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. 1985 Dec 1;101(6):2199–2209. doi: 10.1083/jcb.101.6.2199

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

PMCID: PMC2113985  PMID: 2999159

Abstract

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in- frame ATG codon. A series of deletion mutants constructed from a full- length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N- acetylglucosaminidase H sensitive. In contrast, products of mutants 42- 61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

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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 D. J., Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 1983;96:111–120. doi: 10.1016/s0076-6879(83)96012-3. [DOI] [PubMed] [Google Scholar]
  3. Armstrong J., Niemann H., Smeekens S., Rottier P., Warren G. Sequence and topology of a model intracellular membrane protein, E1 glycoprotein, from a coronavirus. Nature. 1984 Apr 19;308(5961):751–752. doi: 10.1038/308751a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Both G. W., Mattick J. S., Bellamy A. R. Serotype-specific glycoprotein of simian 11 rotavirus: coding assignment and gene sequence. Proc Natl Acad Sci U S A. 1983 May;80(10):3091–3095. doi: 10.1073/pnas.80.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Both G. W., Siegman L. J., Bellamy A. R., Atkinson P. H. Coding assignment and nucleotide sequence of simian rotavirus SA11 gene segment 10: location of glycosylation sites suggests that the signal peptide is not cleaved. J Virol. 1983 Nov;48(2):335–339. doi: 10.1128/jvi.48.2.335-339.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chasey D. Investigation of immunoperoxidase-labelled rotavirus in tissue culture by light and electron microscopy. J Gen Virol. 1980 Sep;50(1):195–200. doi: 10.1099/0022-1317-50-1-195. [DOI] [PubMed] [Google Scholar]
  8. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ericson B. L., Graham D. Y., Mason B. B., Estes M. K. Identification, synthesis, and modifications of simian rotavirus SA11 polypeptides in infected cells. J Virol. 1982 Jun;42(3):825–839. doi: 10.1128/jvi.42.3.825-839.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ericson B. L., Graham D. Y., Mason B. B., Hanssen H. H., Estes M. K. Two types of glycoprotein precursors are produced by the simian rotavirus SA11. Virology. 1983 Jun;127(2):320–332. doi: 10.1016/0042-6822(83)90147-2. [DOI] [PubMed] [Google Scholar]
  11. Fitting T., Kabat D. Evidence for a glycoprotein "signal" involved in transport between subcellular organelles. Two membrane glycoproteins encoded by murine leukemia virus reach the cell surface at different rates. J Biol Chem. 1982 Dec 10;257(23):14011–14017. [PubMed] [Google Scholar]
  12. Florkiewicz R. Z., Smith A., Bergmann J. E., Rose J. K. Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein. J Cell Biol. 1983 Nov;97(5 Pt 1):1381–1388. doi: 10.1083/jcb.97.5.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garoff H., Kondor-Koch C., Pettersson R., Burke B. Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain. J Cell Biol. 1983 Sep;97(3):652–658. doi: 10.1083/jcb.97.3.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guan J. L., Rose J. K. Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface. Cell. 1984 Jul;37(3):779–787. doi: 10.1016/0092-8674(84)90413-6. [DOI] [PubMed] [Google Scholar]
  15. Gunn P. R., Sato F., Powell K. F., Bellamy A. R., Napier J. R., Harding D. R., Hancock W. S., Siegman L. J., Both G. W. Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis. J Virol. 1985 Jun;54(3):791–797. doi: 10.1128/jvi.54.3.791-797.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Klenk H. D., Rott R. Cotranslational and posttranslational processing of viral glycoproteins. Curr Top Microbiol Immunol. 1980;90:19–48. doi: 10.1007/978-3-642-67717-5_2. [DOI] [PubMed] [Google Scholar]
  18. Kondor-Koch C., Riedel H., Söderberg K., Garoff H. Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4525–4529. doi: 10.1073/pnas.79.15.4525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [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. Langone J. J. Radioiodination by use of the Bolton-Hunter and related reagents. Methods Enzymol. 1980;70(A):221–247. doi: 10.1016/s0076-6879(80)70052-6. [DOI] [PubMed] [Google Scholar]
  22. Liscum L., Finer-Moore J., Stroud R. M., Luskey K. L., Brown M. S., Goldstein J. L. Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem. 1985 Jan 10;260(1):522–530. [PubMed] [Google Scholar]
  23. Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ozols J., Heinemann F. S., Johnson E. F. The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450. J Biol Chem. 1985 May 10;260(9):5427–5434. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. J Virol. 1983 Feb;45(2):773–781. doi: 10.1128/jvi.45.2.773-781.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Street J. E., Croxson M. C., Chadderton W. F., Bellamy A. R. Sequence diversity of human rotavirus strains investigated by northern blot hybridization analysis. J Virol. 1982 Aug;43(2):369–378. doi: 10.1128/jvi.43.2.369-378.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sveda M. M., Markoff L. J., Lai C. J. Influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly. J Virol. 1984 Jan;49(1):223–228. doi: 10.1128/jvi.49.1.223-228.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  34. Tartakoff A. M. The confined function model of the Golgi complex: center for ordered processing of biosynthetic products of the rough endoplasmic reticulum. Int Rev Cytol. 1983;85:221–252. doi: 10.1016/S0074-7696(08)62374-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wills J. W., Srinivas R. V., Hunter E. Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products. J Cell Biol. 1984 Dec;99(6):2011–2023. doi: 10.1083/jcb.99.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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