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. 1994 Oct;68(10):6347–6362. doi: 10.1128/jvi.68.10.6347-6362.1994

Improved cell survival by the reduction of immediate-early gene expression in replication-defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function.

P A Johnson 1, M J Wang 1, T Friedmann 1
PMCID: PMC237056  PMID: 8083974

Abstract

Derivatives of herpes simplex virus type 1 (HSV-1) have elicited considerable interest as gene transfer vectors because of their ability to infect a wide range of cell types efficiently, including fully differentiated neurons. However, it has been found that infection of many types of cell with vectors derived from replication-defective mutants of HSV-1 is associated with cytopathic effects (CPE). We have previously shown that viral gene expression played an important role in the induction of CPE caused by an HSV-1 mutant deleted for the essential immediate-early gene 3 (IE 3) (P.A. Johnson, A. Miyanohara, F. Levine, T. Cahill, and T. Friedmann, J. Virol. 66:2952-2965, 1992). We have investigated which viral genes might be responsible for CPE by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a nonproductive infection to inhibit biochemical transformation after cotransfection of BHK or CV-1 cells with a selectable marker gene. Transfection of IE genes 1,2, and 4 individually all caused a marked inhibition of colony formation, while transfection of IE 5 and the large subunit of ribonucleotide reductase had little effect. These results suggested that it would be necessary to mutate or reduce the expression of nearly all HSV-1 IE genes to reduce virus-induced CPE. Therefore, we have used VP16 mutants, which are unable to transinduce IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989), to derive two replication-defective strains: 14H delta 3, which is deleted for both copies of IE 3, and in 1850 delta 42, which has a deletion in the essential early gene UL42. The IE 3-VP16 double mutant, 14H delta 3, is significantly less toxic than a single IE 3 deletion mutant over a range of multiplicities of infection, as measured in a cell-killing assay, and has an enhanced ability to persist in infected cells in a biologically retrievable form. In contrast, the UL42-VP16 double mutant, in 1850 delta 42, showed reduced toxicity only at low multiplicities of infection. To test the role of the virion host shutoff function as an additional candidate to influence virus-induced CPE, we have introduced a large insertion mutation into the virion host shutoff gene of an IE 3 deletion mutant and the double mutant 14H delta 3.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ace C. I., Dalrymple M. A., Ramsay F. H., Preston V. G., Preston C. M. Mutational analysis of the herpes simplex virus type 1 trans-inducing factor Vmw65. J Gen Virol. 1988 Oct;69(Pt 10):2595–2605. doi: 10.1099/0022-1317-69-10-2595. [DOI] [PubMed] [Google Scholar]
  2. Ace C. I., McKee T. A., Ryan J. M., Cameron J. M., Preston C. M. Construction and characterization of a herpes simplex virus type 1 mutant unable to transinduce immediate-early gene expression. J Virol. 1989 May;63(5):2260–2269. doi: 10.1128/jvi.63.5.2260-2269.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Batterson W., Roizman B. Characterization of the herpes simplex virion-associated factor responsible for the induction of alpha genes. J Virol. 1983 May;46(2):371–377. doi: 10.1128/jvi.46.2.371-377.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Block T., Jordan R., Farkas D. H., Hughes R. G., Jr Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes. J Gen Virol. 1991 Jan;72(Pt 1):131–141. doi: 10.1099/0022-1317-72-1-131. [DOI] [PubMed] [Google Scholar]
  5. Breakefield X. O., DeLuca N. A. Herpes simplex virus for gene delivery to neurons. New Biol. 1991 Mar;3(3):203–218. [PubMed] [Google Scholar]
  6. Cai W. Z., Schaffer P. A. Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J Virol. 1989 Nov;63(11):4579–4589. doi: 10.1128/jvi.63.11.4579-4589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campbell M. E., Palfreyman J. W., Preston C. M. Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription. J Mol Biol. 1984 Nov 25;180(1):1–19. doi: 10.1016/0022-2836(84)90427-3. [DOI] [PubMed] [Google Scholar]
  8. Chiocca E. A., Choi B. B., Cai W. Z., DeLuca N. A., Schaffer P. A., DiFiglia M., Breakefield X. O., Martuza R. L. Transfer and expression of the lacZ gene in rat brain neurons mediated by herpes simplex virus mutants. New Biol. 1990 Aug;2(8):739–746. [PubMed] [Google Scholar]
  9. DeLuca N. A., McCarthy A. M., Schaffer P. A. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol. 1985 Nov;56(2):558–570. doi: 10.1128/jvi.56.2.558-570.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeLuca N. A., Schaffer P. A. Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res. 1987 Jun 11;15(11):4491–4511. doi: 10.1093/nar/15.11.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dobson A. T., Margolis T. P., Sedarati F., Stevens J. G., Feldman L. T. A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons. Neuron. 1990 Sep;5(3):353–360. doi: 10.1016/0896-6273(90)90171-b. [DOI] [PubMed] [Google Scholar]
  12. Everett R. D. The regulation of transcription of viral and cellular genes by herpesvirus immediate-early gene products (review). Anticancer Res. 1987 Jul-Aug;7(4A):589–604. [PubMed] [Google Scholar]
  13. Farkas D. H., Block T. M., Hart P. B., Hughes R. G., Jr Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants. J Virol. 1987 Oct;61(10):2989–2996. doi: 10.1128/jvi.61.10.2989-2996.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Fenwick M. L., Everett R. D. Transfer of UL41, the gene controlling virion-associated host cell shutoff, between different strains of herpes simplex virus. J Gen Virol. 1990 Feb;71(Pt 2):411–418. doi: 10.1099/0022-1317-71-2-411. [DOI] [PubMed] [Google Scholar]
  16. Fenwick M. L., Walker M. J. Suppression of the synthesis of cellular macromolecules by herpes simplex virus. J Gen Virol. 1978 Oct;41(1):37–51. doi: 10.1099/0022-1317-41-1-37. [DOI] [PubMed] [Google Scholar]
  17. Fink D. J., Sternberg L. R., Weber P. C., Mata M., Goins W. F., Glorioso J. C. In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer. Hum Gene Ther. 1992 Feb;3(1):11–19. doi: 10.1089/hum.1992.3.1-11. [DOI] [PubMed] [Google Scholar]
  18. Gallo M. L., Dorsky D. I., Crumpacker C. S., Parris D. S. The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase. J Virol. 1989 Dec;63(12):5023–5029. doi: 10.1128/jvi.63.12.5023-5029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gao M., Knipe D. M. Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein. J Virol. 1989 Dec;63(12):5258–5267. doi: 10.1128/jvi.63.12.5258-5267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldstein D. J., Weller S. K. Factor(s) present in herpes simplex virus type 1-infected cells can compensate for the loss of the large subunit of the viral ribonucleotide reductase: characterization of an ICP6 deletion mutant. Virology. 1988 Sep;166(1):41–51. doi: 10.1016/0042-6822(88)90144-4. [DOI] [PubMed] [Google Scholar]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Hardwicke M. A., Vaughan P. J., Sekulovich R. E., O'Conner R., Sandri-Goldin R. M. The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule. J Virol. 1989 Nov;63(11):4590–4602. doi: 10.1128/jvi.63.11.4590-4602.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Harris R. A., Preston C. M. Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814. J Gen Virol. 1991 Apr;72(Pt 4):907–913. doi: 10.1099/0022-1317-72-4-907. [DOI] [PubMed] [Google Scholar]
  24. Imbalzano A. N., Coen D. M., DeLuca N. A. Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene. J Virol. 1991 Feb;65(2):565–574. doi: 10.1128/jvi.65.2.565-574.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jang K. L., Pulverer B., Woodgett J. R., Latchman D. S. Activation of the cellular transcription factor AP-1 in herpes simplex virus infected cells is dependent on the viral immediate-early protein ICPO. Nucleic Acids Res. 1991 Sep 25;19(18):4879–4883. doi: 10.1093/nar/19.18.4879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson P. A., Best M. G., Friedmann T., Parris D. S. Isolation of a herpes simplex virus type 1 mutant deleted for the essential UL42 gene and characterization of its null phenotype. J Virol. 1991 Feb;65(2):700–710. doi: 10.1128/jvi.65.2.700-710.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnson P. A., Miyanohara A., Levine F., Cahill T., Friedmann T. Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1. J Virol. 1992 May;66(5):2952–2965. doi: 10.1128/jvi.66.5.2952-2965.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnson P. A., Yoshida K., Gage F. H., Friedmann T. Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector. Brain Res Mol Brain Res. 1992 Jan;12(1-3):95–102. doi: 10.1016/0169-328x(92)90072-j. [DOI] [PubMed] [Google Scholar]
  29. Johnson P., Friedmann T. Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK. Gene. 1990 Apr 16;88(2):207–213. doi: 10.1016/0378-1119(90)90033-n. [DOI] [PubMed] [Google Scholar]
  30. Kwong A. D., Frenkel N. Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1926–1930. doi: 10.1073/pnas.84.7.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kwong A. D., Frenkel N. The herpes simplex virus virion host shutoff function. J Virol. 1989 Nov;63(11):4834–4839. doi: 10.1128/jvi.63.11.4834-4839.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kwong A. D., Kruper J. A., Frenkel N. Herpes simplex virus virion host shutoff function. J Virol. 1988 Mar;62(3):912–921. doi: 10.1128/jvi.62.3.912-921.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Leib D. A., Coen D. M., Bogard C. L., Hicks K. A., Yager D. R., Knipe D. M., Tyler K. L., Schaffer P. A. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol. 1989 Feb;63(2):759–768. doi: 10.1128/jvi.63.2.759-768.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Leib D. A., Olivo P. D. Gene delivery to neurons: is herpes simplex virus the right tool for the job? Bioessays. 1993 Aug;15(8):547–554. doi: 10.1002/bies.950150808. [DOI] [PubMed] [Google Scholar]
  35. Marchetti M. E., Smith C. A., Schaffer P. A. A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL. J Virol. 1988 Mar;62(3):715–721. doi: 10.1128/jvi.62.3.715-721.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McCarthy A. M., McMahan L., Schaffer P. A. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J Virol. 1989 Jan;63(1):18–27. doi: 10.1128/jvi.63.1.18-27.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McFarlane M., Daksis J. I., Preston C. M. Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65. J Gen Virol. 1992 Feb;73(Pt 2):285–292. doi: 10.1099/0022-1317-73-2-285. [DOI] [PubMed] [Google Scholar]
  38. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  39. McGeoch D. J., Dolan A., Donald S., Rixon F. J. Sequence determination and genetic content of the short unique region in the genome of herpes simplex virus type 1. J Mol Biol. 1985 Jan 5;181(1):1–13. doi: 10.1016/0022-2836(85)90320-1. [DOI] [PubMed] [Google Scholar]
  40. McLauchlan J., Phelan A., Loney C., Sandri-Goldin R. M., Clements J. B. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. J Virol. 1992 Dec;66(12):6939–6945. doi: 10.1128/jvi.66.12.6939-6945.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Miyanohara A., Johnson P. A., Elam R. L., Dai Y., Witztum J. L., Verma I. M., Friedmann T. Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX. New Biol. 1992 Mar;4(3):238–246. [PubMed] [Google Scholar]
  42. Nishioka Y., Silverstein S. Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1. J Virol. 1978 Sep;27(3):619–627. doi: 10.1128/jvi.27.3.619-627.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. O'Hare P., Goding C. R., Haigh A. Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression. EMBO J. 1988 Dec 20;7(13):4231–4238. doi: 10.1002/j.1460-2075.1988.tb03320.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Oroskar A. A., Read G. S. A mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mRNAs. J Virol. 1987 Feb;61(2):604–606. doi: 10.1128/jvi.61.2.604-606.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Oroskar A. A., Read G. S. Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J Virol. 1989 May;63(5):1897–1906. doi: 10.1128/jvi.63.5.1897-1906.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Paterson T., Everett R. D. A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture. J Gen Virol. 1990 Aug;71(Pt 8):1775–1783. doi: 10.1099/0022-1317-71-8-1775. [DOI] [PubMed] [Google Scholar]
  47. Paterson T., Everett R. D. Mutational dissection of the HSV-1 immediate-early protein Vmw175 involved in transcriptional transactivation and repression. Virology. 1988 Sep;166(1):186–196. doi: 10.1016/0042-6822(88)90160-2. [DOI] [PubMed] [Google Scholar]
  48. Perry L. J., Rixon F. J., Everett R. D., Frame M. C., McGeoch D. J. Characterization of the IE110 gene of herpes simplex virus type 1. J Gen Virol. 1986 Nov;67(Pt 11):2365–2380. doi: 10.1099/0022-1317-67-11-2365. [DOI] [PubMed] [Google Scholar]
  49. Preston C. M. Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK. J Virol. 1979 Jan;29(1):275–284. doi: 10.1128/jvi.29.1.275-284.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Preston C. M., Frame M. C., Campbell M. E. A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence. Cell. 1988 Feb 12;52(3):425–434. doi: 10.1016/s0092-8674(88)80035-7. [DOI] [PubMed] [Google Scholar]
  51. Read G. S., Frenkel N. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides. J Virol. 1983 May;46(2):498–512. doi: 10.1128/jvi.46.2.498-512.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rice S. A., Long M. C., Lam V., Spencer C. A. RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection. J Virol. 1994 Feb;68(2):988–1001. doi: 10.1128/jvi.68.2.988-1001.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Roemer K., Friedmann T. Concepts and strategies for human gene therapy. Eur J Biochem. 1992 Sep 1;208(2):211–225. doi: 10.1111/j.1432-1033.1992.tb17176.x. [DOI] [PubMed] [Google Scholar]
  54. Russell J., Stow N. D., Stow E. C., Preston C. M. Herpes simplex virus genes involved in latency in vitro. J Gen Virol. 1987 Dec;68(Pt 12):3009–3018. doi: 10.1099/0022-1317-68-12-3009. [DOI] [PubMed] [Google Scholar]
  55. Sandri-Goldin R. M., Mendoza G. E. A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Genes Dev. 1992 May;6(5):848–863. doi: 10.1101/gad.6.5.848. [DOI] [PubMed] [Google Scholar]
  56. Sears A. E., Halliburton I. W., Meignier B., Silver S., Roizman B. Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice. J Virol. 1985 Aug;55(2):338–346. doi: 10.1128/jvi.55.2.338-346.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sekulovich R. E., Leary K., Sandri-Goldin R. M. The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. J Virol. 1988 Dec;62(12):4510–4522. doi: 10.1128/jvi.62.12.4510-4522.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Smibert C. A., Smiley J. R. Differential regulation of endogenous and transduced beta-globin genes during infection of erythroid cells with a herpes simplex virus type 1 recombinant. J Virol. 1990 Aug;64(8):3882–3894. doi: 10.1128/jvi.64.8.3882-3894.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  60. Spaete R. R., Mocarski E. S. Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol. 1985 Oct;56(1):135–143. doi: 10.1128/jvi.56.1.135-143.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Steiner I., Spivack J. G., Deshmane S. L., Ace C. I., Preston C. M., Fraser N. W. A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia. J Virol. 1990 Apr;64(4):1630–1638. doi: 10.1128/jvi.64.4.1630-1638.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Stow N. D., Stow E. C. Isolation and characterization of a herpes simplex virus type 1 mutant containing a deletion within the gene encoding the immediate early polypeptide Vmw110. J Gen Virol. 1986 Dec;67(Pt 12):2571–2585. doi: 10.1099/0022-1317-67-12-2571. [DOI] [PubMed] [Google Scholar]
  63. Triezenberg S. J., LaMarco K. L., McKnight S. L. Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16. Genes Dev. 1988 Jun;2(6):730–742. doi: 10.1101/gad.2.6.730. [DOI] [PubMed] [Google Scholar]
  64. Weinheimer S. P., Boyd B. A., Durham S. K., Resnick J. L., O'Boyle D. R., 2nd Deletion of the VP16 open reading frame of herpes simplex virus type 1. J Virol. 1992 Jan;66(1):258–269. doi: 10.1128/jvi.66.1.258-269.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. York I. A., Roop C., Andrews D. W., Riddell S. R., Graham F. L., Johnson D. C. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell. 1994 May 20;77(4):525–535. doi: 10.1016/0092-8674(94)90215-1. [DOI] [PubMed] [Google Scholar]

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