Journal of General Virology

Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex

Eric J. Snijder,¹ Hans van Tol,¹ Norbert Roos² and Ketil W. Pedersen²

¹ Department of Virology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands
² Department of Biology, Division of Electron Microscopy, University of Oslo, Norway

The replicase polyproteins of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50 % of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.

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© 2001 SGM

This article is now available in the May 2001 print issue of JGV (vol. 82, 985–994). The complete issue of the journal may be seen in electronic form on JGV Online.