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. 1993 Nov;67(11):6762–6767. doi: 10.1128/jvi.67.11.6762-6767.1993

Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions.

P Bilsel 1, M R Castrucci 1, Y Kawaoka 1
PMCID: PMC238117  PMID: 8411379

Abstract

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.

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Selected References

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  1. Air G. M., Laver W. G. The neuraminidase of influenza virus. Proteins. 1989;6(4):341–356. doi: 10.1002/prot.340060402. [DOI] [PubMed] [Google Scholar]
  2. Aymard-Henry M., Coleman M. T., Dowdle W. R., Laver W. G., Schild G. C., Webster R. G. Influenzavirus neuraminidase and neuraminidase-inhibition test procedures. Bull World Health Organ. 1973;48(2):199–202. [PMC free article] [PubMed] [Google Scholar]
  3. Bos T. J., Davis A. R., Nayak D. P. NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2327–2331. doi: 10.1073/pnas.81.8.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brody B. A., Rhee S. S., Sommerfelt M. A., Hunter E. A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3443–3447. doi: 10.1073/pnas.89.8.3443. [DOI] [PMC free article] [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. Collier N. C., Knox K., Schlesinger M. J. Inhibition of influenza virus formation by a peptide that corresponds to sequences in the cytoplasmic domain of the hemagglutinin. Virology. 1991 Aug;183(2):769–772. doi: 10.1016/0042-6822(91)91008-5. [DOI] [PubMed] [Google Scholar]
  7. Collins P. L., Mink M. A., Stec D. S. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9663–9667. doi: 10.1073/pnas.88.21.9663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colman P. M., Laver W. G., Varghese J. N., Baker A. T., Tulloch P. A., Air G. M., Webster R. G. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. 1987 Mar 26-Apr 1Nature. 326(6111):358–363. doi: 10.1038/326358a0. [DOI] [PubMed] [Google Scholar]
  9. Colman P. M., Ward C. W. Structure and diversity of influenza virus neuraminidase. Curr Top Microbiol Immunol. 1985;114:177–255. doi: 10.1007/978-3-642-70227-3_5. [DOI] [PubMed] [Google Scholar]
  10. Dubay J. W., Roberts S. J., Hahn B. H., Hunter E. Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity. J Virol. 1992 Nov;66(11):6616–6625. doi: 10.1128/jvi.66.11.6616-6625.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Enami M., Sharma G., Benham C., Palese P. An influenza virus containing nine different RNA segments. Virology. 1991 Nov;185(1):291–298. doi: 10.1016/0042-6822(91)90776-8. [DOI] [PubMed] [Google Scholar]
  14. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gabuzda D. H., Lever A., Terwilliger E., Sodroski J. Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins. J Virol. 1992 Jun;66(6):3306–3315. doi: 10.1128/jvi.66.6.3306-3315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gaedigk-Nitschko K., Schlesinger M. J. Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding. Virology. 1991 Jul;183(1):206–214. doi: 10.1016/0042-6822(91)90133-v. [DOI] [PubMed] [Google Scholar]
  17. Gebhardt A., Bosch J. V., Ziemiecki A., Friis R. R. Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly. J Mol Biol. 1984 Apr 5;174(2):297–317. doi: 10.1016/0022-2836(84)90340-1. [DOI] [PubMed] [Google Scholar]
  18. Kundu A., Jabbar M. A., Nayak D. P. Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains. Mol Cell Biol. 1991 May;11(5):2675–2685. doi: 10.1128/mcb.11.5.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lyles D. S., McKenzie M., Parce J. W. Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein. J Virol. 1992 Jan;66(1):349–358. doi: 10.1128/jvi.66.1.349-358.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martin K., Helenius A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell. 1991 Oct 4;67(1):117–130. doi: 10.1016/0092-8674(91)90576-k. [DOI] [PubMed] [Google Scholar]
  25. Metsikkö K., Garoff H. Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro. J Virol. 1990 Oct;64(10):4678–4683. doi: 10.1128/jvi.64.10.4678-4683.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miki T., Nishida Y., Hisajima H., Miyata T., Kumahara Y., Nerome K., Oya A., Fukui T., Ohtsuka E., Ikehara M. The complete nucleotide sequence of the influenza virus neuraminidase gene of A/NJ/8/76 strain and its evolution by segmental duplication and deletion. Mol Biol Med. 1983 Nov;1(4):401–413. [PubMed] [Google Scholar]
  27. 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]
  28. Naim H. Y., Roth M. G. Basis for selective incorporation of glycoproteins into the influenza virus envelope. J Virol. 1993 Aug;67(8):4831–4841. doi: 10.1128/jvi.67.8.4831-4841.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nuss J. M., Air G. M. Transfer of the hemagglutinin activity of influenza virus neuraminidase subtype N9 into an N2 neuraminidase background. Virology. 1991 Aug;183(2):496–504. doi: 10.1016/0042-6822(91)90979-l. [DOI] [PubMed] [Google Scholar]
  30. Palese P., Tobita K., Ueda M., Compans R. W. Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology. 1974 Oct;61(2):397–410. doi: 10.1016/0042-6822(74)90276-1. [DOI] [PubMed] [Google Scholar]
  31. Park K. H., Huang T., Correia F. F., Krystal M. Rescue of a foreign gene by Sendai virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5537–5541. doi: 10.1073/pnas.88.13.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Paterson R. G., Lamb R. A. Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms. J Cell Biol. 1990 Apr;110(4):999–1011. doi: 10.1083/jcb.110.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pattnaik A. K., Brown D. J., Nayak D. P. Formation of influenza virus particles lacking hemagglutinin on the viral envelope. J Virol. 1986 Dec;60(3):994–1001. doi: 10.1128/jvi.60.3.994-1001.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pattnaik A. K., Wertz G. W. Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. J Virol. 1990 Jun;64(6):2948–2957. doi: 10.1128/jvi.64.6.2948-2957.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Perez L. G., Davis G. L., Hunter E. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions. J Virol. 1987 Oct;61(10):2981–2988. doi: 10.1128/jvi.61.10.2981-2988.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shaw M. W., Lamb R. A., Erickson B. W., Briedis D. J., Choppin P. W. Complete nucleotide sequence of the neuraminidase gene of influenza B virus. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6817–6821. doi: 10.1073/pnas.79.22.6817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simpson D. A., Lamb R. A. Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity. J Virol. 1992 Feb;66(2):790–803. doi: 10.1128/jvi.66.2.790-803.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sivasubramanian N., Nayak D. P. Mutational analysis of the signal-anchor domain of influenza virus neuraminidase. Proc Natl Acad Sci U S A. 1987 Jan;84(1):1–5. doi: 10.1073/pnas.84.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sugiura A., Ueda M. Neurovirulence of influenza virus in mice. I. Neurovirulence of recombinants between virulent and avirulent virus strains. Virology. 1980 Mar;101(2):440–449. doi: 10.1016/0042-6822(80)90457-2. [DOI] [PubMed] [Google Scholar]
  41. Varghese J. N., Laver W. G., Colman P. M. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature. 1983 May 5;303(5912):35–40. doi: 10.1038/303035a0. [DOI] [PubMed] [Google Scholar]
  42. Whitt M. A., Chong L., Rose J. K. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J Virol. 1989 Sep;63(9):3569–3578. doi: 10.1128/jvi.63.9.3569-3578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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