Open reading frame 1a-encoded subunits of the arterivirus replicase induceendoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex

The replicase of equine arteritis virus (EAV; family Arteriviridae, order N idovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these p recursors into 12 nonstructural proteins, which direct both genome replicat ion and subgenomic mRNA transcription. Immunofluorescence assays showed tha t most EAV replicase subunits localize to membranes in the perinuclear regi on of the infected cell. Using replicase-specific antibodies and cryoimmuno electron microscopy, unusual double-membrane vesicles (DMVs) were identifie d as the probable site of EAV RNA synthesis. These DMVs were previously obs erved in cells infected with different arteriviruses but,were never implica ted in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicu lar structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase su bunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF 1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.