Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Dec 1;91(3):601–613. doi: 10.1083/jcb.91.3.601

Infectious entry pathway of influenza virus in a canine kidney cell line

PMCID: PMC2112819  PMID: 7328111

Abstract

The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Ash J. F., Louvard D., Singer S. J. Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5584–5588. doi: 10.1073/pnas.74.12.5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ash J. F., Vogt P. K., Singer S. J. Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3603–3607. doi: 10.1073/pnas.73.10.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barazzone P., Lesniak M. A., Gorden P., Van Obberghen E., Carpentier J. L., Orci L. Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study. J Cell Biol. 1980 Nov;87(2 Pt 1):360–369. doi: 10.1083/jcb.87.2.360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blakeslee D., Baines M. G. Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG. J Immunol Methods. 1976;13(3-4):305–320. doi: 10.1016/0022-1759(76)90078-8. [DOI] [PubMed] [Google Scholar]
  5. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  6. Cereijido M., Robbins E. S., Dolan W. J., Rotunno C. A., Sabatini D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol. 1978 Jun;77(3):853–880. doi: 10.1083/jcb.77.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DALES S., CHOPPIN P. W. Attachment and penetration of influenza virus. Virology. 1962 Nov;18:489–493. doi: 10.1016/0042-6822(62)90041-7. [DOI] [PubMed] [Google Scholar]
  8. DAVIES W. L., GRUNERT R. R., HAFF R. F., MCGAHEN J. W., NEUMAYER E. M., PAULSHOCK M., WATTS J. C., WOOD T. R., HERMANN E. C., HOFFMANN C. E. ANTIVIRAL ACTIVITY OF 1-ADAMANTANAMINE (AMANTADINE). Science. 1964 May 15;144(3620):862–863. doi: 10.1126/science.144.3620.862. [DOI] [PubMed] [Google Scholar]
  9. Dales S. Early events in cell-animal virus interactions. Bacteriol Rev. 1973 Jun;37(2):103–135. doi: 10.1128/br.37.2.103-135.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dales S., Pons M. W. Penetration of influenza examined by means of virus aggregates. Virology. 1976 Jan;69(1):278–286. doi: 10.1016/0042-6822(76)90214-2. [DOI] [PubMed] [Google Scholar]
  11. Dourmashkin R. R., Tyrrell D. A. Electron microscopic observations on the entry of influenza virus into susceptible cells. J Gen Virol. 1974 Jul;24(1):129–141. doi: 10.1099/0022-1317-24-1-129. [DOI] [PubMed] [Google Scholar]
  12. EATON M. D., SCALA A. R. Inhibitory effect of glutamine and ammonia on replication of influenza virus in ascites tumor cells. Virology. 1961 Mar;13:300–307. doi: 10.1016/0042-6822(61)90149-0. [DOI] [PubMed] [Google Scholar]
  13. Eagle H. Buffer combinations for mammalian cell culture. Science. 1971 Oct 29;174(4008):500–503. doi: 10.1126/science.174.4008.500. [DOI] [PubMed] [Google Scholar]
  14. Fletcher R. D., Hirschfield J. E., Forbes M. A common mode of antiviral action for ammonium ions and various amines. Nature. 1965 Aug 7;207(997):664–665. doi: 10.1038/207664a0. [DOI] [PubMed] [Google Scholar]
  15. Gaush C. R., Smith T. F. Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl Microbiol. 1968 Apr;16(4):588–594. doi: 10.1128/am.16.4.588-594.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  17. Goldstein J. L., Brunschede G. Y., Brown M. S. Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. J Biol Chem. 1975 Oct 10;250(19):7854–7862. [PubMed] [Google Scholar]
  18. Goudie R. B., Horne C. H., Wilkinson P. C. A simple method for producing antibody specific to a single selected diffusible antigen. Lancet. 1966 Dec 3;2(7475):1224–1226. doi: 10.1016/s0140-6736(66)92305-1. [DOI] [PubMed] [Google Scholar]
  19. Helenius A., Fries E., Kartenbeck J. Reconstitution of Semliki forest virus membrane. J Cell Biol. 1977 Dec;75(3):866–880. doi: 10.1083/jcb.75.3.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Homma M., Ouchi M. Trypsin action on the growth of Sendai virus in tissue culture cells. 3. Structural difference of Sendai viruses grown in eggs and tissue culture cells. J Virol. 1973 Dec;12(6):1457–1465. doi: 10.1128/jvi.12.6.1457-1465.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang R. T., Rott R., Klenk H. D. Influenza viruses cause hemolysis and fusion of cells. Virology. 1981 Apr 15;110(1):243–247. doi: 10.1016/0042-6822(81)90030-1. [DOI] [PubMed] [Google Scholar]
  23. Huet C., Ash J. F., Singer S. J. The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts. Cell. 1980 Sep;21(2):429–438. doi: 10.1016/0092-8674(80)90479-1. [DOI] [PubMed] [Google Scholar]
  24. JENSEN E. M., FORCE E. E., UNGER J. B. Inhibitory effect of ammonium ions on influenza virus in tissue culture. Proc Soc Exp Biol Med. 1961 Jun;107:447–451. doi: 10.3181/00379727-107-26653. [DOI] [PubMed] [Google Scholar]
  25. JENSEN E. M., LIU O. C. Inhibitory effect of simple aliphatic amines on influenza virus in tissue culture. Proc Soc Exp Biol Med. 1963 Feb;112:456–459. doi: 10.3181/00379727-112-28076. [DOI] [PubMed] [Google Scholar]
  26. JENSEN E. M., LIU O. C. Studies of inhibitory effect of ammonium ions in several virus-tissue culture systems. Proc Soc Exp Biol Med. 1961 Aug-Sep;107:834–838. doi: 10.3181/00379727-107-26770. [DOI] [PubMed] [Google Scholar]
  27. Jou W. M., Verhoeyen M., Devos R., Saman E., Fang R., Huylebroeck D., Fiers W., Threlfall G., Barber C., Carey N. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell. 1980 Mar;19(3):683–696. doi: 10.1016/s0092-8674(80)80045-6. [DOI] [PubMed] [Google Scholar]
  28. Kato N., Eggers H. J. Inhibition of uncoating of fowl plague virus by l-adamantanamine hydrochloride. Virology. 1969 Apr;37(4):632–641. doi: 10.1016/0042-6822(69)90281-5. [DOI] [PubMed] [Google Scholar]
  29. King A. C., Hernaez-Davis L., Cuatrecasas P. Lysomotropic amines cause intracellular accumulation of receptors for epidermal growth factor. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3283–3287. doi: 10.1073/pnas.77.6.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kishida Y., Olsen B. R., Berg R. A., Prockop D. J. Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J Cell Biol. 1975 Feb;64(2):331–339. doi: 10.1083/jcb.64.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Klenk H. D., Rott R., Orlich M., Blödorn J. Activation of influenza A viruses by trypsin treatment. Virology. 1975 Dec;68(2):426–439. doi: 10.1016/0042-6822(75)90284-6. [DOI] [PubMed] [Google Scholar]
  32. Koff W. C., Knight V. Inhibition of influenza virus uncoating by rimantadine hydrochloride. J Virol. 1979 Jul;31(1):261–263. doi: 10.1128/jvi.31.1.261-263.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  34. 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]
  35. Lazarowitz S. G., Choppin P. W. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology. 1975 Dec;68(2):440–454. doi: 10.1016/0042-6822(75)90285-8. [DOI] [PubMed] [Google Scholar]
  36. Long W. F., Olusanya J. Adamantanamine and early events following influenza virus infection. Arch Gesamte Virusforsch. 1972;36(1):18–22. doi: 10.1007/BF01250291. [DOI] [PubMed] [Google Scholar]
  37. Maeda T., Ohnishi S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett. 1980 Dec 29;122(2):283–287. doi: 10.1016/0014-5793(80)80457-1. [DOI] [PubMed] [Google Scholar]
  38. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  39. Marshall S., Olefsky J. M. Effects of lysosomotropic agents on insulin interactions with adipocytes. Evidence for a lysosomal pathway for insulin processing and degradation. J Biol Chem. 1979 Oct 25;254(20):10153–10160. [PubMed] [Google Scholar]
  40. Miller D. K., Lenard J. Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step. J Cell Biol. 1980 Feb;84(2):430–437. doi: 10.1083/jcb.84.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Morgan C., Rose H. M. Structure and development of viruses as observed in the electron microscope. 8. Entry of influenza virus. J Virol. 1968 Sep;2(9):925–936. doi: 10.1128/jvi.2.9.925-936.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. NEUMAYER E. M., HAFF R. F., HOFFMAN C. E. ANTIVIRAL ACTIVITY OF AMANTADINE HYDROCHLORIDE IN TISSUE CULTURE AND IN OVO. Proc Soc Exp Biol Med. 1965 Jun;119:393–396. doi: 10.3181/00379727-119-30191. [DOI] [PubMed] [Google Scholar]
  43. Neufeld E. F., Sando G. N., Garvin A. J., Rome L. H. The transport of lysosomal enzymes. J Supramol Struct. 1977;6(1):95–101. doi: 10.1002/jss.400060108. [DOI] [PubMed] [Google Scholar]
  44. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Oxford J. S., Galbraith A. Antiviral activity of amantadine: a review of laboratory and clinical data. Pharmacol Ther. 1980;11(1):181–262. doi: 10.1016/0163-7258(80)90072-8. [DOI] [PubMed] [Google Scholar]
  46. Patterson S., Oxford J. S., Dourmashkin R. R. Studies on the mechanism of influenza virus entry into cells. J Gen Virol. 1979 Apr;43(1):223–229. doi: 10.1099/0022-1317-43-1-223. [DOI] [PubMed] [Google Scholar]
  47. Porter A. G., Barber C., Carey N. H., Hallewell R. A., Threlfall G., Emtage J. S. Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA. Nature. 1979 Nov 29;282(5738):471–477. doi: 10.1038/282471a0. [DOI] [PubMed] [Google Scholar]
  48. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Richardson C. D., Scheid A., Choppin P. W. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology. 1980 Aug;105(1):205–222. doi: 10.1016/0042-6822(80)90168-3. [DOI] [PubMed] [Google Scholar]
  50. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Scheid A., Choppin P. W. Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology. 1974 Feb;57(2):475–490. doi: 10.1016/0042-6822(74)90187-1. [DOI] [PubMed] [Google Scholar]
  52. Seglen P. O., Grinde B., Solheim A. E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem. 1979 Apr 2;95(2):215–225. doi: 10.1111/j.1432-1033.1979.tb12956.x. [DOI] [PubMed] [Google Scholar]
  53. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  54. Simons K., Helenius A., Leonard K., Sarvas M., Gething M. J. Formation of protein micelles from amphiphilic membrane proteins. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5306–5310. doi: 10.1073/pnas.75.11.5306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Skehel J. J., Hay A. J., Armstrong J. A. On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride. J Gen Virol. 1978 Jan;38(1):97–110. doi: 10.1099/0022-1317-38-1-97. [DOI] [PubMed] [Google Scholar]
  56. Stephenson J. R., Hudson J. B., Dimmock N. J. Early events in influenza virus multiplication. II. Penetration of virus into cells at 4 degrees. Virology. 1978 May 1;86(1):264–271. doi: 10.1016/0042-6822(78)90026-0. [DOI] [PubMed] [Google Scholar]
  57. Tolleshaug H., Berg T., Frölich W., Norum K. R. Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes. Biochim Biophys Acta. 1979 Jun 1;585(1):71–84. doi: 10.1016/0304-4165(79)90326-x. [DOI] [PubMed] [Google Scholar]
  58. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  59. White J., Helenius A. pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3273–3277. doi: 10.1073/pnas.77.6.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. White J., Kartenbeck J., Helenius A. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. J Cell Biol. 1980 Oct;87(1):264–272. doi: 10.1083/jcb.87.1.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. White J., Matlin K., Helenius A. Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses. J Cell Biol. 1981 Jun;89(3):674–679. doi: 10.1083/jcb.89.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Willingham M. C., Maxfield F. R., Pastan I. H. alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions. J Cell Biol. 1979 Sep;82(3):614–625. doi: 10.1083/jcb.82.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]
  64. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES