This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oostra, M.
Right arrow Articles by de Haan, C. A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oostra, M.
Right arrow Articles by de Haan, C. A. M.

 Previous Article  |  Next Article 

Journal of Virology, November 2007, p. 12323-12336, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01506-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Localization and Membrane Topology of Coronavirus Nonstructural Protein 4: Involvement of the Early Secretory Pathway in Replication{triangledown}

M. Oostra, E. G. te Lintelo, M. Deijs, M. H. Verheije, P. J. M. Rottier, and C. A. M. de Haan*

Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

Received 10 July 2007/ Accepted 4 September 2007

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].

* Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31 30 253 4195. Fax: 31 30 253 6723. E-mail: C.A.M.deHaan{at}vet.uu.nl

{triangledown} Published ahead of print on 12 September 2007.

Journal of Virology, November 2007, p. 12323-12336, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01506-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Baliji, S., Cammer, S. A., Sobral, B., Baker, S. C. (2009). Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches. J. Virol. 83: 6957-6962 [Abstract] [Full Text]  
  • Oostra, M., Hagemeijer, M. C., van Gent, M., Bekker, C. P. J., te Lintelo, E. G., Rottier, P. J. M., de Haan, C. A. M. (2008). Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning. J. Virol. 82: 12392-12405 [Abstract] [Full Text]  
  • Gadlage, M. J., Graham, R. L., Denison, M. R. (2008). Murine Coronaviruses Encoding nsp2 at Different Genomic Loci Have Altered Replication, Protein Expression, and Localization. J. Virol. 82: 11964-11969 [Abstract] [Full Text]  
  • Neuman, B. W., Joseph, J. S., Saikatendu, K. S., Serrano, P., Chatterjee, A., Johnson, M. A., Liao, L., Klaus, J. P., Yates, J. R. III, Wuthrich, K., Stevens, R. C., Buchmeier, M. J., Kuhn, P. (2008). Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3. J. Virol. 82: 5279-5294 [Abstract] [Full Text]  
  • Posthuma, C. C., Pedersen, K. W., Lu, Z., Joosten, R. G., Roos, N., Zevenhoven-Dobbe, J. C., Snijder, E. J. (2008). Formation of the Arterivirus Replication/Transcription Complex: a Key Role for Nonstructural Protein 3 in the Remodeling of Intracellular Membranes. J. Virol. 82: 4480-4491 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»