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. 1994 May;68(5):3120–3128. doi: 10.1128/jvi.68.5.3120-3128.1994

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

T Horimoto 1, Y Kawaoka 1
PMCID: PMC236802  PMID: 8151777

Abstract

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.

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Selected References

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  1. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  2. Bilsel P., Castrucci M. R., Kawaoka Y. Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions. J Virol. 1993 Nov;67(11):6762–6767. doi: 10.1128/jvi.67.11.6762-6767.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bosch F. X., Garten W., Klenk H. D., Rott R. Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of Avian influenza viruses. Virology. 1981 Sep;113(2):725–735. doi: 10.1016/0042-6822(81)90201-4. [DOI] [PubMed] [Google Scholar]
  4. Bosch F. X., Orlich M., Klenk H. D., Rott R. The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. Virology. 1979 May;95(1):197–207. doi: 10.1016/0042-6822(79)90414-8. [DOI] [PubMed] [Google Scholar]
  5. Castrucci M. R., Bilsel P., Kawaoka Y. Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase. J Virol. 1992 Aug;66(8):4647–4653. doi: 10.1128/jvi.66.8.4647-4653.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Castrucci M. R., Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus. J Virol. 1993 Feb;67(2):759–764. doi: 10.1128/jvi.67.2.759-764.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deshpande K. L., Fried V. A., Ando M., Webster R. G. Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci U S A. 1987 Jan;84(1):36–40. doi: 10.1073/pnas.84.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Enami M., Luytjes W., Krystal M., Palese P. Introduction of site-specific mutations into the genome of influenza virus. Proc Natl Acad Sci U S A. 1990 May;87(10):3802–3805. doi: 10.1073/pnas.87.10.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Enami M., Palese P. High-efficiency formation of influenza virus transfectants. J Virol. 1991 May;65(5):2711–2713. doi: 10.1128/jvi.65.5.2711-2713.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gotoh B., Ogasawara T., Toyoda T., Inocencio N. M., Hamaguchi M., Nagai Y. An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo. EMBO J. 1990 Dec;9(12):4189–4195. doi: 10.1002/j.1460-2075.1990.tb07643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huddleston J. A., Brownlee G. G. The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68. Nucleic Acids Res. 1982 Feb 11;10(3):1029–1038. doi: 10.1093/nar/10.3.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kawahara N., Yang X. Z., Sakaguchi T., Kiyotani K., Nagai Y., Yoshida T. Distribution and substrate specificity of intracellular proteolytic processing enzyme(s) for paramyxovirus fusion glycoproteins. J Gen Virol. 1992 Mar;73(Pt 3):583–590. doi: 10.1099/0022-1317-73-3-583. [DOI] [PubMed] [Google Scholar]
  13. Kawaoka Y., Naeve C. W., Webster R. G. Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology. 1984 Dec;139(2):303–316. doi: 10.1016/0042-6822(84)90376-3. [DOI] [PubMed] [Google Scholar]
  14. Kawaoka Y., Nestorowicz A., Alexander D. J., Webster R. G. Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. Virology. 1987 May;158(1):218–227. doi: 10.1016/0042-6822(87)90256-x. [DOI] [PubMed] [Google Scholar]
  15. Kawaoka Y., Webster R. G. Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin. J Virol. 1989 Aug;63(8):3296–3300. doi: 10.1128/jvi.63.8.3296-3300.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kawaoka Y., Webster R. G. Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. Proc Natl Acad Sci U S A. 1988 Jan;85(2):324–328. doi: 10.1073/pnas.85.2.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khatchikian D., Orlich M., Rott R. Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus. Nature. 1989 Jul 13;340(6229):156–157. doi: 10.1038/340156a0. [DOI] [PubMed] [Google Scholar]
  18. Kido H., Kamoshita K., Fukutomi A., Katunuma N. Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4+ lymphocytes. Purification and characterization. J Biol Chem. 1993 Jun 25;268(18):13406–13413. [PubMed] [Google Scholar]
  19. Kido H., Yokogoshi Y., Sakai K., Tashiro M., Kishino Y., Fukutomi A., Katunuma N. Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein. J Biol Chem. 1992 Jul 5;267(19):13573–13579. [PubMed] [Google Scholar]
  20. Kiefer M. C., Tucker J. E., Joh R., Landsberg K. E., Saltman D., Barr P. J. Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. DNA Cell Biol. 1991 Dec;10(10):757–769. doi: 10.1089/dna.1991.10.757. [DOI] [PubMed] [Google Scholar]
  21. Klenk H. D., Rott R. The molecular biology of influenza virus pathogenicity. Adv Virus Res. 1988;34:247–281. doi: 10.1016/S0065-3527(08)60520-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  23. Lang G., Narayan O., Rouse B. T., Ferguson A. E., Connell M. C. A new influenza A virus infection in turkeys II. A highly pathogenic variant, a/turkey/ontario 772/66. Can Vet J. 1968 Jul;9(7):151–160. [PMC free article] [PubMed] [Google Scholar]
  24. Lazarowitz S. G., Goldberg A. R., Choppin P. W. Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: host cell activation of serum plasminogen. Virology. 1973 Nov;56(1):172–180. doi: 10.1016/0042-6822(73)90296-1. [DOI] [PubMed] [Google Scholar]
  25. Li S. Q., Orlich M., Rott R. Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes. J Virol. 1990 Jul;64(7):3297–3303. doi: 10.1128/jvi.64.7.3297-3303.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li S. Q., Schulman J. L., Moran T., Bona C., Palese P. Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes. J Virol. 1992 Jan;66(1):399–404. doi: 10.1128/jvi.66.1.399-404.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li S., Polonis V., Isobe H., Zaghouani H., Guinea R., Moran T., Bona C., Palese P. Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1. J Virol. 1993 Nov;67(11):6659–6666. doi: 10.1128/jvi.67.11.6659-6666.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li S., Rodrigues M., Rodriguez D., Rodriguez J. R., Esteban M., Palese P., Nussenzweig R. S., Zavala F. Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5214–5218. doi: 10.1073/pnas.90.11.5214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li S., Schulman J., Itamura S., Palese P. Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus. J Virol. 1993 Nov;67(11):6667–6673. doi: 10.1128/jvi.67.11.6667-6673.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu C., Air G. M. Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes. Virology. 1993 May;194(1):403–407. doi: 10.1006/viro.1993.1276. [DOI] [PubMed] [Google Scholar]
  31. Luo G., Chung J., Palese P. Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions. Virus Res. 1993 Aug;29(2):141–153. doi: 10.1016/0168-1702(93)90055-r. [DOI] [PubMed] [Google Scholar]
  32. Lusson J., Vieau D., Hamelin J., Day R., Chrétien M., Seidah N. G. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6691–6695. doi: 10.1073/pnas.90.14.6691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luytjes W., Krystal M., Enami M., Parvin J. D., Palese P. Amplification, expression, and packaging of foreign gene by influenza virus. Cell. 1989 Dec 22;59(6):1107–1113. doi: 10.1016/0092-8674(89)90766-6. [DOI] [PubMed] [Google Scholar]
  34. Muster T., Subbarao E. K., Enami M., Murphy B. R., Palese P. An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5177–5181. doi: 10.1073/pnas.88.12.5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakagawa T., Hosaka M., Torii S., Watanabe T., Murakami K., Nakayama K. Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4. J Biochem. 1993 Feb;113(2):132–135. doi: 10.1093/oxfordjournals.jbchem.a124015. [DOI] [PubMed] [Google Scholar]
  36. Ogasawara T., Gotoh B., Suzuki H., Asaka J., Shimokata K., Rott R., Nagai Y. Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. EMBO J. 1992 Feb;11(2):467–472. doi: 10.1002/j.1460-2075.1992.tb05076.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ohuchi M., Orlich M., Ohuchi R., Simpson B. E., Garten W., Klenk H. D., Rott R. Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2). Virology. 1989 Feb;168(2):274–280. doi: 10.1016/0042-6822(89)90267-5. [DOI] [PubMed] [Google Scholar]
  38. Ohuchi R., Ohuchi M., Garten W., Klenk H. D. Human influenza virus hemagglutinin with high sensitivity to proteolytic activation. J Virol. 1991 Jul;65(7):3530–3537. doi: 10.1128/jvi.65.7.3530-3537.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Orlich M., Khátchikian D., Teigler A., Rott R. Structural variation occurring in the hemagglutinin of influenza virus A/turkey/Oregon/71 during adaptation to different cell types. Virology. 1990 Jun;176(2):531–538. doi: 10.1016/0042-6822(90)90023-k. [DOI] [PubMed] [Google Scholar]
  40. Parvin J. D., Palese P., Honda A., Ishihama A., Krystal M. Promoter analysis of influenza virus RNA polymerase. J Virol. 1989 Dec;63(12):5142–5152. doi: 10.1128/jvi.63.12.5142-5152.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Philpott M., Hioe C., Sheerar M., Hinshaw V. S. Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus. J Virol. 1990 Jun;64(6):2941–2947. doi: 10.1128/jvi.64.6.2941-2947.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rott R., Klenk H. D. Significance of viral glycoproteins for infectivity and pathogenicity. Zentralbl Bakteriol Mikrobiol Hyg A. 1987 Aug;266(1-2):145–154. doi: 10.1016/S0176-6724(87)80028-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rott R., Orlich M., Klenk H. D., Wang M. L., Skehel J. J., Wiley D. C. Studies on the adaptation of influenza viruses to MDCK cells. EMBO J. 1984 Dec 20;3(13):3329–3332. doi: 10.1002/j.1460-2075.1984.tb02299.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rott R., Orlich M., Scholtissek C. Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: nonexculsive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus. J Virol. 1976 Jul;19(1):54–60. doi: 10.1128/jvi.19.1.54-60.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rott R., Reinacher M., Orlich M., Klenk H. D. Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo. Arch Virol. 1980;65(2):123–133. doi: 10.1007/BF01317323. [DOI] [PubMed] [Google Scholar]
  46. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  47. Stieneke-Gröber A., Vey M., Angliker H., Shaw E., Thomas G., Roberts C., Klenk H. D., Garten W. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 1992 Jul;11(7):2407–2414. doi: 10.1002/j.1460-2075.1992.tb05305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tashiro M., Ciborowski P., Klenk H. D., Pulverer G., Rott R. Role of Staphylococcus protease in the development of influenza pneumonia. Nature. 1987 Feb 5;325(6104):536–537. doi: 10.1038/325536a0. [DOI] [PubMed] [Google Scholar]
  49. Van Campen H., Easterday B. C., Hinshaw V. S. Destruction of lymphocytes by a virulent avian influenza A virus. J Gen Virol. 1989 Feb;70(Pt 2):467–472. doi: 10.1099/0022-1317-70-2-467. [DOI] [PubMed] [Google Scholar]
  50. Vey M., Orlich M., Adler S., Klenk H. D., Rott R., Garten W. Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R. Virology. 1992 May;188(1):408–413. doi: 10.1016/0042-6822(92)90775-K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Walker J. A., Kawaoka Y. Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. J Gen Virol. 1993 Feb;74(Pt 2):311–314. doi: 10.1099/0022-1317-74-2-311. [DOI] [PubMed] [Google Scholar]
  52. Walker J. A., Sakaguchi T., Matsuda Y., Yoshida T., Kawaoka Y. Location and character of the cellular enzyme that cleaves the hemagglutinin of a virulent avian influenza virus. Virology. 1992 Sep;190(1):278–287. doi: 10.1016/0042-6822(92)91214-f. [DOI] [PubMed] [Google Scholar]
  53. Webster R. G., Brown L. E., Jackson D. C. Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH. Virology. 1983 Apr 30;126(2):587–599. doi: 10.1016/s0042-6822(83)80015-4. [DOI] [PubMed] [Google Scholar]
  54. Webster R. G., Hinshaw V. S., Bean W. J., Van Wyke K. L., Geraci J. R., St Aubin D. J., Petursson G. Characterization of an influenza A virus from seals. Virology. 1981 Sep;113(2):712–724. doi: 10.1016/0042-6822(81)90200-2. [DOI] [PubMed] [Google Scholar]
  55. Webster R. G., Rott R. Influenza virus A pathogenicity: the pivotal role of hemagglutinin. Cell. 1987 Aug 28;50(5):665–666. doi: 10.1016/0092-8674(87)90321-7. [DOI] [PubMed] [Google Scholar]
  56. White J., Kartenbeck J., Helenius A. Membrane fusion activity of influenza virus. EMBO J. 1982;1(2):217–222. doi: 10.1002/j.1460-2075.1982.tb01150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wood G. W., McCauley J. W., Bashiruddin J. B., Alexander D. J. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes. Arch Virol. 1993;130(1-2):209–217. doi: 10.1007/BF01319010. [DOI] [PubMed] [Google Scholar]

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