Abstract
To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.
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Selected References
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- Auperin D. D., Sasso D. R., McCormick J. B. Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA. Virology. 1986 Oct 15;154(1):155–167. doi: 10.1016/0042-6822(86)90438-1. [DOI] [PubMed] [Google Scholar]
- Binns M. M., Boursnell M. E., Cavanagh D., Pappin D. J., Brown T. D. Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV. J Gen Virol. 1985 Apr;66(Pt 4):719–726. doi: 10.1099/0022-1317-66-4-719. [DOI] [PubMed] [Google Scholar]
- 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]
- Both G. W., Sleigh M. J., Cox N. J., Kendal A. P. Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites. J Virol. 1983 Oct;48(1):52–60. doi: 10.1128/jvi.48.1.52-60.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Collins P. L., Huang Y. T., Wertz G. W. Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7683–7687. doi: 10.1073/pnas.81.24.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis A. R., Bos T. J., Nayak D. P. Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3976–3980. doi: 10.1073/pnas.80.13.3976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Elango N., Satake M., Coligan J. E., Norrby E., Camargo E., Venkatesan S. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit. Nucleic Acids Res. 1985 Mar 11;13(5):1559–1574. doi: 10.1093/nar/13.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Fuller R. S., Sterne R. E., Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol. 1988;50:345–362. doi: 10.1146/annurev.ph.50.030188.002021. [DOI] [PubMed] [Google Scholar]
- Furie B., Furie B. C. The molecular basis of blood coagulation. Cell. 1988 May 20;53(4):505–518. doi: 10.1016/0092-8674(88)90567-3. [DOI] [PubMed] [Google Scholar]
- Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
- Garten W., Bosch F. X., Linder D., Rott R., Klenk H. D. Proteolytic activation of the influenza virus hemagglutinin: The structure of the cleavage site and the enzymes involved in cleavage. Virology. 1981 Dec;115(2):361–374. doi: 10.1016/0042-6822(81)90117-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Klenk H. D., Garten W., Rott R. Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187. EMBO J. 1984 Dec 1;3(12):2911–2915. doi: 10.1002/j.1460-2075.1984.tb02231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Klenk H. D., Wöllert W., Rott R., Scholtissek C. Association of influenza virus proteins with cytoplasmic fractions. Virology. 1974 Jan;57(1):28–41. doi: 10.1016/0042-6822(74)90105-6. [DOI] [PubMed] [Google Scholar]
- Kuroda K., Gröner A., Frese K., Drenckhahn D., Hauser C., Rott R., Doerfler W., Klenk H. D. Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J Virol. 1989 Apr;63(4):1677–1685. doi: 10.1128/jvi.63.4.1677-1685.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
- 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]
- 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]
- Paterson R. G., Shaughnessy M. A., Lamb R. A. Analysis of the relationship between cleavability of a paramyxovirus fusion protein and length of the connecting peptide. J Virol. 1989 Mar;63(3):1293–1301. doi: 10.1128/jvi.63.3.1293-1301.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pipas J. M., Peden K. W., Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):203–213. doi: 10.1128/mcb.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
- Rice C. M., Strauss J. H. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2062–2066. doi: 10.1073/pnas.78.4.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
- Spaete R. R., Saxena A., Scott P. I., Song G. J., Probert W. S., Britt W. J., Gibson W., Rasmussen L., Pachl C. Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. J Virol. 1990 Jun;64(6):2922–2931. doi: 10.1128/jvi.64.6.2922-2931.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toyoda T., Sakaguchi T., Imai K., Inocencio N. M., Gotoh B., Hamaguchi M., Nagai Y. Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus. Virology. 1987 May;158(1):242–247. doi: 10.1016/0042-6822(87)90261-3. [DOI] [PubMed] [Google Scholar]
- Yoshimasa Y., Seino S., Whittaker J., Kakehi T., Kosaki A., Kuzuya H., Imura H., Bell G. I., Steiner D. F. Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. Science. 1988 May 6;240(4853):784–787. doi: 10.1126/science.3283938. [DOI] [PubMed] [Google Scholar]
- de Curtis I., Simons K. Isolation of exocytic carrier vesicles from BHK cells. Cell. 1989 Aug 25;58(4):719–727. doi: 10.1016/0092-8674(89)90106-2. [DOI] [PubMed] [Google Scholar]