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

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.

[1]  C. Bona,et al.  Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1 , 1993, Journal of virology.

[2]  P. Palese,et al.  Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus , 1993, Journal of virology.

[3]  Y. Kawaoka,et al.  Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions , 1993, Journal of virology.

[4]  P. Palese,et al.  Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions. , 1993, Virus research.

[5]  M Chrétien,et al.  cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  N. Katunuma,et al.  Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4+ lymphocytes. Purification and characterization. , 1993, The Journal of biological chemistry.

[7]  P. Palese,et al.  Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  G. Air,et al.  Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes. , 1993, Virology.

[9]  T Watanabe,et al.  Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4. , 1993, Journal of biochemistry.

[10]  Y. Kawaoka,et al.  Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. , 1993, The Journal of general virology.

[11]  Y. Kawaoka,et al.  Biologic importance of neuraminidase stalk length in influenza A virus , 1993, Journal of virology.

[12]  Y. Kawaoka,et al.  Location and character of the cellular enzyme that cleaves the hemagglutinin of a virulent avian influenza virus. , 1992, Virology.

[13]  Y. Kawaoka,et al.  Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase , 1992, Journal of virology.

[14]  N. Katunuma,et al.  Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein. , 1992, The Journal of biological chemistry.

[15]  M. Vey,et al.  Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin‐like endoprotease. , 1992, The EMBO journal.

[16]  H. Klenk,et al.  Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R☆ , 1992, Virology.

[17]  T. Yoshida,et al.  Distribution and substrate specificity of intracellular proteolytic processing enzyme(s) for paramyxovirus fusion glycoproteins. , 1992, The Journal of general virology.

[18]  K. Shimokata,et al.  Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. , 1992, The EMBO journal.

[19]  C. Bona,et al.  Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes , 1992, Journal of virology.

[20]  Richard I. Joh,et al.  Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. , 1991, DNA and cell biology.

[21]  P. Barr,et al.  Mammalian subtilisins: The long-sought dibasic processing endoproteases , 1991, Cell.

[22]  H. Klenk,et al.  Human influenza virus hemagglutinin with high sensitivity to proteolytic activation , 1991, Journal of virology.

[23]  B. Murphy,et al.  An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Palese,et al.  High-efficiency formation of influenza virus transfectants , 1991, Journal of virology.

[25]  M. Hamaguchi,et al.  An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo. , 1990, The EMBO journal.

[26]  R. Rott,et al.  Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes , 1990, Journal of virology.

[27]  V. Hinshaw,et al.  Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus , 1990, Journal of virology.

[28]  R. Rott,et al.  Structural variation occurring in the hemagglutinin of influenza virus A/turkey/Oregon/71 during adaptation to different cell types. , 1990, Virology.

[29]  P. Palese,et al.  Introduction of site-specific mutations into the genome of influenza virus. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Parvin,et al.  Amplification, expression, and packaging of a foreign gene by influenza virus , 1989, Cell.

[31]  J. Parvin,et al.  Promoter analysis of influenza virus RNA polymerase , 1989, Journal of virology.

[32]  R. Webster,et al.  Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin , 1989, Journal of virology.

[33]  R. Rott,et al.  Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus , 1989, Nature.

[34]  H. Klenk,et al.  Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2). , 1989, Virology.

[35]  V. Hinshaw,et al.  Destruction of lymphocytes by a virulent avian influenza A virus. , 1989, The Journal of general virology.

[36]  H. Klenk,et al.  The Molecular Biology of Influenza Virus Pathogenicity , 1988, Advances in Virus Research.

[37]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[38]  R. Webster,et al.  Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Webster,et al.  Influenza virus a pathogenicity: The pivotal role of hemagglutinin , 1987, Cell.

[40]  H. Klenk,et al.  Significance of viral glycoproteins for infectivity and pathogenicity* , 1987, Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene. Series A: Medical Microbiology, Infectious Diseases, Virology, Parasitology.

[41]  R. Webster,et al.  Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. , 1987, Virology.

[42]  H. Klenk,et al.  Role of Staphylococcus protease in the development of influenza pneumonia , 1987, Nature.

[43]  R. Webster,et al.  Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Skehel,et al.  Studies on the adaptation of influenza viruses to MDCK cells. , 1984, The EMBO journal.

[46]  C. Naeve,et al.  Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? , 1984, Virology.

[47]  R. Webster,et al.  Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH. , 1983, Virology.

[48]  G. Brownlee,et al.  The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68. , 1982, Nucleic acids research.

[49]  A. Helenius,et al.  Membrane fusion activity of influenza virus. , 1982, The EMBO journal.

[50]  W. J. Bean,et al.  Characterization of an influenza A virus from seals. , 1981, Virology.

[51]  H. Klenk,et al.  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. , 1981, Virology.

[52]  H. Klenk,et al.  The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. , 1979, Virology.

[53]  C. Scholtissek,et al.  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 , 1976, Journal of virology.

[54]  P. Choppin,et al.  Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: host cell activation of serum plasminogen. , 1973, Virology.

[55]  B. Rouse,et al.  A new influenza A virus infection in turkeys II. A highly pathogenic variant, a/turkey/ontario 772/66. , 1968, The Canadian veterinary journal = La revue veterinaire canadienne.