The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Cover Page

Abstract


The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.


O. V. Orlova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

V. L. Drutsa

Moscow State University

Email: belj@eimb.ru

Russian Federation

P. V. Spirin

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

A. V. Ivanov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

V. S. Prasolov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

P. M. Rubtsov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

S. N. Kochetkov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: belj@eimb.ru

Russian Federation

S. N. Beljelarskaya

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: belj@eimb.ru

Russian Federation

  1. Hnatyszyn H.J. // Antiviral Therapy 2005, V.10, P.1-11
  2. Baumert T.F., Ito S., Wong D.T., Liang T.J. // Virology Journal 1998, V.72, P.3827-3836
  3. Reed K.E., Rice C.M. // Curr. Top. Microbiol. Immunol. 2000, V.242, P.55-84
  4. Ivanov A.V., Kuzyakin A.O., Kochetkov C.N. // Uspehi biokhimii. (Advances Biochemistry). 2005, V.45, P.37-86
  5. Choo Q.L., Kuo G., Weiner A.J., Overby L.R., Bradley D.W., Houghton M. // Science. 1989, V.244, P.359-362
  6. Burda P., Aebi M. // Biochim. Biophys. Acta. 1999, V.1426, P.239-257
  7. Choukhi A., Ung S., Wychowski C., Dubuisson J. // Virology Journal 1998, V.72, P.3851-3858
  8. Goffard A., Dubuisson J. // Biochimie. 2003, V.85, P.295-301
  9. Op de Beeck A., Cocquerel L., Dubuisson J. // J. Gen. Virol. 2001, V.82, P.2589-2595
  10. Montreuil J., Vliegenthart J.F., Schachter H. // Glycoproteins. Elsevier. 1995, P.1-12
  11. Chapel C., Garcia C., Roingeard Ph. // J. Gen. Virol. 2006, V.87, P.861-871
  12. Helle F., Goffard A., Morel V., Duverlie G., McKeating J., Keck Z.Y., Foung S., Penin F., Dubuisson J., Voisset C. // Virology Journal 2007, V.81, P.8101-8111
  13. Xiang J., Wunschmann S., George S.L., Klinzman D., Schmidt W.N., LaBrecque D.R., Stapleton J.T. // J. Med. Virol. 2002, V.68, P.537-543
  14. Brazzoli M., Helenius A., Foung S.K., Houghton M., Abrignani S., Merola M. // Virol. 2005, V.332, P.438-453
  15. Cocquerel L., Meunier J.C., Op de Beeck A., Bonte D., Wychowski C., Dubuisson J. // J. Gen. Virol. 2001, V.82, P.1629-1635
  16. Mohonov V.V., Novikov E.I., Samohvalov E.I. // Voprocy virusologii. (Problems of Virology). 2002, V.47, P.9-12
  17. Jones D.M., McLauchlan J. // J. Biol. Chem. 2010, V.285, P.22733-22739
  18. Maniatis T., Fritsch E.F., Sambrook J. // Molecular cloning. A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Lab. Press, 1982 1982
  19. // Bac-to-Bac Baculovirus Expression System. Instruction Manual. St. Louis, MO: Life Technologies, Inc., Monsanto Corp. Res. 1993 1993
  20. Beljelarskaya S.N., Koroleva N.N., Popenko V.I., Drutsa V.L., Orlova O.V., Rubtsov P.M., Kochetkov S.N. // Molekulyar. biologiya. (Molecular biology). 2010, V.44, P.107-119
  21. Drutsa V.L., Kaberdin V.R., Koroleva O.N., Shilov I.O. // Bioorgan. khimiya. (Bioorganic chemistry). 1991, V.17, P.1487-1493
  22. Timohova A.V., Bakinovskii L.V., Zinin A.I., Popenko V.I., Ivanov A.V., Rubtsov P.M., Kochetkov S.N., Beljelarskaya S.N. // Moleculyar. biologiya. (Molecular biology). 2012, V.46, P.644-653
  23. Orlova O.V., Drutsa V.L., Spirin P.V., Popenko V.I., Prasolov V.S., Rubtsov P.M., Kochetkov S.N., Beljelarskaya S.N. // Moleculyar. biologiya. (Molecular biology). 2013, V.47, P.147-156
  24. Clayton R.F., Owsianka A., Aitken J., Graham S., Bhella D., Patel A.H. // Virology Journal 2002, V.76, P.7672-7682
  25. McEwen C.R. // Anal. Biochem. 1967, V.20, P.114-149
  26. Helle F., Vieyres G., Elkrief L., Popescu C.I., Wychowski C., Descamps V., Castelain S., Roingeard P., Duverlie G., Dubuisson J. // Virology Journal 2010, V.84, P.11905-11915
  27. Tryatni M., Saunier B., Maruvada P., Davis A.R., Ulianich L., Heller T., Patel A., Kohn L.D., Liang T.J. // Virology Journal 2002, V.76, P.9335-9344

Views

Abstract - 218

PDF (English) - 77

PDF (Russian) - 83

Cited-By


PlumX

Dimensions

Refbacks

  • There are currently no refbacks.

Copyright (c) 2015 Orlova O.V., Drutsa V.L., Spirin P.V., Prasolov V.S., Rubtsov P.M., Kochetkov S.N., Beljelarskaya S.N.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies