus p10 gene function using insertion-deletion mutants.J. Gen. Virol. 70, 187–202.

    PubMed  CAS  Google Scholar 

  • Wilson J. A., Hill J. E., Kuzio J. and Faulkner P. 1995 Characterization of the baculovirusChoristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain.J. Gen. Virol. 76, 2923–2932.

    Article  PubMed  CAS  Google Scholar 

  • Yang C. L., Stetler D. A. and Weaver R. F. 1991 Structural comparisons of theAutographa californica nuclear polyhedrosis virus-induced RNA polymerase and the 3 nuclear RNA polymerases from the host,Spodoptera frugiperda.Virus Res. 20, 251–264.

    Article  PubMed  CAS  Google Scholar 

  • Yaozhou Z. 1992 Studies on the p10 gene ofBombyx mori nuclear polyhedrosis virus. Ph.D. thesis, Graduate School of Chinese Academy of Agricultural Sciences, Shanghai Institute of Biochemistry, Academia Sinica, Shanghai, China.

    Google Scholar 

  • Zanotto P. M. de A., Sampaio M. J. A., Johnson D. W., Rocha T. L. and Marunaik J. E. 1992 TheAnticarsia gemmatalis nuclear polyhedrosis virus polyhedrin gene region: sequence analysis, gene product and structural comparisons.J. Gen. Virol. 73, 1049–1056.

    PubMed  CAS  Google Scholar 

  • Zuidema D., Van Oers M. M., Van Strien E. A., Caballero P. C., Klok E. J., Goldbach R. W. and Vlak J. M. 1993 Nucleotide sequence and transcriptional analysis of the p10 gene ofSpodoptera exigua nuclear polyhedrosis virus.J. Gen. Virol. 74, 1017–1024.

    PubMed  CAS  Google Scholar 

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  1. Department of Microbiology and Cell Biology, Indian Institute of Science, 560 012, Bangalore, India

    Vikas B. Palhan & Karumathil P. Gopinathan

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  1. Vikas B. Palhan
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  2. Karumathil P. Gopinathan
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Correspondence to Karumathil P. Gopinathan.

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Palhan, V.B., Gopinathan, K.P. Thep10 gene ofBombyx mori nucleopolyhedrosis virus encodes a 7.5-kDa protein and is hypertranscribed from a TAAG motif. J Genet 79, 33–40 (2000). https://doi.org/10.1007/BF02728943

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