Downloaded from www.microbiologyresearch.org by IP: 54.174.126.203 On: Tue, 29 Aug 2017 06:20:13 Journal of General Virology (2001), 82, 985–994. Printed in Great Britain ................................................................................................................................................................................................................................................................................... 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 Roos 2 and Ketil W. Pedersen 2 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. Introduction The replicative proteins (or ‘ replicases ’) of many positive- stranded RNA (RNA) viruses associate with host cell membrane compartments and modify these to establish a complex that is specialized for viral RNA synthesis (see Carette et al., 2000; Chen & Ahlquist, 2000; Egger et al., 2000; Mackenzie et al., 1999 ; Pedersen et al., 1999; Schaad et al., 1997 ; Schlegel et al., 1996; van der Meer et al., 1999; and references therein). Among the intracellular changes that have been documented are membrane proliferation and the modi- fication of different cellular organelles, like those of the endo- and exocytotic pathways, the nucleus, peroxisomes and mitochondria. Although such changes appear to be a general feature of RNA virus replication in eukaryotic cells, little is Author for correspondence : Eric Snijder. Fax 31 71 5266761. e-mail E.J.SnijderLUMC.nl known about the exact role of membranes in viral RNA synthesis. The membranes may play a structural andor functional role by offering a suitable microenvironment for viral RNA synthesis or they may facilitate the recruitment of membrane-associated host cell proteins for the purpose of virus transcription. In many RNA viruses, including all major groups of animal RNA viruses, the membrane association of virus non-structural proteins (nsps) is linked to their generation from large polyprotein precursors by pro- teolytic processing. Consequently, membrane association or translocation, polyprotein cleavage and the initiation of viral RNA synthesis probably occur in a highly co-ordinated fashion during the initial stages of the replication of these viruses. Equine arteritis virus (EAV) (Doll et al., 1957) is the prototype of the family Arteriviridae (Snijder & Meulenberg, 1998), which has been placed in the order Nidovirales, together with the family Coronaviridae (Cavanagh, 1997). The ancestral relationship between the two virus groups is most evident 0001-7499  2001 SGM JIF