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Journal of Virology, December 2008, p. 12392-12405, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01219-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning{triangledown} ,{dagger}

Monique Oostra, Marne C. Hagemeijer, Michiel van Gent, Cornelis P. J. Bekker, Eddie G. te Lintelo, Peter J. M. Rottier, and Cornelis A. M. de Haan*

Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Received 12 June 2008/ Accepted 30 September 2008

Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps—nsp3, nsp4, and nsp6—multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.

* Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases & 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}uu.nl

{triangledown} Published ahead of print on 8 October 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, December 2008, p. 12392-12405, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01219-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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