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. 2002 Nov 15;368(Pt 1):159–169. doi: 10.1042/BJ20020456

Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus.

Seema Sehrawat 1, Narayanaswamy Srinivasan 1, Karumathil P Gopinathan 1
PMCID: PMC1222975  PMID: 12169097

Abstract

Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330+/-20 microM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.

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Selected References

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  1. Beniya H., Funk C. J., Rohrmann G. F., Weaver R. F. Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro. Virology. 1996 Feb 1;216(1):12–19. doi: 10.1006/viro.1996.0029. [DOI] [PubMed] [Google Scholar]
  2. Cong P., Shuman S. Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. Mol Cell Biol. 1995 Nov;15(11):6222–6231. doi: 10.1128/mcb.15.11.6222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
  4. Fischer D., Rice D., Bowie J. U., Eisenberg D. Assigning amino acid sequences to 3-dimensional protein folds. FASEB J. 1996 Jan;10(1):126–136. doi: 10.1096/fasebj.10.1.8566533. [DOI] [PubMed] [Google Scholar]
  5. Fuchs L. Y., Woods M. S., Weaver R. F. Viral Transcription During Autographa californica Nuclear Polyhedrosis Virus Infection: a Novel RNA Polymerase Induced in Infected Spodoptera frugiperda Cells. J Virol. 1983 Dec;48(3):641–646. doi: 10.1128/jvi.48.3.641-646.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glocker B., Hoopes R. R., Jr, Hodges L., Rohrmann G. F. In vitro transcription from baculovirus late gene promoters: accurate mRNA initiation by nuclear extracts prepared from infected Spodoptera frugiperda cells. J Virol. 1993 Jul;67(7):3771–3776. doi: 10.1128/jvi.67.7.3771-3776.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gomi S., Majima K., Maeda S. Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol. 1999 May;80(Pt 5):1323–1337. doi: 10.1099/0022-1317-80-5-1323. [DOI] [PubMed] [Google Scholar]
  8. Gross C. H., Shuman S. RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein. J Virol. 1998 Dec;72(12):10020–10028. doi: 10.1128/jvi.72.12.10020-10028.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guarino L. A., Jin J., Dong W. Guanylyltransferase activity of the LEF-4 subunit of baculovirus RNA polymerase. J Virol. 1998 Dec;72(12):10003–10010. doi: 10.1128/jvi.72.12.10003-10010.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guarino L. A., Xu B., Jin J., Dong W. A virus-encoded RNA polymerase purified from baculovirus-infected cells. J Virol. 1998 Oct;72(10):7985–7991. doi: 10.1128/jvi.72.10.7985-7991.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hashimoto Y., Hayakawa T., Ueno Y., Fujita T., Sano Y., Matsumoto T. Sequence analysis of the Plutella xylostella granulovirus genome. Virology. 2000 Sep 30;275(2):358–372. doi: 10.1006/viro.2000.0530. [DOI] [PubMed] [Google Scholar]
  12. Huh N. E., Weaver R. F. Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus. J Gen Virol. 1990 Jan;71(Pt 1):195–201. doi: 10.1099/0022-1317-71-1-195. [DOI] [PubMed] [Google Scholar]
  13. Hâkansson K., Wigley D. B. Structure of a complex between a cap analogue and mRNA guanylyl transferase demonstrates the structural chemistry of RNA capping. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1505–1510. doi: 10.1073/pnas.95.4.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jin J., Dong W., Guarino L. A. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities. J Virol. 1998 Dec;72(12):10011–10019. doi: 10.1128/jvi.72.12.10011-10019.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones D. T. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J Mol Biol. 1999 Apr 9;287(4):797–815. doi: 10.1006/jmbi.1999.2583. [DOI] [PubMed] [Google Scholar]
  16. Kelley L. A., MacCallum R. M., Sternberg M. J. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J Mol Biol. 2000 Jun 2;299(2):499–520. doi: 10.1006/jmbi.2000.3741. [DOI] [PubMed] [Google Scholar]
  17. Kool M., Ahrens C. H., Goldbach R. W., Rohrmann G. F., Vlak J. M. Identification of genes involved in DNA replication of the Autographa californica baculovirus. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11212–11216. doi: 10.1073/pnas.91.23.11212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuzio J., Pearson M. N., Harwood S. H., Funk C. J., Evans J. T., Slavicek J. M., Rohrmann G. F. Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar. Virology. 1999 Jan 5;253(1):17–34. doi: 10.1006/viro.1998.9469. [DOI] [PubMed] [Google Scholar]
  19. Lu A., Miller L. K. Differential requirements for baculovirus late expression factor genes in two cell lines. J Virol. 1995 Oct;69(10):6265–6272. doi: 10.1128/jvi.69.10.6265-6272.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lu A., Miller L. K. Identification of three late expression factor genes within the 33.8- to 43.4-map-unit region of Autographa californica nuclear polyhedrosis virus. J Virol. 1994 Oct;68(10):6710–6718. doi: 10.1128/jvi.68.10.6710-6718.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maeda S. Expression of foreign genes in insects using baculovirus vectors. Annu Rev Entomol. 1989;34:351–372. doi: 10.1146/annurev.en.34.010189.002031. [DOI] [PubMed] [Google Scholar]
  22. Maeda S., Kawai T., Obinata M., Fujiwara H., Horiuchi T., Saeki Y., Sato Y., Furusawa M. Production of human alpha-interferon in silkworm using a baculovirus vector. Nature. 1985 Jun 13;315(6020):592–594. doi: 10.1038/315592a0. [DOI] [PubMed] [Google Scholar]
  23. Martins A., Shuman S. Mutational analysis of baculovirus capping enzyme Lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity. J Biol Chem. 2001 Sep 11;276(49):45522–45529. doi: 10.1074/jbc.M107615200. [DOI] [PubMed] [Google Scholar]
  24. Merrington C. L., Kitts P. A., King L. A., Possee R. D. An Autographa californica nucleopolyhedrovirus lef-2 mutant: consequences for DNA replication and very late gene expression. Virology. 1996 Mar 1;217(1):338–348. doi: 10.1006/viro.1996.0121. [DOI] [PubMed] [Google Scholar]
  25. Miyajima A., Schreurs J., Otsu K., Kondo A., Arai K., Maeda S. Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3. Gene. 1987;58(2-3):273–281. doi: 10.1016/0378-1119(87)90382-9. [DOI] [PubMed] [Google Scholar]
  26. Mizuguchi K., Deane C. M., Blundell T. L., Johnson M. S., Overington J. P. JOY: protein sequence-structure representation and analysis. Bioinformatics. 1998;14(7):617–623. doi: 10.1093/bioinformatics/14.7.617. [DOI] [PubMed] [Google Scholar]
  27. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  28. Palhan V. B., Sumathy S., Gopinathan K. P. Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae. Biotechniques. 1995 Jul;19(1):97-8, 100, 102-4. [PubMed] [Google Scholar]
  29. Passarelli A. L., Todd J. W., Miller L. K. A baculovirus gene involved in late gene expression predicts a large polypeptide with a conserved motif of RNA polymerases. J Virol. 1994 Jul;68(7):4673–4678. doi: 10.1128/jvi.68.7.4673-4678.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rapp J. C., Wilson J. A., Miller L. K. Nineteen baculovirus open reading frames, including LEF-12, support late gene expression. J Virol. 1998 Dec;72(12):10197–10206. doi: 10.1128/jvi.72.12.10197-10206.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sehgal D., Gopinathan K. P. Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein. Biotechniques. 1998 Dec;25(6):997-1000, 1002, 1004 passim. doi: 10.2144/98256st02. [DOI] [PubMed] [Google Scholar]
  32. Shuman S., Hurwitz J. Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate. Proc Natl Acad Sci U S A. 1981 Jan;78(1):187–191. doi: 10.1073/pnas.78.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
  34. Shuman S., Surks M., Furneaux H., Hurwitz J. Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme). J Biol Chem. 1980 Dec 10;255(23):11588–11598. [PubMed] [Google Scholar]
  35. Silva E., Ullu E., Kobayashi R., Tschudi C. Trypanosome capping enzymes display a novel two-domain structure. Mol Cell Biol. 1998 Aug;18(8):4612–4619. doi: 10.1128/mcb.18.8.4612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Srinivasan N., Bax B., Blundell T. L., Parker P. J. Structural aspects of the functional modules in human protein kinase-C alpha deduced from comparative analyses. Proteins. 1996 Oct;26(2):217–235. doi: 10.1002/(SICI)1097-0134(199610)26:2<217::AID-PROT11>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  37. Srinivasan N., Blundell T. L. An evaluation of the performance of an automated procedure for comparative modelling of protein tertiary structure. Protein Eng. 1993 Jul;6(5):501–512. doi: 10.1093/protein/6.5.501. [DOI] [PubMed] [Google Scholar]
  38. Sriram S., Gopinathan K. P. The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication. Virology. 1998 Nov 10;251(1):108–122. doi: 10.1006/viro.1998.9404. [DOI] [PubMed] [Google Scholar]
  39. Sutcliffe M. J., Haneef I., Carney D., Blundell T. L. Knowledge based modelling of homologous proteins, Part I: Three-dimensional frameworks derived from the simultaneous superposition of multiple structures. Protein Eng. 1987 Oct-Nov;1(5):377–384. doi: 10.1093/protein/1.5.377. [DOI] [PubMed] [Google Scholar]
  40. Sutcliffe M. J., Hayes F. R., Blundell T. L. Knowledge based modelling of homologous proteins, Part II: Rules for the conformations of substituted sidechains. Protein Eng. 1987 Oct-Nov;1(5):385–392. doi: 10.1093/protein/1.5.385. [DOI] [PubMed] [Google Scholar]
  41. Todd J. W., Passarelli A. L., Miller L. K. Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J Virol. 1995 Feb;69(2):968–974. doi: 10.1128/jvi.69.2.968-974.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Topham C. M., McLeod A., Eisenmenger F., Overington J. P., Johnson M. S., Blundell T. L. Fragment ranking in modelling of protein structure. Conformationally constrained environmental amino acid substitution tables. J Mol Biol. 1993 Jan 5;229(1):194–220. doi: 10.1006/jmbi.1993.1018. [DOI] [PubMed] [Google Scholar]
  43. Venkatesan S., Gershowitz A., Moss B. Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus. J Biol Chem. 1980 Feb 10;255(3):903–908. [PubMed] [Google Scholar]
  44. Wang S. P., Deng L., Ho C. K., Shuman S. Phylogeny of mRNA capping enzymes. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9573–9578. doi: 10.1073/pnas.94.18.9573. [DOI] [PMC free article] [PubMed] [Google Scholar]

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