The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.The replication or replication/transcription complexes (RTCs) of a wide variety of eukaryotic positive-strand RNA viruses have been found to be associated with (modified) intracellular membranes (for recent reviews, see references 1, 2, 22, 24, 25, and 31). Membrane association of the RTC is thought to be important for creating a suitable (micro)environment for viral RNA synthesis and may also aid in preventing the activation of host defense mechanisms that can be triggered by double-stranded RNA replication intermediates. For several virus groups, replicase subunits have been identified that are involved in targeting the RTC to membrane compartments and/or modifying these membranes, often resulting in vesiculation or the formation of invaginations. Frequently, parts of these nonstructural proteins, which often contain multiple hydrophobic segments, are known or thought to be embedded in the membrane. All major groups of mammalian positive-strand RNA viruses produce their replicative machinery from replicase polyproteins containing both these hydrophobic subunits and the enzymes directly involved in RNA synthesis. Consequently, both the correct proteolytic processing and the membrane association of replicase subunits are important and probably highly coordinated events during the initial stages of the viral life cycle.Equi...