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Journal of Virology, September 2003, p. 9931-9942, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9931-9942.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Plasma Membrane Budding as an Alternative Release Mechanism of the Extracellular Enveloped Form of Vaccinia Virus from HeLa Cells

Andrea Meiser, Carmen Sancho, and Jacomine Krijnse Locker*

Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Received 14 March 2003/ Accepted 19 June 2003

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [35S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.

* Corresponding author. Mailing address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Phone: 49-6221-387508. Fax: 49-6221-387306. E-mail: krijnse{at}embl.de.

Journal of Virology, September 2003, p. 9931-9942, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9931-9942.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Okeke, M. I., Nilssen, O., Traavik, T. (2006). Modified vaccinia virus Ankara multiplies in rat IEC-6 cells and limited production of mature virions occurs in other mammalian cell lines. J. Gen. Virol. 87: 21-27 [Abstract] [Full Text]  
  • Senkevich, T. G., Moss, B. (2005). Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion. J. Virol. 79: 4744-4754 [Abstract] [Full Text]  
  • Senkevich, T. G., Ward, B. M., Moss, B. (2004). Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene. J. Virol. 78: 2357-2366 [Abstract] [Full Text]  


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