Membrane Topology of Pestiviral Nonstructural Protein 2 and Determination of the Minimal Autoprotease Domain

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae. A distinctive feature of the Flaviviridae is the importance of nonstructural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is tem-porally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes. In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the substituted cysteine accessibility method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the endoplasmic reticulum (ER) lumen and is followed by three transmembrane segments (TMs) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine map-ping of the minimal autoprotease domain. Only one TM was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to that of hepatitis C virus (HCV) NS2, our data also reveal poten-tially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future func-tional and structural studies on pestiviral NS2. IMPORTANCE Pestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular chaperone DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports vi-rion morphogenesis. For a better understanding of the underlying molecular interac-tions, topological and structural data are required. The topology-based determina-tion of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as nonstructural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

Automated summary

Pestiviral NS2 plays a central role in regulating RNA replication and virion morphogenesis through its autoprotease activity, which is temporally controlled during the viral life cycle. Experimental determination of the membrane topology of NS2 provides essential insights for understanding its structural and functional properties. This knowledge will support future studies on the role of NS2 in pestiviral RNA replication and virion morphogenesis.