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. 1997 Nov;71(11):8268–8278. doi: 10.1128/jvi.71.11.8268-8278.1997

Rotavirus is released from the apical surface of cultured human intestinal cells through nonconventional vesicular transport that bypasses the Golgi apparatus.

N Jourdan 1, M Maurice 1, D Delautier 1, A M Quero 1, A L Servin 1, G Trugnan 1
PMCID: PMC192285  PMID: 9343179

Abstract

Rotaviruses are nonenveloped viruses that infect enterocytes of the small intestine and cause severe infantile gastroenteritis. It was previously thought that rotavirus exits cells by lysis, but this behavior does not match the local pathogenesis of the virus. In this study, we have investigated the release of the simian rotavirus strain (RRV) from the polarized intestinal Caco-2 cells. We found that RRV is released almost exclusively from the apical pole of Caco-2 cells before any cells lyse. Using confocal laser scanning microscopy and drugs that inhibit vesicular transport, we studied the RRV transport route from the endoplasmic reticulum (ER) to the apical side of intestinal cells. We demonstrated that RRV exits from the ER through a carbonyl cyanide m-chlorophenylhydrazone-sensitive vesicular transport. RRV staining was never found within the Golgi apparatus or lysosomes, suggesting that the RRV intracellular pathway does not involve these organelles. This finding was confirmed by treatment with monensin or NH4Cl, which do not affect release of RRV. Electron microscopic analysis revealed RRV containing small smooth vesicles in the apical area and free virions outside the cell in the brush border, consistent with a vesicular vectorial transport of virus. These results may provide, for the first time, a cellular explanation of the pathogenesis of rotavirus.

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Selected References

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  1. Altenburg B. C., Graham D. Y., Estes M. K. Ultrastructural study of rotavirus replication in cultured cells. J Gen Virol. 1980 Jan;46(1):75–85. doi: 10.1099/0022-1317-46-1-75. [DOI] [PubMed] [Google Scholar]
  2. Anderson G. W., Jr, Smith J. F. Immunoelectron microscopy of Rift Valley fever viral morphogenesis in primary rat hepatocytes. Virology. 1987 Nov;161(1):91–100. doi: 10.1016/0042-6822(87)90174-7. [DOI] [PubMed] [Google Scholar]
  3. Bacher A., Griebl K., Mackamul S., Mitreiter R., Mückter H., Ben-Shaul Y. Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines. Exp Cell Res. 1992 May;200(1):97–104. doi: 10.1016/s0014-4827(05)80076-x. [DOI] [PubMed] [Google Scholar]
  4. Baricault L., Garcia M., Cibert C., Sapin C., Geraud G., Codogno P., Trugnan G. Forskolin blocks the apical expression of dipeptidyl peptidase IV in Caco-2 cells and induces its retention in lamp-1-containing vesicles. Exp Cell Res. 1993 Dec;209(2):277–287. doi: 10.1006/excr.1993.1312. [DOI] [PubMed] [Google Scholar]
  5. Bishop R. F., Davidson G. P., Holmes I. H., Ruck B. J. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet. 1973 Dec 8;2(7841):1281–1283. doi: 10.1016/s0140-6736(73)92867-5. [DOI] [PubMed] [Google Scholar]
  6. Bridger J. C., Burke B., Beards G. M., Desselberger U. The pathogenicity of two porcine rotaviruses differing in their in vitro growth characteristics and genes 4. J Gen Virol. 1992 Nov;73(Pt 11):3011–3015. doi: 10.1099/0022-1317-73-11-3011. [DOI] [PubMed] [Google Scholar]
  7. Carlsson S. R., Roth J., Piller F., Fukuda M. Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem. 1988 Dec 15;263(35):18911–18919. [PubMed] [Google Scholar]
  8. Chen S. Y., Matsuoka Y., Compans R. W. Assembly and polarized release of Punta Toro virus and effects of brefeldin A. J Virol. 1991 Mar;65(3):1427–1439. doi: 10.1128/jvi.65.3.1427-1439.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark S. M., Roth J. R., Clark M. L., Barnett B. B., Spendlove R. S. Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. J Virol. 1981 Sep;39(3):816–822. doi: 10.1128/jvi.39.3.816-822.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clayson E. T., Brando L. V., Compans R. W. Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis. J Virol. 1989 May;63(5):2278–2288. doi: 10.1128/jvi.63.5.2278-2288.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elliott G., O'Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell. 1997 Jan 24;88(2):223–233. doi: 10.1016/s0092-8674(00)81843-7. [DOI] [PubMed] [Google Scholar]
  12. Fantini J., Baghdiguian S., Yahi N., Chermann J. C. Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells. Virology. 1991 Dec;185(2):904–907. doi: 10.1016/0042-6822(91)90570-2. [DOI] [PubMed] [Google Scholar]
  13. Fogh J., Fogh J. M., Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst. 1977 Jul;59(1):221–226. doi: 10.1093/jnci/59.1.221. [DOI] [PubMed] [Google Scholar]
  14. Garcia M., Mirre C., Quaroni A., Reggio H., Le Bivic A. GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells. J Cell Sci. 1993 Apr;104(Pt 4):1281–1290. doi: 10.1242/jcs.104.4.1281. [DOI] [PubMed] [Google Scholar]
  15. Gorvel J. P., Ferrero A., Chambraud L., Rigal A., Bonicel J., Maroux S. Expression of sucrase-isomaltase and dipeptidylpeptidase IV in human small intestine and colon. Gastroenterology. 1991 Sep;101(3):618–625. doi: 10.1016/0016-5085(91)90517-o. [DOI] [PubMed] [Google Scholar]
  16. Gottlieb T. A., Gonzalez A., Rizzolo L., Rindler M. J., Adesnik M., Sabatini D. D. Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells. J Cell Biol. 1986 Apr;102(4):1242–1255. doi: 10.1083/jcb.102.4.1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jourdan N., Cotte Laffitte J., Forestier F., Servin A. L., Quéro A. M. Infection of cultured human intestinal cells by monkey RRV and human Wa rotavirus as a function of intestinal epithelial cell differentiation. Res Virol. 1995 Sep-Oct;146(5):325–331. doi: 10.1016/0923-2516(96)80595-4. [DOI] [PubMed] [Google Scholar]
  18. Kabcenell A. K., Atkinson P. H. Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11. J Cell Biol. 1985 Oct;101(4):1270–1280. doi: 10.1083/jcb.101.4.1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kitamoto N., Ramig R. F., Matson D. O., Estes M. K. Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2). Virology. 1991 Oct;184(2):729–737. doi: 10.1016/0042-6822(91)90443-f. [DOI] [PubMed] [Google Scholar]
  20. Kundu A., Avalos R. T., Sanderson C. M., Nayak D. P. Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. J Virol. 1996 Sep;70(9):6508–6515. doi: 10.1128/jvi.70.9.6508-6515.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McQueen N., Nayak D. P., Stephens E. B., Compans R. W. Polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus G protein. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9318–9322. doi: 10.1073/pnas.83.24.9318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mengaud J., Ohayon H., Gounon P., Mege R-M, Cossart P. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell. 1996 Mar 22;84(6):923–932. doi: 10.1016/s0092-8674(00)81070-3. [DOI] [PubMed] [Google Scholar]
  23. Mirazimi A., von Bonsdorff C. H., Svensson L. Effect of brefeldin A on rotavirus assembly and oligosaccharide processing. Virology. 1996 Mar 15;217(2):554–563. doi: 10.1006/viro.1996.0150. [DOI] [PubMed] [Google Scholar]
  24. Mirre C., Monlauzeur L., Garcia M., Delgrossi M. H., Le Bivic A. Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin. Am J Physiol. 1996 Sep;271(3 Pt 1):C887–C894. doi: 10.1152/ajpcell.1996.271.3.C887. [DOI] [PubMed] [Google Scholar]
  25. Musalem C., Espejo R. T. Release of progeny virus from cells infected with simian rotavirus SA11. J Gen Virol. 1985 Dec;66(Pt 12):2715–2724. doi: 10.1099/0022-1317-66-12-2715. [DOI] [PubMed] [Google Scholar]
  26. Ménard R., Dehio C., Sansonetti P. J. Bacterial entry into epithelial cells: the paradigm of Shigella. Trends Microbiol. 1996 Jun;4(6):220–226. doi: 10.1016/0966-842X(96)10039-1. [DOI] [PubMed] [Google Scholar]
  27. Owens R. J., Dubay J. W., Hunter E., Compans R. W. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3987–3991. doi: 10.1073/pnas.88.9.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Petrie B. L., Graham D. Y., Hanssen H., Estes M. K. Localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry. J Gen Virol. 1982 Dec;63(2):457–467. doi: 10.1099/0022-1317-63-2-457. [DOI] [PubMed] [Google Scholar]
  29. Petrie B. L., Greenberg H. B., Graham D. Y., Estes M. K. Ultrastructural localization of rotavirus antigens using colloidal gold. Virus Res. 1984;1(2):133–152. doi: 10.1016/0168-1702(84)90069-8. [DOI] [PubMed] [Google Scholar]
  30. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  31. Rodriguez Boulan E., Pendergast M. Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell. 1980 May;20(1):45–54. doi: 10.1016/0092-8674(80)90233-0. [DOI] [PubMed] [Google Scholar]
  32. Rodriguez Boulan E., Sabatini D. D. Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5071–5075. doi: 10.1073/pnas.75.10.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rolsma M. D., Gelberg H. B., Kuhlenschmidt M. S. Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition. J Virol. 1994 Jan;68(1):258–268. doi: 10.1128/jvi.68.1.258-268.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rossen J. W., Bekker C. P., Voorhout W. F., Strous G. J., van der Ende A., Rottier P. J. Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells. J Virol. 1994 Dec;68(12):7966–7973. doi: 10.1128/jvi.68.12.7966-7973.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rossen J. W., Voorhout W. F., Horzinek M. C., van der Ende A., Strous G. J., Rottier P. J. MHV-A59 enters polarized murine epithelial cells through the apical surface but is released basolaterally. Virology. 1995 Jun 20;210(1):54–66. doi: 10.1006/viro.1995.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roth M. G., Compans R. W., Giusti L., Davis A. R., Nayak D. P., Gething M. J., Sambrook J. Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA. Cell. 1983 Jun;33(2):435–443. doi: 10.1016/0092-8674(83)90425-7. [DOI] [PubMed] [Google Scholar]
  37. Roth M. G., Gundersen D., Patil N., Rodriguez-Boulan E. The large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells. J Cell Biol. 1987 Mar;104(3):769–782. doi: 10.1083/jcb.104.3.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roth M. G., Srinivas R. V., Compans R. W. Basolateral maturation of retroviruses in polarized epithelial cells. J Virol. 1983 Mar;45(3):1065–1073. doi: 10.1128/jvi.45.3.1065-1073.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schwarz K. B., Moore T. J., Willoughby R. E., Jr, Wee S. B., Vonderfecht S. L., Yolken R. H. Growth of group A rotaviruses in a human liver cell line. Hepatology. 1990 Oct;12(4 Pt 1):638–643. doi: 10.1002/hep.1840120403. [DOI] [PubMed] [Google Scholar]
  40. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  41. Srinivas R. V., Balachandran N., Alonso-Caplen F. V., Compans R. W. Expression of herpes simplex virus glycoproteins in polarized epithelial cells. J Virol. 1986 May;58(2):689–693. doi: 10.1128/jvi.58.2.689-693.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stein M. A., Mathers D. A., Yan H., Baimbridge K. G., Finlay B. B. Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells. Infect Immun. 1996 Nov;64(11):4820–4825. doi: 10.1128/iai.64.11.4820-4825.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Suomalainen M., Hultenby K., Garoff H. Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding. J Cell Biol. 1996 Dec;135(6 Pt 2):1841–1852. doi: 10.1083/jcb.135.6.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Superti F., Donelli G. Characterization of SA-11 rotavirus receptorial structures on human colon carcinoma cell line HT-29. J Med Virol. 1995 Dec;47(4):421–428. doi: 10.1002/jmv.1890470421. [DOI] [PubMed] [Google Scholar]
  45. Superti F., Donelli G. Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells. J Gen Virol. 1991 Oct;72(Pt 10):2467–2474. doi: 10.1099/0022-1317-72-10-2467. [DOI] [PubMed] [Google Scholar]
  46. Suzuki H., Kitaoka S., Konno T., Sato T., Ishida N. Two modes of human rotavirus entry into MA 104 cells. Arch Virol. 1985;85(1-2):25–34. doi: 10.1007/BF01317003. [DOI] [PubMed] [Google Scholar]
  47. Svensson L., Finlay B. B., Bass D., von Bonsdorff C. H., Greenberg H. B. Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells. J Virol. 1991 Aug;65(8):4190–4197. doi: 10.1128/jvi.65.8.4190-4197.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tartakoff A. M. Temperature and energy dependence of secretory protein transport in the exocrine pancreas. EMBO J. 1986 Jul;5(7):1477–1482. doi: 10.1002/j.1460-2075.1986.tb04385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tartakoff A. M., Vassalli P. Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides. J Cell Biol. 1983 Oct;97(4):1243–1248. doi: 10.1083/jcb.97.4.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tashiro M., Pritzer E., Khoshnan M. A., Yamakawa M., Kuroda K., Klenk H. D., Rott R., Seto J. T. Characterization of a pantropic variant of Sendai virus derived from a host range mutant. Virology. 1988 Aug;165(2):577–583. doi: 10.1016/0042-6822(88)90601-0. [DOI] [PubMed] [Google Scholar]
  51. Tashiro M., Yamakawa M., Tobita K., Seto J. T., Klenk H. D., Rott R. Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells. J Virol. 1990 Oct;64(10):4672–4677. doi: 10.1128/jvi.64.10.4672-4677.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Theil K. W., Bohl E. H., Cross R. F., Kohler E. M., Agnes A. G. Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs. Am J Vet Res. 1978 Feb;39(2):213–220. [PubMed] [Google Scholar]
  53. Tian P., Ball J. M., Zeng C. Q., Estes M. K. The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity. J Virol. 1996 Oct;70(10):6973–6981. doi: 10.1128/jvi.70.10.6973-6981.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Trugnan G., Rousset M., Chantret I., Barbat A., Zweibaum A. The posttranslational processing of sucrase-isomaltase in HT-29 cells is a function of their state of enterocytic differentiation. J Cell Biol. 1987 May;104(5):1199–1205. doi: 10.1083/jcb.104.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tucker S. P., Compans R. W. Virus infection of polarized epithelial cells. Adv Virus Res. 1993;42:187–247. doi: 10.1016/S0065-3527(08)60086-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tucker S. P., Thornton C. L., Wimmer E., Compans R. W. Vectorial release of poliovirus from polarized human intestinal epithelial cells. J Virol. 1993 Jul;67(7):4274–4282. doi: 10.1128/jvi.67.7.4274-4282.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Willoughby R. E., Yolken R. H., Schnaar R. L. Rotaviruses specifically bind to the neutral glycosphingolipid asialo-GM1. J Virol. 1990 Oct;64(10):4830–4835. doi: 10.1128/jvi.64.10.4830-4835.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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