Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Apr;81(8):2327–2331. doi: 10.1073/pnas.81.8.2327

NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation.

T J Bos, A R Davis, D P Nayak
PMCID: PMC345052  PMID: 6326121

Abstract

Influenza virus neuraminidase (NA), unlike the majority of integral membrane proteins, does not contain a cleavable signal sequence. It contains an NH2-terminal hydrophobic domain that functions as an anchor. We have investigated the signal function for translocation of this NH2-terminal hydrophobic domain of NA by constructing chimeric cDNA clones in which the DNA coding for the first 40 NH2-terminal hydrophobic amino acids of NA was joined to the DNA coding for the signal-minus hemagglutinin (HA) of influenza virus. The chimeric HA (N4OH) containing the NH2 terminus of NA was expressed in CV1 cells by using a simian virus 40 late-expression vector. The chimeric HA is synthesized, translocated into the rough endoplasmic reticulum, and glycosylated, whereas HA lacking the signal sequence is present only in small amounts and is unglycosylated. These results clearly show that the NH2 terminus of NA, in addition to its anchor function, also provides the signal function in translocation. However, the acquisition of complex oligosaccharides and the transport of N4OH to the cell surface are greatly retarded. To determine if the presence of two anchor sequences, one provided by NA at the NH2 terminus and the other provided by HA at the COOH terminus of N4OH, was responsible for the slow transport, the NH2 terminus of NA was fused to an "anchorless" HA. The resulting chimeric HA (N4OH482) contains the hydrophobic domain of NA at the NH2 terminus but lacks the HA anchor at the COOH terminus. N4OH482 was synthesized and glycosylated; however, as with N4OH, the acquisition of complex oligosaccharides and the migration to the cell surface are greatly retarded. Immunofluorescence data also support that, compared to the native HA, only a small amount of chimeric HA proteins is transported to the cell surface. Thus, the hydrophobic NH2 terminus of NA, although capable of providing the signal function in translocation across the rough endoplasmic reticulum, interferes with the transport of the chimeric HA to the cell surface.

Full text

PDF
2327

Images in this article

2329Image
on p.2329
2329Image
on p.2329
2331Image
on p.2331

Selected References

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

  1. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blok J., Air G. M., Laver W. G., Ward C. W., Lilley G. G., Woods E. F., Roxburgh C. M., Inglis A. S. Studies on the size, chemical composition, and partial sequence of the neuraminidase (NA) from type A influenza viruses show that the N-terminal region of the NA is not processed and serves to anchor the NA in the viral membrane. Virology. 1982 May;119(1):109–121. doi: 10.1016/0042-6822(82)90069-1. [DOI] [PubMed] [Google Scholar]
  3. Colman P. M., Varghese J. N., Laver W. G. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature. 1983 May 5;303(5912):41–44. doi: 10.1038/303041a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Davis A. R., Hiti A. L., Nayak D. P. Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus. Gene. 1980 Aug;10(3):205–218. doi: 10.1016/0378-1119(80)90050-5. [DOI] [PubMed] [Google Scholar]
  6. Fields S., Winter G., Brownlee G. G. Structure of the neuraminidase gene in human influenza virus A/PR/8/34. Nature. 1981 Mar 19;290(5803):213–217. doi: 10.1038/290213a0. [DOI] [PubMed] [Google Scholar]
  7. Gething M. J., Bye J., Skehel J., Waterfield M. Cloning and DNA sequence of double-stranded copies of haemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nature. 1980 Sep 25;287(5780):301–306. doi: 10.1038/287301a0. [DOI] [PubMed] [Google Scholar]
  8. Gething M. J., Sambrook J. Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene. Nature. 1981 Oct 22;293(5834):620–625. doi: 10.1038/293620a0. [DOI] [PubMed] [Google Scholar]
  9. Gething M. J., Sambrook J. Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature. 1982 Dec 16;300(5893):598–603. doi: 10.1038/300598a0. [DOI] [PubMed] [Google Scholar]
  10. Hartman J. R., Nayak D. P., Fareed G. C. Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors. Proc Natl Acad Sci U S A. 1982 Jan;79(2):233–237. doi: 10.1073/pnas.79.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hiti A. L., Davis A. R., Nayak D. P. Complete sequence analysis shows that the hemagglutinins of the H0 and H2 subtypes of human influenza virus are closely related. Virology. 1981 May;111(1):113–124. doi: 10.1016/0042-6822(81)90658-9. [DOI] [PubMed] [Google Scholar]
  12. Hiti A. L., Nayak D. P. Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33. J Virol. 1982 Feb;41(2):730–734. doi: 10.1128/jvi.41.2.730-734.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krystal M., Elliott R. M., Benz E. W., Jr, Young J. F., Palese P. Evolution of influenza A and B viruses: conservation of structural features in the hemagglutinin genes. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4800–4804. doi: 10.1073/pnas.79.15.4800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laver W. G., Kilbourne E. D. Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology. 1966 Nov;30(3):493–501. doi: 10.1016/0042-6822(66)90125-5. [DOI] [PubMed] [Google Scholar]
  15. Markoff L., Lai C. J. Sequence of the influenza A/Udorn/72 (H3N2) virus neuraminidase gene as determined from cloned full-length DNA. Virology. 1982 Jun;119(2):288–297. doi: 10.1016/0042-6822(82)90089-7. [DOI] [PubMed] [Google Scholar]
  16. McQueen N. L., Nayak D. P., Jones L. V., Compans R. W. Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface. Proc Natl Acad Sci U S A. 1984 Jan;81(2):395–399. doi: 10.1073/pnas.81.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mertz J. E., Berg P. Defective simian virus 40 genomes: isolation and growth of individual clones. Virology. 1974 Nov;62(1):112–124. doi: 10.1016/0042-6822(74)90307-9. [DOI] [PubMed] [Google Scholar]
  18. Palese P. The genes of influenza virus. Cell. 1977 Jan;10(1):1–10. doi: 10.1016/0092-8674(77)90133-7. [DOI] [PubMed] [Google Scholar]
  19. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  20. Rose J. K., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sveda M. M., Lai C. J. Functional expression in primate cells of cloned DNA coding for the hemagglutinin surface glycoprotein of influenza virus. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5488–5492. doi: 10.1073/pnas.78.9.5488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sveda M. M., Markoff L. J., Lai C. J. Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences. Cell. 1982 Sep;30(2):649–656. doi: 10.1016/0092-8674(82)90261-6. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  26. Wiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981 Jan 29;289(5796):373–378. doi: 10.1038/289373a0. [DOI] [PubMed] [Google Scholar]
  27. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES